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1
Gobessi, Stefania, Francesca Belfiore, Sara Bennardo, Brendan Doe, Luca Laurenti та Dimitar G. Efremov. "Expression of ZAP-70 Does Not Accelerate Leukemia Development and Progression in the Eμ-TCL1 Transgenic Mouse Model of Chronic Lymphocytic Leukemia". Blood 120, № 21 (2012): 925. http://dx.doi.org/10.1182/blood.v120.21.925.925.
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Abstract Abstract 925 One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered. We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower. Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011). Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth. To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024). In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses. Disclosures: No relevant conflicts of interest to declare.
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Larsson, Connie A., Kensuke Kojima, Yong Wang та ін. "BET Bromodomain Inhibition Reduces Leukemic Burden and Prolongs Survival In The Eμ-TCL1 Transgenic Mouse Model Of Chronic Lymphocytic Leukemia (CLL) Independent Of TP53 Mutation Status". Blood 122, № 21 (2013): 876. http://dx.doi.org/10.1182/blood.v122.21.876.876.
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Abstract Introduction Inhibition of Brd4, a bromodomain and extra-terminal (BET) protein, results in antiproliferative effects and terminal differentiation in the MYC-driven B-cell malignancies multiple myeloma and Burkitt's lymphoma by selectively repressing MYC. Elevated MYC expression correlates with progression of B-cell chronic lymphocytic leukemia (B-CLL) marking Brd4 as a potential therapeutic target. 17p deletion or somatic TP53 mutations are the poorest prognostic factors in B-CLL, resulting refractoriness to conventional therapy. The recent identification of a Brd4-interacting protein as a validated target in CLL led us to evaluate the potential of BET inhibition, using jq1, for the treatment of B-CLL. Results Jq1 remarkably reduced proliferation of the immortalized human B-CLL cell lines, Mec-1, Mec-2, and WaC3 (IC50 values: 1.12, 0.68, and 0.64 μM, respectively). These results were validated in 13 frozen and 7 fresh primary whole peripheral blood B-CLL samples (median EC50 0.66 μM [range, 0.051 to 2.169 μM]) using a FACS-based proprietary platform (Vivia Biothech). Eμ-TCL1 (n=13) & Eμ-TCL1:p53R172H/+ (n=9) mice treated with intraperitoneal (IP) injections of jq1 at 50 mg/kg/day for 21 consecutive days had a significant reduction in lymphocytes and white blood cells (WBC) (with excellent tolerance and no off-target effects on hemoglobin level or platelet counts) compared to vehicle-treated mice. The median lymphocyte count at baseline and after 2 weeks of jq1 therapy was 13.4 & 5.92 x10^3/μL (p=0.008) for Eμ-TCL1 mice and 13.2 & 7.2 x10^3/μL (p=0.009) for Eμ-TCL1:p53R172H/+ mice. jq1 therapy significantly prolonged survival in both Eμ-TCL1 (428 vs 377 days, p=0.05) and Eμ-TCL1:p53R172H/+ (382.5 vs 300 days, p=0.0003) mice compared to mice receiving vehicle. FACs analysis was performed on blood taken from Eμ-TCL1 (n=5) and Eμ-TCL1:p53R172H/+ (n=6) mice at baseline and after 7 daily IP jq1 doses. Eμ-TCL1:p53R172H/+ mice showed a significant reduction in CD5+/CD19+ lymphocytes (mean difference, 14.37 [95% CI, 2.4 to 26.3]; p=0.03), which was not observed in Eμ-TCL1 mice. Cell cycle analysis of cultured Eμ-TCL1 splenocytes treated with jq1 showed a significant increase (p<0.0005) in the number of sub-G1 cells compared to untreated splenocytes, regardless of p53 status. Sensitivity against fludarabine was also tested in these samples and we show that 10μM fludarabine did not elicit significant changes in the number of cells in sub-G1 compared to untreated cells but a significant difference (p=0.009) was observed between splenocytes treated with 10μM fludarabine and 1μM jq1. In depth analyses of RNA sequencing data of splenocytes from 10 matched-pairs samples (5 Eμ-TCL1 & 5 Eμ-TCL1:p53R172H/+) treated in culture with 0 or 1μM jq1 for 24 hours are currently underway to identify BET targets in B-CLL, but preliminary gene expression analysis reveal that the sensitivity of B-CLL to BET inhibition is not Myc-dependent despite elevated Myc levels. Notably, c-Fos was one of the most significant differentially expressed genes, regardless of p53 status. Interestingly, AP-1 complex, a heterodimer of c-Fos and c-Jun, is inhibited by direct interaction with Tcl-1. We are currently validating these findings in additional samples and performing functional assays to gain a better understanding of BET targets in B-CLL. Conclusion BET bromodomain inhibition markedly reduced proliferation in both human and mouse B-CLL samples. Furthermore, the loss of wild-type p53 activity did not desensitize CLL cells to jq1, which was also effective at inducing massive cell death in fludarabine resistant CLL cells, regardless of p53 status. Mice receiving jq1 exhibited a significant reduction in leukemic burden and prolonged survival compared to control mice, with a more pronounced effect observed in mice with mutant p53. These data demonstrate the potent effect of BET inhibition in B-CLL and more importantly, provide evidence as an effective treatment for aggressive forms of B-CLL with 17p deletion or TP53 mutation or chemoresistant refractory disease. Disclosures: Primo: Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Martinez:Vivia Biotech: Employment. Bradner:Tensha Therapeutics: Dr. Bradner is a scientific founder of Tensha Therapeutics, which is developing drug-like derivatives of the JQ1 bromodomain inhibitor as cancer therapeutics, through a license from the Dana-Farber Cancer Institute. Other. Ballesteros:Vivia Biotech: Employment.
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Bennardo, Sara, Stefano Iacovelli, Stefania Gobessi та ін. "The Nature of the Antigen Determines Leukemia Development and Behavior in the Eμ-TCL1 Transgenic Mouse Model of CLL". Blood 120, № 21 (2012): 181. http://dx.doi.org/10.1182/blood.v120.21.181.181.
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Abstract Abstract 181 Studies conducted over the past decade have revealed a strong association between the mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes and clinical course in patients with chronic lymphocytic leukemia (CLL). In patients with aggressive CLL, the leukemic cells typically express B cell receptors (BCRs) encoded by unmutated IGHV genes, whereas these genes are most often mutated in leukemic cells from patients with indolent disease. The mutational status of the IGHV genes reflects features of the antigen, such as antigen structure, form, presentation and affinity, indicating that the difference in the clinical course between IGHV-unmutated and IGHV-mutated CLL could be due to recognition of different types of antigens. In line with this possibility, recent studies have shown that IGHV-unmutated CLL (U-CLL) cells frequently express polyreactive BCRs that bind with low affinity to both microbial antigens and autoantigens translocated or exposed on apoptotic cells, whereas such reactivity is infrequent in IGHV-mutated CLL (M-CLL). To further explore the possibility that the clinical course in CLL is determined by the availability of particular types of antigenic stimuli, we investigated the impact of different antigen/BCR interactions on leukemia development and behavior in the Eμ-TCL1 transgenic mouse model of CLL. We initially established three cohorts of Eμ-TCL1 transgenic mice that expressed transgenic BCRs with different antigen specificity. Two of these cohorts expressed low-affinity unmutated transgenic BCRs reactive with the antigens phosphatidylcholine (PtC) and Sm (IgPtC and IgSm, respectively), whereas the third cohort expressed a high-affinity mutated transgenic BCR (IgHEL) specific for the antigen hen egg lysozyme (HEL). Of note, Sm is a ribonucleoprotein complex that is translocated to the surface of apoptotic cells and has been shown to be recognized by certain human U-CLL BCRs, whereas PtC is a cell membrane component that is exposed on senescent red blood cells and gut bacteria. Because no data are currently available regarding the reactivity of the M-CLL BCRs, we subdivided the cohort of Eμ-TCL1/IgHEL double transgenic mice into four additional cohorts. These included a cohort without antigen (Eμ-TCL1/IgHEL), a cohort in which HEL was provided as a foreign antigen (Eμ-TCL1/IgHEL double transgenic mice repetitively immunized with particles coated with HEL and CpG oligonucleotides), a cohort in which HEL was provided as a soluble autoantigen (Eμ-TCL1/IgHEL/sHEL triple transgenic mice) and a cohort in which HEL was provided as a membrane-bound autoantigen exposed on apoptotic cells (Eμ-TCL1/IgHEL/mHEL-KK triple transgenic mice). Each cohort consisted of 12–14 animals, of which at least 8 have been followed for >1 year. Animals from all cohorts developed CD5-positive B cell leukemias, but only in Eμ-TCL1/IgSm and Eμ-TCL1/IgPtC mice the leukemic cells expressed a transgenic BCR. In Eμ-TCL1/IgHEL mice the leukemias were always derived from the small percentage of B cells that express an endogenous BCR, whereas B cells that express the transgenic IgHEL BCR were never transformed. Interestingly, leukemia development and progression was more rapid in Eμ-TCL1/IgPtC than Eμ-TCL1/IgSm transgenic mice (7/14 at 6 months of age and 2/10 at 8 months of age, respectively). Since PtC is expressed as both a foreign- (gut flora) and self- (senescent red blood cells) antigen, we investigated whether suppression of gut flora will affect the growth of adoptively transferred Eμ-TCL1/IgPtC leukemias. Pretreatment of syngeneic recipient mice with a three-week course of broad-spectrum antibiotics significantly delayed leukemia growth, suggesting that PtC is more potent in driving the expansion of the leukemic clone when expressed as a foreign than self antigen. To summarize, these data demonstrate that U-CLL can be induced by both microbial antigens and autoantigens exposed on apoptotic cells, including autoantigens that are recognized by human CLL cells, such as Sm. In contrast, M-CLL can not be induced by chronic or repetitive antigen stimulation, regardless whether the antigen is provided as a foreign antigen, as a soluble autoantigen, or as a membrane-bound autoantigen exposed on apoptotic cells. Collectively, these data suggest that the mechanisms that drive U-CLL and M-CLL are different and indicate that only U-CLL is an antigen-driven disease. Disclosures: No relevant conflicts of interest to declare.
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Wu, Qingli, Zierold Claudia, and Erik A. Ranheim. "Dysregulation of Frizzled 6 Is a Critical Component of B Cell Leukemogenesis in a Mouse Model of Chronic Lymphocytic Leukemia." Blood 110, no. 11 (2007): 347. http://dx.doi.org/10.1182/blood.v110.11.347.347.
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Abstract Wnt/Fzd signaling is known to play a key role in development, tissue specific stem cell maintenance, and tumorigenesis, particularly through the canonical pathway involving stabilization of β-catenin. We have previously shown that Fzd9−/− mice exhibit a decrease in pre-B cells at a stage when self-renewing division is occurring in preference to further differentiation, prior to light chain immunoglobulin recombination. To determine whether pathologic usurpation of this pathway plays a role in B cell leukemogenesis, we examined the expression of Wnt/Fzd pathway genes in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia (CLL). We find that in the course of leukemogenesis, the expression of Wnt16, Wnt10b, Fzd1, and most dramatically, Fzd6, are progressively upregulated in the transformed CD5+ B cells of these mice, as are β-catenin protein levels. Fzd6 expression is increased an average of 35-fold in tumor B cells in comparison to CD5- normal B cells in the same mouse. In 3 animals we were able to compare oligoclonal, preleukemic CD5+ B cells with monoclonally transformed CD5+ B cells and noted between 4 and 340 fold incremental increases in Fzd6 expression. Elimination of Fzd6 gene expression by crossing Eμ-TCL1 into Fzd6−/− mice significantly delays or eliminates development of CLL in this model, while crossing into Fzd9−/− mice has no effect. Greater than 50% of Fzd6+/+ X Eμ-TCL1 mice have evidence of CLL in the peripheral blood at 5 months of age, while in Fzd6−/− X Eμ-TCL1 mice, this occurs at 8 months (p < 0.001). Even at 1 yr. of age, >30% of Fzd6−/− X Eμ-TCL1 mice remain disease free while >95% of Fzd6+/+ X Eμ-TCL1 mice have leukemia at 7 months of age. Tumors that do arise in Fzd6−/− mice are morphologically similar to the usual Eμ-TCL1 CLL cells, but do not show upregulation of β-catenin protein by flow cytometry, suggesting that Fzd6 is responsible for this aberrant expression. Further, rather than upregulating Wnt16, Wnt10b, and Fzd1, these genes are down-modulated in tumor cells lacking Fzd6, suggesting a balance between positive and negative signals in CLL B cells mediated by the Wnt/Fzd pathway with Fzd6 acting as an oncogene and other family members potentially as tumor suppressors. We and others have noted increases in LEF-1, Wnt10b, Wnt16, and Fzd3 in human CLL B cells as well, mirroring the findings in the mouse model. Our findings suggest that the self-renewal signals mediated by Wnt/Fzd that are enlisted during B cell development may be pathologically reactivated in the neoplastic transformation of mature B cells; and that agents targeting specific members of the beta-catenin signaling pathway may have therapeutic impact that could be tested in the Eμ-TCL1 model.
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Bellone, Matteo, Paolo Dellabona, Arianna Calcinotto, et al. "CD4+ T Cells Sustain Aggressive Chronic Lymphocytic Leukemia through a CD40L-Independent Mechanism." Blood 134, Supplement_1 (2019): 683. http://dx.doi.org/10.1182/blood-2019-128246.
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In Chronic Lymphocytic Leukemia (CLL), mature CD5+ B cells accumulate in lymphoid organs such as bone marrow and lymph nodes where they proliferate and expand within localized proliferation centers. In vitro and in vivo data suggest that survival and proliferation of CLL cells within proliferation centers may be also dependent on microenvironmental interactions originating from the surrounding cellular elements (e.g. monocyte-derived nurse-like cells, mesenchymal stromal cells, or CD4+ T lymphocytes), that deliver both membrane-bound and soluble signals to CLL cells. In particular, the role of CD4+ T cells in vivo is less defined and data in animal models are conflicting as they appear to sustain CLL clone expansion and survival through CD40L-CD40 interactions, though in approximately 40% of patients with CLL, aggressive leukemic clones appear to be independent of CD40 stimulation. We aimed at clarifying the role of CD4+ T lymphocytes taking advantage of the Eμ-TCL1 mouse model, which develops a disease that mimics aggressive, human CLL. To this aim, we generated genetically modified Eμ-TCL1 mice lacking either CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-). In these mice, disease appearance and progression were monitored in lymphoid organs and blood by flow cytometry and immunohistochemistry analyses. Findings were confirmed by adoptive transfer of leukemic clones into mice either lacking CD4+ T cells, or CD40L, or treated with monoclonal antibodies depleting selected T cell populations, or blocking CD40L-CD40 interactions. Interestingly, we observed that CLL clones did not expand in mice either lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development in Eμ-TCL1 mice. On the contrary, in TCL1+/+TAP-/- mice, lacking CD8+ T cells, disease progression was accelerated, suggesting an anti-tumor activity exerted by this subset of T cells. Specificity of CD4+ T cells was marginal for CLL development, as leukemic clones developed regularly in transgenic mice whose CD4+ T cells had TCR with CLL-unrelated specificities. Similarly, TCL1+/+CD40-/- mice developed frank CLL with no differences compared to controls, as well as leukemic clones expanded when transferred into wild type mice treated with monoclonal antibodies blocking CD40, or into CD40L-/- mice, suggesting a dispensable role for CD40/CD40L stimulation in the development of murine CLL. Analysis of peritoneal fluid, spleen, lymph nodes and bone marrow showed similar CLL development in Eμ-TCL1 and TCL1+/+CD40-/- mice. In conclusion, our data demonstrates that CD8+ and CD4+ T cells exert opposite roles in CLL: CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the expansion of CLL clones in Eμ-TCL1 mice through CD40-indipendent, and apparently non-cognate mechanisms. Further studies are warranted to dissect the nature of the molecules, either soluble or membrane-bound, responsible for the interactions occurring between CD4+ T cells and CLL B cells fueling the onset and expansion of CLL cells. Disclosures Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy.
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Alhakeem, Sara S., Mary K. McKenna, Sunil K. Nooti, et al. "Suppression of Anti-Tumor Immunity in Chronic Lymphocytic Leukemia Via Interleukin-10 Production." Blood 128, no. 22 (2016): 3215. http://dx.doi.org/10.1182/blood.v128.22.3215.3215.
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Abstract The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which is characterized by a progressive accumulation of abnormal B-lymphocytes in blood, bone marrow and secondary lymphoid organs. Typically disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study the basis of this immunoregulation, we used cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL due to a B-cell specific expression of the oncogene, Tcl1. Previously we showed that Eμ-Tcl1 CLL cells constitutively produce an anti-inflammatory cytokine, IL-10. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not affect the survival of CLL cells in vitro. On the other hand, adoptively transferred Eμ-Tcl1 cells grew at a slower rate in IL-10R KO mice vs. wild type (WT) mice. There was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO compared to WT mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice compared to those purified from CLL-injected IL-10R KO mice. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production. This led us to investigate possible mechanisms by which IL-10 is produced. We found a novel role of B-cell receptor (BCR) signaling pathway in constitutive IL-10 secretion. Inhibition of Src or Syk family kinases reduces the constitutive IL-10 production by Eμ-Tcl1 cells in a dose dependent manner. In addition, we found that Eμ-Tcl1 CLL cells exhibit clonal variation in their IL-10 production in response to BCR cross-linking. Further studies are being performed to understand the mechanisms by which BCR signaling affects IL-10 production. Disclosures No relevant conflicts of interest to declare.
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McKenna, Mary Kathryn, Sunil K. Nooti, Sara Samir Alhakeem, et al. "Role of Prostate apoptosis response-4 tumor suppressor in the survival and growth of Chronic Lymphocytic Leukemia." Journal of Immunology 196, no. 1_Supplement (2016): 72.15. http://dx.doi.org/10.4049/jimmunol.196.supp.72.15.
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Abstract Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. Prostate apoptosis response-4 (Par-4) is a pro-apoptotic tumor suppressor protein, which is silenced by promoter methylation in more than 30% of all cancers. It is also secreted and induces apoptosis selectively in many types of cancer cells but not in normal cells. Here we characterized the role of Par-4 in CLL cells using a murine model. The Eμ-Tcl1 mouse serves as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age, due to B cell specific over-expression of the oncogene, T cell Leukemia 1 (Tcl1). Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 CLL mice into recipient syngeneic mice leads to the development of a CLL like disease within 3–5 weeks of transfer. Surprisingly Eμ-Tcl1 CLL cells constitutively express more Par-4 than normal B-1 or B-2 cells in mice. These CLL cells also secrete Par-4 which is functional in being cytotoxic to prostate cancer cells. We showed that Eμ-Tcl1 CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling causes a decrease in Par-4 expression and increases apoptosis. Interestingly, we have found that shRNA mediated knockdown of Par-4 in human CLL cell lines, results in their reduced growth. These results suggest that intrinsic Par-4 may play a pro-survival rather than pro-apoptotic role in CLL. We are currently investigating the mechanisms underlying this novel role of Par-4 and regulation of its expression in CLL cells using CRISPR system to knockout Lyn and Btk in CLL cells.
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McClanahan, Fabienne, Cristina Ghirelli, Paul Greaves та ін. "Inhibitory Ligands CD200, CD270, CD274 and CD276 Are Expressed On Eμ-TCL1 Transgenic Mouse Splenocytes and Are of Potential Relevance to Impaired T-Cell Function in Vivo". Blood 120, № 21 (2012): 313. http://dx.doi.org/10.1182/blood.v120.21.313.313.
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Abstract Abstract 313 Background: We have previously demonstrated that CD4 and CD8 T-cells from CLL patients show profound dysfunctions in multiple gene pathways, including the actin cytoskeleton, which impairs the formation of functional immunologic synapses between T cells and APCs. Functional screening assays on Mec-1 cells have identified CD200, CD270, CD274, and CD276 as inhibitory ligands which induce impaired actin synapse formation in both allogeneic and autologous T cells. We also demonstrated that the Eμ-TCL1 transgenic mouse model of CLL closely resembles the T-cell defects observed in humans, validating it as a valuable preclinical tool to examine changes in the microenvironment alongside the development of leukaemia. The aim of the current study is to investigate the role of CD200, CD270, CD274, and CD276 in the Eμ-TCL1 model. Methods: We used multiparameter flow cytometry to establish the expression of inhibitory ligands on CD19+/CD5+ unpurified splenocytes from Eμ-TCL1 mice on both the C57Bl/6 (B6) and the C3HB6-F1 background and compared this to unpurified splenocytes from age matched wild-type (WT) controls of the respective coisogenic strain. Results: A total of 19 leukemic Eμ-TCL1 (n=10 C57Bl/6 and n=9 C3HB6-F1 background) and 11 WT mice (n=6 C57Bl/6 and n=5 C3HB6-F1 background) were examined. CD19+/CD5+ CLL cells constituted 92% (range 62–97%) of the DAPI-negative lymphocyte population. On CD19+/CD5+ CLL cells, CD274 (mean 98% ± SEM 0.4) and CD200 (mean 84% ± SEM 2.9 were uniformly strongly expressed, while CD270 (mean 74% ± SEM 4.7) and CD276 (mean 50% ± SEM 6.6) showed a weaker and more diverse expression, with no significant differences between the two backgrounds (all p>.05). Similar expression patterns were observed in Eμ-TCL1 mice with spontaneously occurring CLL and transplanted transgenic mice, with no differences between spontaneous and induced CLL (all p>.05). We then compared transgenic CD19+/CD5+ CLL cells to the WT CD19+ and the WT CD19+/CD5+ B1a-like cell population. Eμ-TCL1 CLL splenocytes showed a significant higher expression of CD274 and CD276 compared to expression on WT CD19+ (p<.0001, p=.00349) splenocytes. When compared to WT B1a-like splenocytes, only CD274 was significantly higher expressed (p<.0001). To clarify the impact of genetic strain, B6 and C3HB6-F1 were investigated separately: transgenic mice on the B6 background showed significantly higher expression of CD274 compared to WT B6 CD19+ (p=.0015) and WT B6 B1a-like (p<.0001) splenocytes. In contrast, transgenic mice on the C3HB6-F1 background showed a significant higher expression of CD274 and CD276 compared to WT CD19+ (p=.0002, p=.00354) and WT B1a-like (p=.0005, p=.00384) splenocytes. These patterns substantiate differences of the expression of inhibitory ligands between the WT strains, but of note, these were not mirrored in TCL1 mice. In previous experiments, we used the Eμ-TCL1 model to investigate the polarization of F-actin and phosphotyrosine at the immune synapse between splenic autologous T-cells and APCs and subsequent effector function. Age-matched WT mice had a significantly higher accumulation than transgenic mice. To assess the functional role of inhibitory ligands, knock-down experiments using lentiviral shRNA and blocking antibodies are currently under way to assess if this restores immune synapse formation and T cell effector function in vivo. Conclusions: The inhibitory ligands CD200, CD270, CD274 and CD276 are expressed in vivo and appear to be of functional relevance for the anti-cancer immune response. They therefore represent attractive targets to restore T-cell effector function, which might be achieved by gene therapy approaches and blocking antibodies. Disclosures: Gribben: Celgene: Honoraria.
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Kriss, Crystina L., Javier A. Pinilla-Ibarz, Adam W. Mailloux, et al. "Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice." Blood 120, no. 5 (2012): 1027–38. http://dx.doi.org/10.1182/blood-2011-11-394346.
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Abstract Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ∼ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eμ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eμ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igβ, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.
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Enzler, Thomas, Arnon P. Kater, Weizhou Zhang та ін. "Chronic lymphocytic leukemia of Eμ-TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression". Blood 114, № 20 (2009): 4469–76. http://dx.doi.org/10.1182/blood-2009-06-230169.
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AbstractResults of heavy-water labeling studies have challenged the notion that chronic lymphocytic leukemia (CLL) represents an accumulation of noncycling B cells. We examined leukemia cell turnover in Eμ-TCL1 transgenic (TCL1-Tg) mice, which develop a CLL-like disease at 8 to 12 months of age. We found that leukemia cells in these mice not only had higher proportions of proliferating cells but also apoptotic cells than did nonleukemic lymphocytes. We crossed TCL1-Tg with BAFF-Tg mice, which express high levels of CD257. TCL1×BAFF-Tg mice developed CLL-like disease at a significantly younger age and had more rapid disease progression and shorter survival than TCL1-Tg mice. Leukemia cells of TCL1×BAFF-Tg mice had similar proportions of proliferating cells, but fewer proportions of dying cells, than did the CLL cells of TCL1-Tg mice. Moreover, leukemia cells from either TCL1×BAFF-Tg or TCL1-Tg mice produced more aggressive disease when transferred into BAFF-Tg mice than into wild-type (WT) mice. Neutralization of CD257 resulted in rapid reduction in circulating leukemia cells. These results indicate that the leukemia cells of TCL1-Tg mice undergo high levels of spontaneous apoptosis that is offset by relatively high rates of leukemia cell proliferation, which might allow for acquisition of mutations that contribute to disease evolution.
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Nganga, Vincent K., Victoria L. Palmer, Hina Naushad та ін. "Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1". Blood 121, № 19 (2013): 3855–66. http://dx.doi.org/10.1182/blood-2012-08-446732.
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Key Points Expressing dominant-negative RAG1 to inhibit BCR editing of autoreactivity in CLL-prone Eμ-TCL1 mice accelerates disease onset. Gene expression profiling studies provide evidence of distinct but convergent pathways for CLL development.
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Grioni, Matteo, Arianna Brevi, Elena Cattaneo та ін. "CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism". Blood Advances 5, № 14 (2021): 2817–28. http://dx.doi.org/10.1182/bloodadvances.2020003795.
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Abstract Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.
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Wu, Qingli, and Erik A. Ranheim. "Upregulation of Genes Involved in the Beta-Catenin Signaling Pathway in a Mouse Model of Chronic Lymphocytic Leukemia." Blood 104, no. 11 (2004): 1121. http://dx.doi.org/10.1182/blood.v104.11.1121.1121.
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Abstract Binding of Wnt ligands to Frizzled (Fzd) family and LRP5/6 receptors results in stabilization of beta-catenin protein, its translocation to the nucleus, and in association with LEF/TCF family transcription factors, expression of target genes. This canonical Wnt signaling pathway plays a critical role in development and in the maintenance of tissue specific stem cell populations in the skin, gut, and bone marrow. Dysregulation of the beta-catenin signaling pathway has been described in numerous human malignancies, including chronic lymphocytic leukemia (CLL). Using the Eμ-TCL1 transgenic mouse model of CLL developed by C. Croce et al., we have examined expression of various components of the canonical Wnt pathway during lymphomagenesis by RT-PCR. The Eμ-TCL1 mouse spontaneously develops a hyperplasia of CD5+ B cells in the peripheral blood and peritoneum which progresses towards a monoclonal B cell leukemia/lymphoma with infiltration of spleen, bone marrow, and other organs. This provides the ability, largely lacking in human subjects, to analyze changes in gene expression during development of a lymphoid malignancy. We have FACS sorted normal CD5- B cells as well as CD5+ B cells from the hyperplastic, oligoclonal and malignant, monoclonal phases of leukemia development in Eμ-TCL1 mice and assessed their expression of various members of the Wnt, Fzd, LRP coreceptor, and LEF/TCF transcription factor families. Coreceptor LRP6 is expressed in all populations in approximately equal amounts as determined by quantitative RT-PCR. We find no expression of Fzd receptors 1, 2, 3, 4, or 9, nor Wnt4 or 5a in any population. We find mRNA for Fzd6 and LEF-1 are increased in hyperplastic and malignant CD5+ B cells in Eμ-TCL1 mice as compared to CD5- polyclonal B cells from the same animals. Of interest, both LEF-1 and Fzd6 expression also have been shown to be upregulated in human CLL B cells. To further assess Fzd6 expression during malignant transformation, we took advantage of the fact that only 3–5% of mouse B cells express lambda light chain. Therefore, large expansions of lambda expressing CD5+ B cells are most likely to be monoclonal in Eμ-TCL1 mice. In three separate mice, oligoclonal expansions of CD5+lambda- B cells coexisted with a clonal expansion of CD5+lambda+ cells, as confirmed by PCR analysis of germline immunoglobulin DNA. These two populations, as well as polyclonal CD5- B cells, were purified by FACS; and Fzd6 expression was quantified by real time RT-PCR. Fzd6 mRNA was increased in the oligoclonal CD5+ B cells by an average of 15.9 fold over the normal CD5- B cells (range 4.1-36.9). In the monoclonal lambda expressing subset, Fzd6 expression was increased 41.7 fold (range 23.7–68.3) over CD5- B cells, and increased over that seen in the oligoclonal CD5+ B cells of the same animal by 4.1 fold (range 1.9–5.7). These findings show that progressively increased expression of Fzd6 correlates with malignant transformation in B cell leukemogenesis in the Eμ-TCL1 mouse model and parallels the findings in fully transformed CLL B cells in humans. The functional consequences of enhanced Fzd6 and other components of the canonical Wnt signaling pathway in neoplastic B cells are currently being investigated.
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Ramsay, Alan G., Gullu Gorgun, Tobias A. W. Holderried, et al. "A Mouse Model for Immunotherapeutic Reversal of Leukemia-Induced T Cell Dysfunction." Blood 112, no. 11 (2008): 30. http://dx.doi.org/10.1182/blood.v112.11.30.30.
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Abstract Understanding the elusive mechanisms of tumor-driven immune evasion will aid the refinement of existing cancer immunotherapy strategies and identify novel treatments. To date, pre-clinical animal models that closely model human cancer, including the immune suppressive mechanisms utilized by cancer cells, have been under-characterized. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in non-malignant cells by cancer, we examined T cell function in Eμ-TCL1 transgenic mice as they developed leukemia from 12-months of age. Transgenic expression of TCL1 in B cells had no demonstrable effect on T cells, however, mice with leukemia had decreased in vivo antigen specific T cell activation, suppressed T cell mitogenic proliferation and impaired induction of idiotype specific CD8 T cells capable of killing CLL cells compared to control WT mice (age-matched throughout study) or Eμ-TCL1 transgenic mice without CLL. Leukemic mice also had dysfunctional T cell lymphokine production (Th2-preponderant). To understand the molecular basis for the observed functional defects and to compare changes seen in mice and patients with CLL we performed gene expression profiling. Analysis of highly purified CD4 and CD8 T cells in CLL mice demonstrated altered gene expression profiles compared to WT mice or to young Eμ-TCL1 mice without disease. Of note, infusion of CLL cells into young Eμ-TCL1 mice induced gene expression changes comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T cell defects. Analysis of gene expression changes in T cells in CLL mice compared with those in patients was performed using RESOURCERER, a database for annotating and linking microarray resources within and across species and identified 50 overlapping genes in CD4 T cells and 45 overlapping genes in CD8 T cells. The majority of differentially expressed genes in CD4 T cells from both mice and patients with CLL were involved in cell proliferation and activation pathways with increase in Lck. Multiple defects within the actin cytoskeletal formation pathways were identified in both CD4 and CD8 T cells including Cdc42. Integrity of the T cell cytoskeleton is essential to regulate the dynamic signaling required for T cell activation and effector function in response to immunological recognition of antigen-presenting cells (APCs). T cell conjugates from mice with leukemia had suppressed antigen-dependent F-actin accumulation and early T cell signaling at the immune synapse with CLL cells (APCs) compared to WT mice conjugates. Moreover, we have demonstrated that infusion of CLL cells into young mice induces this T cell defect, demonstrating an in vivo immunomodulating mechanism utilized by tumor cells. Treatment of both CLL cells and autologous T cells from leukemic mice with lenalidomide (0.5 μM for 24 h) enhanced the formation of the F-actin immune synapse and recruitment of tyrosine-phosphorylated proteins irrespective of the presence of exogenous antigen. Of note, the capacity to repair immunological recognition with this agent was associated with increased recruitment of the cytoskeletal signaling molecules Lck and Cdc42 to the immunological synapse, regardless of whether the gene was increased or decreased on gene expression profiling. These results demonstrate that leukemia cells induce changes in multiple T cell pathways regulating antigen recognition and effector function. The similarities with human CLL including reversible immunological synapse dysfunction with an immunomodulating drug validates the use of Eμ-TCL1 mice as a model for further analyses of ways to prevent and reverse cancer-induced immune dysfunction. The use of this model to understand and reverse the molecular changes in T cells induced by leukemia will likely have broad applications to maximize immune responses in patients.
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Reinart, Nina, Malgorzata Ciesla, Cornelia Rudolph, et al. "Macrophage Migration Inhibitory Factor (MIF) Promotes the Development of Murine Chronic Lymphocytic Leukemia (CLL)." Blood 112, no. 11 (2008): 27. http://dx.doi.org/10.1182/blood.v112.11.27.27.
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Abstract Introduction: Tumor formation results from a complex interplay between genetic/epigenetic alterations, cell cycle dysregulation and promotion by the tumor environment. Stimulation by extracellular survival factors is important for chronic lymphocytic leukemia (CLL), since the leukemic cells undergo spontaneous apoptosis when removed from their normal milieu. Since preliminary experiments demonstrated that macrophage migration inhibitory factor (MIF), a chemokine-like proinflammatory mediator and an intracellular regulator of growth and apoptosis, is overexpressed in human CLL, we investigated whether MIF participates in the pathogenesis of murine CLL. Methods: We studied the role of MIF in CLL by crossing the Eμ-TCL1-transgenic mouse model with MIF knockout (MIF−/−) mice. B-cell-specific overexpression of T cell leukemia-1 (TCL1) leads to accumulation and proliferation of IgM+/CD5+ mature B-cells via activation of AKT. This results in a CLL-like disease with peripheral lymphocytic leukemia, lymphadenopathy, splenomegaly, BM infiltration and premature death after 8–15 months. TCL1+/wtMIF−/− and TCL1+/wtMIF+/+ mice were compared with respect to leukemia development, tumor burden, cytogenetics and survival. Results: The MIF receptors CD74/CD44 and CXCR2 are expressed on murine B-cells. TCL1+/wtMIF+/+ mice exhibited increased numbers of IgM+/CD5+ B-cells already in the preleukemic phase at month 3 and developed overt leukemia (WBC &gt; 20G/l) 3 months earlier than their MIF−/− counterparts (p = 0.02). Leukemia load at 12 months of age as measured by hepatosplenomegaly was increased in TCL1+/wtMIF+/+ animals and lymphatic organs were densely infiltrated by small, mature lymphocytes. The accelerated disease progression in the presence of MIF translated into a median survival which was 60 days shorter than in the absence of MIF (TCL1+/wtMIF+/+ 400 days, TCL1+/wtMIF−/− 460 days, p = 0.04). SKY analysis in leukemic splenocytes yielded various complex genetic aberrations with trisomies (e.g. +15), tetraploidy, translocations and deletions. Overexpression of tp53 due to the presence of an inactivating mutation in the p53 gene was found more frequently in TCL1+/wtMIF+/+ than in TCL1+/wtMIF−/− animals. Although the rates of DNA-damage-induced apoptosis in pre-leukemic and leukemic mice ex vivo were not significantly different between the genotypes, this defect in the p53-dependent apoptosis pathway corresponded with a reduced rate of spontaneous apoptosis in spleens of leukemic TCL1+/wtMIF+/+ animals. Conclusions: Our experience with the Eμ-TCL-1-transgenic mice shows that this model is suitable for the identification of novel regulators of CLL-like disease. We provide genetic proof that MIF acts to promote the early preleukemic and the leukemic phase of TCL1-induced CLL and thereby identify MIF as a novel regulator of CLL pathogenesis. Ongoing efforts are focussing on further characterizing the differences in pathology, the activation of the AKT pathway and cell cycle control between TCL1+/wtMIF−/− and TCL1+/wtMIF+/+ mice.
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Scielzo, Cristina, Maria T. S. Bertilaccio, Giorgia Simonetti, et al. "HS1 has a central role in the trafficking and homing of leukemic B cells." Blood 116, no. 18 (2010): 3537–46. http://dx.doi.org/10.1182/blood-2009-12-258814.
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Abstract The function of the intracellular protein hematopoietic cell–specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1−/− mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2−/−γc−/− mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eμ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eμ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.
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Ferrer, Gerardo, Xiao-Jie Yan, Brendan Franca, et al. "Chronic Lymphocytic Leukemia Patients and Eµ-TCL1 Mice Share a Phenotype of Functional Granulocyte-like and Dysfunctional Monocyte-like Myeloid Derived Suppressor Cells." Blood 126, no. 23 (2015): 614. http://dx.doi.org/10.1182/blood.v126.23.614.614.
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Abstract Immune imbalance is a common characteristic of patients with chronic lymphocytic leukemia (CLL). This feature is shared with Eμ-TCL1 transgenic mice that, like CLL patients, exhibit an expansion of CD5+ B cells with associated non-B-cell defects. In patients and in mice, T-cell responses are often ineffective. This alteration is generally considered due to the direct effects of the leukemic cells. The expansion of myeloid derived suppressor cells (MDSCs), which play a major role in helping tumor cells escape immune surveillance by inhibiting T-cell responses, is promoted by many cancers. MDSCs are a heterogeneous population of cells that are subdivided into monocyte-like (m-MDSC) and granulocyte-like (g-MDSC) subsets, both in humans and mice. There we have investigated the extent that patients with CLL have expansions of MDSCs, what are their types and functions, and how these correlate with the Eμ-TCL1 mice model. Using flow cytometry on cryopreserved PBMCs, we found that the absolute numbers of MDSCs (HLA-DRlo/CD11b+/CD33+) in 49 untreated CLL patients were significantly higher than 15 healthy controls (HCs) (966 446 vs. 163 578 cells/ml, P<0.001). Moreover, we observed that the absolute numbers of MDSCs significantly correlated with CLL B-cell counts in the blood (P=0.005, Spearman r=0.423). Of note, the distribution between m-MDSCs (CD14+) and g-MDSCs (CD15+) was dramatically different, with CLL patients exhibiting significantly higher numbers and percentages of g-MDSCs than HCs (702 296 vs. 26 818 cells/ml, P<0.001; 50.89 vs. 16.98%, P<0.001).In line with these results, when we explored the MDSC populations (CD11b+/GR1+) in Eμ-TCL1 mice of 5-16 months of age with leukemia cell blood counts ranging from 0.1 to 100 x 106 cell/ml. This analysis indicated a positive correlation between MDSCs and leukemic CD19+ CD5+ cells (P=0.003; Spearman r=0.328). Furthermore, the dot-plot analysis of GR1 and CD11b showed three well defined cell populations: one monocytic (Ly6-C+) and two granulocytic (Ly6-G+ CD11blo and Ly6-G+ CD11bhi). As in patients, the g-MDSC population was larger than the m-MDSC population (884 100 vs. 454 700,P=0.016). However in this case, the m-MDSCs correlated with the numbers of circulating leukemic cells (P<0.001; Spearman r=0.463) and the g-MDSCs did not. The latter was the case even when they were subdivided into both CD11blo and CD11bhi subgroups. A similar pattern was observed when analyzing single cell suspensions from murine spleens. When we evaluated the ability of MDSCs to inhibit autologous T-cell proliferation in CLL patients (n=7), we observed a consistent reduction of proliferation only when co-culturing with g-MDSCs(P=0.034). In contrast, the effects of m-MDSCs on T-cell expansion were varied and insignificant statistically. In 5 CLL samples, we induced m-MDSCs (im-MDSCs) from purified CD33+ cells in vitro with GM-CSF, IL10, and IL6; the im-MDSCs effectively suppressed T-cell proliferation in 4 of 5 cases at an average inhibition of 33% (range: 10-79%). Thus, dysfunctional m-MDSC suppression was not inherent and functional suppression could be achieved by stimulation of CLL precursor cells. Similarly in 3 independent experiments performed with MDSCs from Eμ-TCL1 mice (12-14 months of age), we observed a reduction of in vitro proliferation with g-MDSCs (P=0.049) and not with m-MDSCs. In addition, for those Eμ-TCL1 animals for which sufficient sample was available, we subdivided the g-MDSC population into the two subpopulations based on CD11b density; the CD11blo subset present less nuclear segmentation and higher suppressive activity. In summary, absolute numbers of MDSCs in the blood of CLL patients and Eμ-TCL1 mice are elevated and correlate with the levels of expansion of the leukemia. The major subtype in both situations was g-MDSCs.These g-MDSCs were functionally competent suppressors, whereas m-MDSCs were impaired in this function. In CLL patients, this m-MDSC suppressor defect could be corrected by in vitro stimulation with growth factors that support monocyte differentiation. The high similarity between CLL patients and Eμ-TCL1 mice in relation to MDSC number and function suggest that an imbalance in g-MDC vs. m-MDSC function may affect CLL development and expansion, altering interactions with members of the microenvironment such as T cells. Disclosures No relevant conflicts of interest to declare.
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Alhakeem, Sara Samir, Mary Kathryn McKenna, Beth W. Gachuki, et al. "The role of IL-10 in B-cell chronic lymphocytic leukemia cell survival." Journal of Immunology 196, no. 1_Supplement (2016): 211.17. http://dx.doi.org/10.4049/jimmunol.196.supp.211.17.
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Abstract B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of abnormal lymphocytes in the periphery. Typically the disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study B-CLL we use cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL. Previously we showed that Eμ-Tcl1 CLL cells produce an anti-inflammatory cytokine, IL-10, constitutively. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not effect the survival of CLL cells in vitro. When we adoptively transferred Eμ-Tcl1 cells into IL-10R KO mice, we found that cells grew at a slower rate in IL-10R KO mice vs. wild type mice. We also found that there was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice in compare to the IL-10R KO injected mice. In current in vivo studies, CLL growth will be monitored in WT mice after IL-10 neutralization using anti-IL-10 monoclonal antibody to further elucidate the role of IL-10 in CLL development. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production.
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Enzler, Thomas, Weizhou Zhang, Arnon P. Kater, et al. "Constitutive Baff Signalling Plays a Key Role in CLL Development by Promoting Tumor Cell Survival." Blood 112, no. 11 (2008): 28. http://dx.doi.org/10.1182/blood.v112.11.28.28.
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Abstract The B cell-activating factor of the tumor necrosis factor family (BAFF) is a potent B-cell survival factor. We recently found that nurselike cells, which presumably reside in the leukemia-microenvironment, express BAFF, which promotes survival of leukemia cells of patients with chronic lymphocytic leukemia (CLL) cells through activation of the classical NF-kB pathway. To study the influence of BAFF on leukemogenesis, we crossed BAFF transgenic (Tg) mice with either Eμ-TCL-Tg mice, which develop a lymphoproliferative disease resembling human CLL at about 12 months of age, or Ea-MYC-Tg mice, which experience higher rates of apoptosis in their mature B cells, but do not develop overt lymphoproliferative disease per se. We found that BAFF/TCL1-Tg mice had a shorter mean survival than either TCL1-Tg or BAFF-Tg mice due to the early development of a CD5+CD3−CD45Rlo leukemia B-cell population resembling human CLL at the age of about 3–4 months as compared to 7–9 months in TCL1-Tg mice. In contrast none of the BAFF-Tg or wt mice developed lymphoproliferative disease over the 26-month period of observation. The CD5+CD3−CD45Rlo cell population increased in BAFF/TCL1-Tg mice relatively rapidly, coming to represent &gt;99% of the total blood mononuclear cells of 9-month-old double Tg animals. At this age, these mice also developed massive splenomegaly and their spleens were heavily infiltrated with leukemia B cells. Southern blot analyses of splenocytes harvested at various ages detected oligoclonal/monoclonal splenic B cell populations at approximately 4 months of age in the BAFF/TCL1-Tg mice compared to 9–12 months in the TCL1-Tg mice. Similarly, Ea-MYC-Tg mice, generated by inserting a single copy of MYC into the mouse Ig heavy-chain Ca locus, were crossed with the BAFF-Tg mice in order to obtain BAFF/MYC-Tg mice. In contrast to Ea-MYC-Tg mice, the BAFF/MYC-Tg mice developed expansions of CD5+CD3−CD45Rlo CLL-like leukemia B cells in the blood and spleen starting at 4 months of age, resulting in splenomegaly. These cells could be adoptively transferred into syngeneic BAFF-Tg mice, allowing for continued proliferation of such CD5+CD3−CD45Rlo leukemia B-cells and development lymphoproliferative disease. Staining of splenic sections for proliferating-cell nuclear antigen (PCNA) demonstrated similar high-rates of cell-proliferation in Ea-MYC-Tg mice and BAFF/MYC-Tg mice, which were twice greater than those of BAFF-Tg mice or wt mice. In contrast, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of splenic sections revealed that Ea-MYC-Tg mice had 4 times greater proportions of apoptotic splenocytes than did BAFF/MYC-Tg mice, suggesting that BAFF promoted B cell expansion by inhibiting B cell turnover. Consistent with this, we found that BAFF induced in TCL1-Tg, as well as MYC-Tg mice, high-level B cell expression of anti-apoptotic proteins such as Bcl-XL, Bcl-2, A1/Bfl1, and Pim-2 in leukemia B cells. Also, these leukemia B cells transferred more efficiently into syngeneic BAFF-Tg mice than in mice lacking expression of BAFF. We treated BAFF-Tg mice with intraperitoneal (i.p.) injections of BAFF-neutralizing BR3-Fc or control protein prior to adoptive transfer of leukemia cells of Eμ-TCL1-Tg mice. We found that i.p. injection of 200 mg BR3-Fc into the recipient animals reduced the numbers of circulating CLL cells relative to that found in control-treated mice by nearly 20% (18.2%±5.3%; n=3) within 6 days. These findings indicate that BAFF accelerates development of leukemia in Eμ-TCL1-Tg and Ea-MYC-Tg mice primarily by enhancing leukemia B-cell survival, but not leukemia-cell proliferation, resulting in accelerated rates of tumor development and apparent enhancement of leukemia B cell proliferation in vivo. These findings suggest that targeting BAFF or BAFF-expressing cells within the leukemia microenvironment might be an effective strategy for treatment of CLL and related B-cell malignancies.
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Gobessi, Stefania, Sara Bennardo, Pablo G. Longo, Brendan Doe, and Dimitar G. Efremov. "Development of a Transgenic Mouse Model to Study the Role of ZAP-70 in the Development and Progression of Chronic Lymphocytic Leukemia." Blood 118, no. 21 (2011): 2830. http://dx.doi.org/10.1182/blood.v118.21.2830.2830.
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Abstract Abstract 2830 The protein tyrosine kinase ZAP-70 is an important prognostic factor in chronic lymphocytic leukemia (CLL). Patients that are considered ZAP-70-positive typically express 30–100% of the levels of ZAP-70 in T-cells, whereas in the remaining patients ZAP-70 is either not expressed or is expressed at lower levels. ZAP-70-positive patients have more aggressive disease and shorter survival than patients with low or absent ZAP-70. In vitro experiments with human lymphoma cell lines and primary CLL B-cells have shown that ZAP-70 is involved in B cell receptor (BCR) signaling, indicating that overexpression of ZAP-70 could affect the capacity of the leukemic cells to respond to antigen stimulation. Despite the strong association between ZAP-70 expression and prognosis, it is still not clear whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of more aggressive CLL. To further address this issue, we generated transgenic (tg) mice that express different levels of ZAP-70 in B cells. In these mice expression of the murine ZAP-70 transgene is targeted to the B cell compartment by a VH or a CD19 promoter (VH-ZAP70 and CD19-ZAP70 tg mice, respectively). B cells in CD19-ZAP70 tg mice express the same levels of ZAP-70 as normal murine T cells, whereas the levels of ZAP-70 in B cells of VH-ZAP70 tg mice are approximately 10 times lower. Immunophenotyping analysis of spleen and peritoneal cavity samples from wild type, VH-ZAP70 and CD19-ZAP70 tg mice did not reveal significant differences in the percentage of follicular (FO), marginal zone (MZ) and B1 B cells, indicating that ectopic expression of ZAP-70 does not affect normal B cell development and maturation. In terms of BCR signal transduction, no abnormalities were detected in VH-ZAP70 tg mice, suggesting that low levels of ZAP-70 do not affect BCR signaling. In contrast, B cells from CD19-ZAP70 tg mice showed altered phosphorylation of several molecules downstream of the BCR, such as Syk and BLNK, whereas phosphorylation of Cbl was not affected. To investigate the impact of ZAP-70 expression on leukemia development and progression, we crossed VH-ZAP70 and CD19-ZAP70 tg mice with Eμ-TCL1 tg mice. The latter mice develop leukemias that are considered a mouse model of human CLL. These leukemias are CD5+, express unmutated IGHV genes and stereotyped polyreactive BCRs, but are always ZAP-70-negative. VH-ZAP70/Eμ-TCL1 tg mice (n=11) have been followed for over a year and did not show any differences with respect to their Eμ-TCL1 littermates (n=10). Both groups, starting from the age of 7–8 months, developed leukemias with a similar rate of progression and impact on survival, suggesting that low levels of ZAP-70 do not affect the behavior of the disease. The cohort of CD19-ZAP70/Eμ-TCL1 tg mice was more recently established. These animals are currently 4 months old and still do not show signs of leukemia development. Data from the extended follow-up of these mice will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.
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Lee, Avery C., Sai R. Pingali, Javier A. Pinilla-Ibarz, Chih-Hang A. Tang, and Chih-Chi A. Hu. "Abstract 142: Loss of AID exacerbates the malignant progression of CLL." Cancer Research 82, no. 12_Supplement (2022): 142. http://dx.doi.org/10.1158/1538-7445.am2022-142.
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Abstract Activation-induced cytidine deaminase (AID) is an enzyme that has been implicated as both a positive and a negative factor in the progression of B cell chronic lymphocytic leukemia (CLL), but the role that it plays in the development and progression of this disease is still unclear. Higher AID expression in patients has been correlated with a worse prognosis, but patients with mutated immunoglobulin (IgHV) genes, which requires AID activity, have a better prognosis. To elucidate the role of AID in leukemic progression, we generated an AID-deficient mouse model of CLL, AID-/-/Eμ-TCL1, and found that these mice die significantly earlier than their AID-proficient counterparts. AID-/-/Eμ-TCL1 mice have higher percentages of germinal center B cells and increased infiltration of CLL cells into the lungs, liver, and peritoneal cavity. Both AID-deficient B cells and CLL cells downregulated the tumor suppressive Smad1/S1PR2 signaling pathway, which normally restrains expansion of the germinal center, potentially leading to altered proliferation and homing of leukemia cells. Furthermore, AID-/-/Eμ-TCL1 CLL cells exhibited higher levels of B cell receptor (BCR) signaling, supporting their growth and survival. Upregulated BCR signaling led us to investigate the IRE1α/XBP1s pathway in these cells, which is necessary to support proper BCR signaling and is upregulated in CLL cells. IRE1α is an endoplasmic reticulum (ER) stress sensor that cleaves XBP1 mRNA to produce the functional transcription factor XBP1s. XBP1s promotes synthesis of lipids and chaperones necessary for BCR signaling, and inhibition of this pathway compromises BCR signaling and CLL survival. Compared to Eμ-TCL1 controls, AID-/-/Eμ-TCL1 CLL cells indeed respond to various ER stressors with a higher ER stress response, particularly through the IRE1α/XBP1s pathway, resulting in cells that are more resistant to the stressed conditions within the tumor microenvironment. Mechanistically, this upregulation in the ER stress response cannot be solely attributed to an accelerated leukemic progression, as B cells from AID-/- mice also express increased levels of XBP1s and are more responsive to treatment with pharmacological ER stressors. These studies are translatable to human CLL, as shRNA knockdown of AID in the CLL cell line WaC3 resulted in an increased ER stress response, and CLL cells from patients with IgHV-unmutated disease express higher levels of XBP1 mRNA than those from patients with mutated CLL. Additionally, treatment of both mouse and human CLL cells with a combination of the S1PR2 agonist CYM-5520 and the IRE1 inhibitor B-I09 led to improved induction of apoptosis, revealing a novel way to treat CLL. Overall, these results demonstrate multiple pathways through which the loss of AID leads to worsened CLL and may explain why IgHV-unmutated CLL has such a high proliferative rate that requires therapeutic intervention. Citation Format: Avery C. Lee, Sai R. Pingali, Javier A. Pinilla-Ibarz, Chih-Hang A. Tang, Chih-Chi A. Hu. Loss of AID exacerbates the malignant progression of CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 142.
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Bondada, Subbarao, James P. Collard, Mary Kathryn McKenna, et al. "The role of the splenic microenvironment in Chronic Lymphocytic Leukemia." Journal of Immunology 204, no. 1_Supplement (2020): 163.6. http://dx.doi.org/10.4049/jimmunol.204.supp.163.6.
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Abstract Chronic Lymphocytic Leukemia (CLL), the most prevalent adult leukemia, is characterized by the clonal expansion of CD5+ CD19+ B cells within lymphoid organs and accumulation of quiescent cells in the blood. Eμ-Tcl1 transgenic mice expressing the Tcl1 oncogene in a B cell-specific manner develop CLL and adoptive transfer of CLL cells from these mice induces CLL growth in C57BL/6 mice. These models typically cause splenomegaly as the CLL burden increases, and CLL cells localize mainly in the spleen within the first 10 days post-injection. We hypothesized that the splenic microenvironment is vital for CLL expansion. Accordingly, we found that splenectomy significantly delayed and decreased CD5+ CD19+ CLL cell burden. The role of the splenic microenvironment was further explored by isolating splenic stromal cells from Eμ-Tcl1 mice and establishing stable cell lines (EMST cells). In cell culture, CLL cells divide poorly and remain mostly quiescent. The EMST cells induced proliferation in a majority of mouse and human CLL cells without the addition of any cytokines or growth factors commonly used to accomplish in vitro proliferation of CLL cells. They do not express CD40L, an important stimulatory molecule for B cell proliferation. EMST cells lack conventional immune cell markers, but they express the cytokines and chemokines BAFF, CCL2, CXCL12, and Wnt4. CCL2 levels dramatically increased upon co-culture with CLL. EMST cells also enhance IL-10 secretion by the CLL cells and thus may enhance an immunosuppressive environment to prevent anti-CLL immunity from developing. Our studies support the hypothesis that the spleen is vital for the establishment of CLL and EMST cells produce specific factors that may play a role in CLL growth.
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Märklin, Melanie, Stefanie Bugl, Jonas S. Heitmann, et al. "Genetic Loss of NFAT2 Induces Profound Acceleration of CLL in the TCL1 Mouse Model." Blood 120, no. 21 (2012): 862. http://dx.doi.org/10.1182/blood.v120.21.862.862.
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Abstract Abstract 862 NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated and relocated to the cytoplasm by a network of several kinases. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells. Several recent studies have demonstrated that Calcineurin/NFAT signaling is involved in the pathogenesis of a wide array of hematological malignancies including diffuse large B cell lymphoma, CLL as well as Burkitt and Burkitt-like lymphomas. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific CD19 promoter. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eμ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eμ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). Mice with NFAT2 knock out exhibited a significantly accelerated accumulation of CD5+CD19+ CLL cells as compared to control animals. Flow cytometric analysis at distinct time points showed a tremendous infiltration by CD5+ B cells in the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. At approximately 26 weeks of age, NFAT2 ko mice showed an approximately 40 fold increased lymphocyte count in the peripheral blood than their litter mate controls (1500/μL vs. 60000/μL). Splenomegaly and lymphatic adenopathy was also significantly increased in the NFAT ko population. Furthermore, NFAT2 ko mice showed a dramatically reduced median survival (200 vs. 325 days) and maximum survival (265 vs. 398 days) in comparison to regular TCL1 transgenic mice. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko at an age of approximately 7 months exhibited significantly higher rates of proliferation than control animals. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells implicating Ca2+/NFAT signaling as a potential target for the treatment of this disease. Disclosures: No relevant conflicts of interest to declare.
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McKenna, Mary Kathryn, Sunil K. Noothi, Sara Samir Alhakeem, et al. "Splenic microenvironment is important in the survival and growth of Chronic Lymphocytic Leukemia in mice." Journal of Immunology 198, no. 1_Supplement (2017): 130.20. http://dx.doi.org/10.4049/jimmunol.198.supp.130.20.
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Abstract Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in the peripheral blood and secondary lymphoid organs. CLL cell survival is dependent on B cell receptor signaling and cross-talk within the leukemia microenvironment. CLL presents with enlargement of the spleen, and lymph nodes due to accumulation of long-lived lymphocytes with impaired apoptotic mechanisms. The Eμ-Tcl1 mice, with a B cell specific overexpression of the oncogene, T cell Leukemia 1 (Tcl1), serve as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age. They exhibit splenomegaly, enlarged lymph nodes and a high peripheral blood white count. Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 mice into syngeneic recipients leads to rapid disease development. Ultrasonography of recipient mice showed a dramatic enlargement of the spleen, which preceded detection of CLL cells in the blood. Unlike various genetic modifications, which can only delay the onset of CLL, splenectomy in these models, cures or significantly delays disease development from 30–35 days to 250–270 days. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for prostate apoptotic response-4 (Par-4) compared to C57BL/6 wild type mice. Par-4 is a known tumor suppressor protein that can be secreted and induce apoptosis selectively in cancer cells but not in normal cells. Par-4 appears to regulate a specific microenvironment required for CLL growth. Presently we are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.
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Woyach, Jennifer A., Matthew R. Stefanovski, Virginia Goettl, et al. "Global Inhibition of Bruton's Tyrosine Kinase (BTK) Delays the Development and Expansion of Chronic Lymphocytic Leukemia (CLL) in the TCL1 Mouse Model of Disease." Blood 120, no. 21 (2012): 183. http://dx.doi.org/10.1182/blood.v120.21.183.183.
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Abstract Abstract 183 CLL is a common leukemia which demonstrates variable reactivity of the B cell receptor (BCR) to antigen ligation, but constitutive activation of this pathway. Bruton's Tyrosine Kinase (BTK) is a member of this pathway which shows transcriptional upregulation and constitutive activity in CLL. Ongoing trials of ibrutinib, an orally bioavailable inhibitor of BTK, have shown outstanding preliminary activity in CLL. However results with other known reversible and more selective irreversible BTK inhibitors have had variable results. As ibrutinib potentially has other targets in addition to BTK, this raises the question of whether BTK is an important target in CLL. To answer this question and determine the role of BTK in the development and expansion of CLL, we have used Eμ-TCL1 (TCL1) transgenic mouse model of CLL, where the TCL1 oncogene is under the control of the VH promoter-IgH-Eμ enhancer and adult transgenic mice spontaneously develop CLL. To determine whether BTK is an important target in CLL as opposed to an alternative target of ibrutinib, we crossed the B6/TCL1 mouse with the XID mouse, which harbors a mutation in the PH domain of BTK, rendering it kinase-inactive. In B-cells derived from these mice we observed continued TCL1 expression and diminished BCR signaling with BCR ligation. XID/TCL1 mice (n=65) had a significantly prolonged overall survival (OS) compared to those with wild-type (WT) BTK (n=79) (Median not reached versus (vs) 13.4 months respectively, p<0.0001), and at similar timepoints, XID/TCL1 mice were less likely to be leukemic as measured by CD5/CD19+ B-cells in blood than a WT TCL1 cohort. This study confirms both that kinase functional Btk is critical for leukemia development in this model, and that BTK is an important target of ibrutinib. Next, we wanted to determine whether ibrutinib was effective in this mouse model, and whether ibrutinib could inhibit CLL development similar to genetic manipulation of BTK. To evaluate the efficacy of ibrutinib, we transplanted leukemic TCL1 spleen lymphocytes into CB17/SCID mice, and at the time of leukemia diagnosis by flow cytometry, treated mice with vehicle or ibrutinib administered continuously via drinking water giving an approximate daily dose of 25 mg/kg. Mice treated with ibrutinib had significantly prolonged OS from the time of leukemia diagnosis compared to vehicle treated mice (46 days versus 24 days respectively, p=0.003), confirming that ibrutinib inhibits the expansion phase of CLL in this model. To evaluate the effects of ibrutinib on the development of CLL, we treated B6/TCL1 (n=24) and C3H/B6/TCL1 (n=24) mice from the time of weaning with continuous vehicle or ibrutinib administered via drinking water. We found that mice treated with ibrutinib had a significantly prolonged time to leukemia development compared to vehicle (p<0.0001 for B6/TCL1 mice and p=0.001 for C3H/B6/TCL1 mice), and that OS as well favors ibrutinib-treated mice (p=0.016). Because ibrutinib inhibits Btk in the tumor microenvironment as well as the tumor cells, we set to determine whether global BTK inhibition was necessary to inhibit leukemia development, or whether inhibition in the tumor cells or the microenvironment alone was sufficient. As ibrutinib is an irreversible inhibitor of BTK, to evaluate BTK inhibition in tumor cells alone, we treated TCL1 spleen lymphocytes with vehicle, 1uM ibrutinib, or 1uM fludarabine for 1 hour in vitro prior to engraftment into CB17/SCID mice (n=124 total). The three conditions were not significantly different in terms of leukemia free or overall survival (p=0.26, p=0.98 respectively), suggesting that inhibition of BTK in the CLL cell alone is not sufficient to prevent tumor engraftment following transplant. To determine whether inhibition of BTK in the microenvironment alone was sufficient to inhibit leukemia development, we transplanted leukemic B6/TCL1 lymphocytes into B6 or XID mice (n=46 per group). There was no difference in the time to development of leukemia between B6 mice and XID mice, demonstrating that BTK inhibition in the tumor microenvironment alone is not sufficient to inhibit leukemia development. In conclusion, our data show that active BTK is critical for the development and expansion of CLL in the TCL1 mouse model of CLL. Continuous global inhibition of this kinase through mutation or pharmacologic inhibition delays the development of CLL, confirming that BTK is an important target for drug development. Disclosures: Chang: Pharmacyclics, Inc.: Employment. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.
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Schrage, Matthew I., David Kim, Jeffrey Calimlim, et al. "The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model." Blood 112, no. 11 (2008): 1806. http://dx.doi.org/10.1182/blood.v112.11.1806.1806.
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Abstract Previously, we identified and validated PIM-1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM-1 to be a significant prognostic biomarker in MCL. PIM-1 is an oncogenic serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. It cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM-1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-type of DLBCL. Here we tested the in vivo cooperation of PIM-1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM-1 transgenic mice overexpress murine PIM-1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ -B29-TCL1) fail to downregulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies, including Burkitt-like lymphoma (BLL), DLBCL, marginal zone lymphoma, and B-cell chronic lymphocytic leukemia. We hypothesized that PIM-1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and flow cytometry and classified according to the mouse models of human cancer consortium (MMHCC) ‘Bethesda’ classification scheme. Forty double transgenic mice (PIM-1/TCL1) have been generated and observed for a median follow-up of 9 months. To date, 8/40 (20 %) of the PIM-1/TCL1 mice developed lymphomas, in contrast to 9/88 (10%) PIM-1 and 11/49 (22%) TCL1 transgenic mice, with a median follow-up of 7 and 15 months, respectively. A Kaplan-Meyer plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM-1/TCL1 transgenic mice when compared with single TCL1 transgenic mice (p=0.037). PIM-1 transgenic mice developed early (&lt; 7 months of age) T-cell lymphoblastic lymphomas and late (&gt; 20 months of age) DLBCL. TCL1 transgenic mice developed DLBCL, with single occurrences of lymphoblastic, lymphocytic and Burkitt lymphomas. PIM-1/TCL1 transgenic mice developed DLBCL, frequently with extranodal involvement (spleen, liver and lung). A single case of follicular lymphoma was seen. In addition, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM-1, PIM-2, and also PIM-3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM-1 overexpression in TCL1 transgenic mice accelerated the development of mature B-cell lymphomas. To date, the classification of lymphomas in PIM-1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. The expression of all 3 PIM kinase family members in lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted family members.
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Minden, Marcus Dühren-von, Thomas Wossning, Hassan Jumaa та Hendrik Veelken. "The Role of the BCR Class Expressed by Eμ-TCL1tg Mice and Human CLL". Blood 120, № 21 (2012): 182. http://dx.doi.org/10.1182/blood.v120.21.182.182.
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Abstract Abstract 182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL. By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all developmental stages, we demonstrate a significantly lower frequency of CD19+CD5+IgM+IgDlow lymphoma cells in heterozygous IgM+/−TCL1 mice compared to conventional IgM+/+TCL1 mice (p=0.007). Furthermore, IgM+/−TCL1 mice show a delayed or slowed disease progression compared to TCL1tg mice carrying both IgM alleles. In both TCL1tg mice strains, lymphoma development was exclusively linked to expression of surface IgM, since no IgD single positive lymphoma was detected in IgM+/−TCL1 mice (n=12). TCL1tg mice that lack both μCH alleles showed an accumulation of CD19+CD5+ cells in the spleen at the age of 6 months. According to the genotype of these mice, this population was indeed IgD single positive. However, no further progression could be observed during follow-up to an age of 8 months, indicating a benign form of lymphoproliferation. In contrast to BCRs derived from Eμ-TCL1 mice, analysis of the signaling properties of BCRs derived from IgM−/−TCL1 mice failed to show any autonomous signaling capacity, even when they were expressed as IgM. To address whether IgD in general is able to mediate autonomous signaling reported for TCL1tg- and CLL-derived BCRs, we tested these receptors for autonomous signaling capacity when expressed as IgD. However, expression as IgD led to a complete loss of autonomous signaling capacity in all cases (n=10). In conclusion, whereas autonomous signaling is a characteristic feature of TCL1tg- and CLL-derived BCRs, the pathogenesis of CLL is dependent on the expression of their BCR as the IgM isotype. Expression of IgD-BCRs leads to loss of autonomous signaling capacity, and mice that lack μCH fail to develop malignant lymphoproliferation. To address the question if differential expression of IgD and IgM also had an impact on the clinical behavior of human CLL, we measured the relative expression levels of surface IgD and IgM on circulating lymphoma cells from 67 CLL patients by flow cytometry with simultaneous staining. According to previous reports (Mockridge et al., Blood 2007), unmutated (UM-CLL) cases (n=22) had a higher level of total surface Ig compared to mutated CLL (M-CLL) cases (n=45). Based on our results that IgM is more potent to drive lymphoproliferation, we calculated the ratio of mean fluorescence intensities for IgD over IgM, further called DvM-Score, for every case. A significant difference in the expression pattern as represented by the DvM-Score was observed for UM-CLL and M-CLL (p=0.003) as well as for ZAP70+ and ZAP70- cases (p=0.0002). Both UM-CLL cases as well as ZAP70+ cases show a higher amount of IgM compared to IgD represented by a DvM-Score of <1, whereas the majority of M-CLL and ZAP70− cases express less IgM than IgD and show a DvM-Score of >1. Based on receiver operating characteristics, an DvM cut-off value of 1.15 was identified to optimally discriminate mutational status (AUC: 0.72) and ZAP70 expression (AUC: 0.78). Preliminary data show that CLL samples with a DvM-Score <1.15 had a more aggressive disease course as indicated by a median time to first treatment (TTFT) of 39 months, whereas cases with a DvM-Score of >1.15 show a median TTFT of 154 months (log rank test, p=0.0014). In summary, our results demonstrate an important role of the BCR class, especially with respect to the pathogenetic role of autonomously active IgM BCRs expressed by CLL B cells, for the outcome of the disease. In addition, the DvM score may represent a convenient and novel prognostic marker for CLL. Disclosures: No relevant conflicts of interest to declare.
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Motiwala, Tasneem, Nicola Zanesi, Jharna Datta, et al. "AP-1 elements and TCL1 protein regulate expression of the gene encoding protein tyrosine phosphatase PTPROt in leukemia." Blood 118, no. 23 (2011): 6132–40. http://dx.doi.org/10.1182/blood-2011-01-323147.
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Abstract We previously demonstrated that the gene encoding PTPROt, the truncated form of protein tyrosine phosphatase receptor type O expressed predominantly in hematopoietic cells, is a candidate tumor suppressor and is down-regulated in chronic lymphocytic leukemia (CLL). Here, we show that PTPROt expression is significantly reduced in CD19+ spleen B cells from Eμ-T cell leukemia 1 (TCL1) transgenic mice relative to the wild-type mice. Strikingly, as much as a 60% decrease in PTPROt expression occurs at 7 weeks independently of promoter methylation. To elucidate the potential mechanism for this early suppression of PTPROt in these mice, we explored the role of activating protein-1 (AP-1) in its expression. We first demonstrate that AP-1 activation by 12-O-tetradecanoylphorbol-13-acetate induces PTPROt expression with concurrent recruitment of c-fos and c-jun to its promoter. The PTPROt promoter is also responsive to over- and underexpression of AP-1, confirming the role of AP-1 in PTPROt expression. Next, we demonstrate that TCL1 can repress the PTPROt promoter by altering c-fos expression and c-jun activation state. Finally, using primary CLL cells we have shown an inverse relationship between TCL1 and PTPROt expression. These findings further substantiate the role of TCL1 in PTPROt suppression and its importance in the pathogenesis of CLL.
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Hertlein, Erin K., Timothy L. Chen, David M. Lucas, et al. "NFkB p50 (Nfkb1) Contributes to Disease in the Eu-TCL1 Mouse Model of Chronic Lymphocytic Leukemia." Blood 126, no. 23 (2015): 1248. http://dx.doi.org/10.1182/blood.v126.23.1248.1248.
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Abstract Chronic lymphocytic leukemia (CLL) is a disease of mature B-cells that are resistant to apoptosis and accumulate in the blood over time. Due to the slowly progressing nature of this disease, studies to determine the molecular events leading to defective apoptosis are challenging. Our group has previously reported that during disease progression in the Eμ-TCL1 CLL mouse model, genes become silenced in a progressive manner over the course of the disease. This silencing is due in part to a high degree of methylation at the promoters of key tumor suppressors. However, even though a high degree of methylation is observed in both this mouse model and in CLL patient samples, agents targeting methylation such as decitabine have proven ineffective in CLL therapy. Therefore other novel methods to reverse gene silencing in CLL are an attractive therapeutic option. In our previous study, we observed early transcriptional mechanisms of gene silencing (occurring prior to methylation) involving NF-κB p50 homodimers. NF-κB is a family of transcription factors which is known to play an important role in the progression of CLL. Advances in next generation sequencing have recently identified loss of function mutations in NFKBIE,a negative regulator of NF-κB signaling,in CLL. These mutations contribute to increased nuclear localization (and hence increased activation) of NF-κB subunits. In addition, a mutagenesis screen in the Eμ-TCL1 mouse found that several of the most frequently occurring mutations that contribute to disease progression occur in genes related to the NF-κB family, particularly in the p50 (Nfkb1) gene. In the present study, we have generated a new mouse model to further study the role of p50 in CLL pathogenesis. The Eμ-TCL1 mouse was crossed to the p50-/- mouse. The p50-/- mice are fertile with normal growth and development under sterile conditions; however they do exhibit a defective response to infection and decreased antibody production. As such, animals in this study are housed under pathogen free conditions. The initial cross resulted in mice that were all heterozygous for p50 (p50+/-), and either positive or negative for the TCL1 transgene. The p50+/-; TCL1+ animals were subsequently crossed with one another to generate three genotypes: p50+/+. p50+/- and p50-/-. Animals are monitored for disease by monthly flow cytometry analysis of CD19 and CD5 in whole blood, and leukemia is defined as >10% double positive cells. The p50-/- mice have a significantly lower incidence of leukemia compared to p50+/+ mice (Chi-square p <0.001), and p50+/- mice show an intermediate phenotype (p=0.024 compared to p50-/- mice). Despite pathogen free conditions, some mice within the p50-/- group die at an early age with no evidence of disease. Therefore, competing risks regression was performed to take into account the mice that die without leukemia via the CIF (cumulative incidence function) based on the Fine and Gray model. Differences in time to leukemia between the p50+/+ and p50-/- mice remained significant (Subdistribution Hazard Ratio; SHR = 8.45; 95% CI: 1.86 - 38.47; p=0.006). The p50+/- mice still exhibit an intermediate phenotype, with more separation between the p50+/- and p50-/- (SHR = 4.39; 95% CI: 1.00 - 19.29; p=0.050) than the p50+/- and p50+/+ groups (SHR = 0.52; 95% CI: 0.26 - 1.06; p=0.069). Finally, spleens from p50-/- mice are notably smaller than the p50+/+ littermates (average spleen score of 0-1 for p50-/- versus 2-3 for p50+/+, using a 0-4 scale), indicating that disease in the secondary lymphoid tissues is also affected by the loss of p50 in this model. In conclusion, these data genetically demonstrate the significant contribution of the p50 (Nfkb1) gene to disease progression in the Eµ-TCL1 mouse model. These studies highlight the importance of the NF-κB family in CLL, and suggest that p50 is a promising therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.
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Alhakeem, Sara, Mary McKenna, Beth Gachuki, et al. "Constitutive IL-10 production by normal and malignant B-1 cells is dependent on B cell receptor signaling (IRM10P.620)." Journal of Immunology 194, no. 1_Supplement (2015): 131.18. http://dx.doi.org/10.4049/jimmunol.194.supp.131.18.
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Abstract The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which can have immunosuppressive effects. CLL cells share multiple phenotypic markers with B-1 B cells such as their surface expression of CD5. This study identifies some functional similarities between B-1 and CLL cells. B-1 B cells have been shown to produce a high level of interleukin-10 (IL-10) constitutively and upon toll-like receptor stimulation, a characteristic property of regulatory B cells. Eμ-Tcl1 mice, which express the Tcl1 oncogene in a B cell specific manner, spontaneously develop B-CLL. We show that Eμ-Tcl1 mouse B-CLL cells also produce IL-10 constitutively. The constitutively produced IL-10 was not required for CLL survival in vitro. This study describes a novel role of B cell receptor (BCR) signaling pathway in constitutive IL-10 secretion by normal and malignant B-1 cells. Thus inhibition of Src family kinases, spleen tyrosine kinase (Syk) or Bruton’s tyrosine kinase (Btk), all of which are downstream of BCR, reduces constitutive IL-10 production by both normal and malignant B-1 cells. Analysis of a published microarray study showed that IL-10 levels were higher in peripheral blood of CLL patients than normal human blood samples, which correlated positively with STAT3 levels. Interestingly, we found that BCR signaling induced IL-10 production is independent of STAT3. Current studies are directed at understanding the mechanisms by which BCR signaling affects IL-10 production.
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Dong, Shuai, John C. Byrd, and Amy J. Johnson. "Genetic Inhibition of PI3K p110delta Antagonizes Survival Signals and Induces Immune Activation in Chronic Lymphocytic Leukemia (CLL)." Blood 126, no. 23 (2015): 1711. http://dx.doi.org/10.1182/blood.v126.23.1711.1711.
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Abstract Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) p110δ pathway. In addition, immune tolerance, a critical mechanism for cancer immune invasion, remains a major barrier for effective anti-cancer therapy. PI3K p110δ also plays critical roles in T cell development and function. Studies using genetic p110δ kinase-dead mice have showed that p110δ inhibition can reverse the tumor induced immune tolerance, impairs the expansion of regulatory T cells (Treg) thereby enhancing the cytotoxic CD8+ T cells to induce tumor regression in various solid tumor models. Methods: Here we use a PI3K p110δ kinase-dead (δD910A/D910A) mouse model and a well-characterized Eμ-TCL1 mouse CLL model to delineate the role of p110δ signaling in CLL leukemia pathogenesis and microenvironmental immune tolerance. The δD910A/D910A has a point mutation on the p110δ kinase domain, which results in the complete inactivation of p110δ activity without affecting the expression of p110δ or the other isoforms. TCL1 mice exhibit B-cell leukemia similar to that in humans beginning around 4-6 months. Adoptive transfer experiments are performed by tail-vein injection of 5e6 splenic lymphocytes from a leukemic Eμ-TCL1 mouse. Leukemia progression is measure by flow cytometry for the CD5+ CD19+ % lymphocytes in the peripheral blood. Whole blood cytotoxicity assay was performed by incubating whole blood from recipient mice with cryopreserved cells from the original donor to assess the their cytotoxic effects towards the donor cells. Results: δD910A/D910A mice were crossed with TCL1 mice to examine the effect of systemic p110δ inactivation in CLL. We show that global inactivation of PI3K p110δ significantly prevented spontaneous leukemia development. Both δWT /TCL1 and δWT/D910A /TCL1 mice developed CLL like disorder starting 4 months whereas δD910A/D910A /TCL1 mice did not show any sign of disease up to 14 months. This indicates that p110δ is a critical kinase for CLL disease initiation and expansion. We then performed adoptive transfer of TCL1 leukemic cells into p110δ mice to examine the role of p110δ in the microenvironment. We show that inhibiting p110δ in the microenvironment impairs leukemia engraftment. On day 31 after engraftment, 7 out of 10 mice from the δWT group developed leukemia while no mice from δD910A/D910A group had detectable leukemia. On day 43, δD910A/D910A group started to have leukemia incidence (2 out of 7) when δWT group continued to progress. δWT/D910A group showed intermediate leukemia incidence and disease development, which were different from the phenotype we saw in δWT/D910A /TCL1. This suggests that p110δ plays a different role outside of the leukemia compartment. To identify potential effector cells, we performed adoptive transfer of ovalbumin (OVA) -expressing TCL1 leukemia cells into p110δ mice. The TCR (OT1) specific SIINFEKL peptide of OVA is used to induce the expansion of OVA antigen specific CD8+ T cells. Interestingly, upon engraftment, δWT and δWT/D910A mice showed similar levels (4% and 3.5%, respectively) of OVA specific CD8+ T cells whereas the δD910A/D910A mice only have 2%. This appears to be contradicting to the less leukemia development we observed previously. However, in vitro whole blood killing assay showed that whole blood from δD910A/D910A mice exhibited higher cytotoxicity to TCL1 leukemia cells despite their impaired development of the antigen specific anti-tumor CD8+ T cells. Conclusions: This study demonstrates that blocking p110δ in CLL not only abrogates survival signals in leukemic cells but also has the potential to reverse immune suppression in the micoenvironment. Studies are ongoing to pinpoint the effector cell subsets. The finding from this study will suggest that idelalisib, the PI3K p110δ inhibitor recently approved by FDA for the treatment of CLL, or other inhibitors of this pathway can potentially be used to treat a wide range of cancers by unleashing host anti-tumor immunity. Disclosures Byrd: Acerta Pharma BV: Research Funding.
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Ontiveros, Evelena, David Dae-Young Kim, Jeffrey M. Calimlim, et al. "The PIM1 Oncogene Accelerates TCL1 Driven Lymphomagenesis in a Double-Transgenic Murine Model." Blood 114, no. 22 (2009): 2968. http://dx.doi.org/10.1182/blood.v114.22.2968.2968.
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Abstract Abstract 2968 Poster Board II-944 Previously, we identified and validated PIM1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM1 to be a significant prognostic biomarker in MCL. PIM1 is a serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. PIM1 cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-subtype of diffuse large B-cell lymphomas (DLBCL). Here, we tested the in vivo cooperation of PIM1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM1 transgenic mice overexpress murine PIM1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ-B29-TCL1) fail to down-regulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies. We hypothesized that PIM1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and classified by a hematopathologist according to “Mouse Models of Human Blood Cancers” (Li et al., 2008). A Kaplan-Meier plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM1/TCL1 transgenic mice when compared with the single transgenic strains. The median lymphoma-free survival for TCL1 single transgenic mice, PIM1 single transgenic mice, or PIM1/TCL double transgenic mice were 10.0 months, 16.0 months, and 8.5 months, respectively. The results were statistically significant: TCL1 vs. PIM1/TCL1 (p=0.0008), PIM1 vs. PIM1/TCL1 (p<0.0001). PIM1 transgenic mice developed rare [1 of 31 (3.2%)] and early (< 7 months of age) T-cell lymphoblastic lymphomas and more commonly developed late (>12 months of age) B-cell lymphomas. The most common lymphomas in PIM1 single transgenic were low-grade B-cell lymphomas [12 of 31 (38.7%)], mainly follicular lymphomas. A minority of PIM1 single transgenic mice developed aggressive lymphomas [6 of 31 (19.4%)], including DLBCL and Burkitt's lymphoma. In contrast, the majority of TCL1 transgenic mice developed aggressive B-cell lymphomas [21 of 36 (58.3%)], mainly DLBCL, lymphohistiocytic subtype. The majority of PIM1/TCL1 double transgenic mice also developed aggressive B-cell lymphomas [20 of 34 (58.8%)], mainly DLBCL, lymphohistiocytic subtype. The low-grade lymphomas that developed in PIM1/TCL1 mice included 5 cases of lymphoplasmacytic lymphoma (LPL); one of these cases had transformed in addition to a DLBCL. Further, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM1, PIM2, and PIM3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM1 overexpression in TCL1 transgenic mice accelerated the development of mature, aggressive B-cell lymphomas. The classification of lymphomas in PIM1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. Single transgenic PIM1 mice developed low-grade B-cell lymphomas after prolonged observation time. The expression of all 3 PIM kinase family members in human lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of drug resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted Pim family members. A clinical trial with a pan-PIM inhibitor is currently ongoing. Disclosures: No relevant conflicts of interest to declare.
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Suljagic, Mirza, Pablo G. Longo, Luca Laurenti, and Dimitar G. Efremov. "The Syk Inhibitor R788 (FosD) Inhibits Tumor Growth in the TCL1 Transgenic Mouse Model of CLL by Blocking Antigen-Dependent BCR Signaling." Blood 114, no. 22 (2009): 887. http://dx.doi.org/10.1182/blood.v114.22.887.887.
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Abstract Abstract 887 CLL B-cells depend on various signals from the microenvironment for survival and proliferation. Among these, antigenic stimuli that are propagated through the B-cell receptor (BCR) are considered particularly important for the development and progression of CLL, suggesting that the BCR signaling pathway could be an important target for therapeutic intervention. We have previously characterized some of the critical components of the signaling pathway downstream of the BCR in CLL B cells and identified the protein tyrosine kinase Syk as a promising therapeutic target. In a recent study we showed that CLL B-cells frequently have increased basal/constitutive Syk activity and are moderately sensitive to the cytotoxic effect of the selective Syk inhibitor R406 [Gobessi et al, Leukemia 2009]. More importantly, the survival signal induced by sustained BCR engagement was completely abolished by R406, suggesting that this compound may exert an even greater effect in vivo by inhibiting antigen-dependent Syk activation. We have now tested this possibility in the Eμ-TCL1 transgenic mouse model of CLL. Aged Eμ-TCL1 mice develop CD5+ B-cell leukemias that, similar to aggressive human CLL, show features of an antigen-driven process, including expression of stereotyped BCRs and reactivity with common autoantigens and microbial agents [Yan et al, Proc Natl Acad Sci USA 2006]. For our experiments we used a TCL1 leukemia (TCL1-002) that does not grow in vitro, but can be propagated in syngeneic recipients in vivo. TCL1-002 cells express an unmutated stereotyped BCR encoded by the VH12/VK4 combination, which reacts with phosphatidylcholine, an autoantigen exposed on the surface of senescent erythrocytes. In vitro experiments showed that R406 is not cytotoxic for TCL1-002 cells, although it completely inhibited both the basal and BCR-induced activation of signaling pathways downstream of Syk. The absence of a direct cytotoxic effect provided a unique opportunity to investigate whether inhibition of BCR signaling will affect leukemia growth in vivo. For this purpose, 1×107 TCL1-002 cells were injected intraperitoneally in 18 syngeneic mouse recipients. Three days later treatment was started in 8 mice with R788, which is the water-soluble prodrug of R406, at a daily dose of 80mg/kg during 18 consecutive days. Because of the rapid clearance of the drug (serum half-life <2 hours) R788 was administered in 3 divided doses at 4 hour intervals. Two weeks after the end of treatment leukemia developed in all mice from the control group (median WBC counts 131×106/ml, range 12-300×106/ml), whereas all R788-treated mice showed normal WBC numbers (median 6×106/ml, range 3-8×106/ml, P<0.001). Three weeks later all mice in the control group had died (median survival 46 days), whereas all mice in the R788 group were still alive and only two of them had detectable leukemic cells. R788 also showed some efficacy in the treatment of mice with overt TCL1-002 leukemias (WBC >50×106/ml). Whereas all mice from the control group (n=9) died between 6 and 18 days from the beginning of therapy, 4 out of 9 mice from the R788 group survived for more than 33 days. The mechanism of R788 activity was primarily related to inhibition of leukemic cell proliferation, as evidenced by a substantial decrease in the percentage of Ki67-positive cells after 7 days of treatment (30% before, 5% after therapy, P<0.001). To investigate whether R788 will also be effective against other TCL1 tumors we treated five TCL1 mice with preleukemic mono- or oligoclonal B-cell expansions during a four week period. R788 reduced the percentage of CD5+/B220+ cells in 2 cases, whereas in 2 other cases the percentage increased. Interestingly, the pattern of clonal Ig gene rearrangements changed during therapy, suggesting that only certain TCL1 clones are sensitive to R788 treatment. In summary, this study shows that R788 can effectively inhibit the growth of certain TCL1 tumors and provides the first in vivo experimental evidence suggesting that inhibition of antigen-dependent BCR signaling could be an effective therapeutic approach in CLL. Disclosures: No relevant conflicts of interest to declare.
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Rößner, Philipp M., Bola S. Hanna, Thorsten Zenz, Stephan Stilgenbauer, Peter Lichter, and Martina Seiffert. "The role of CXCR3 in the microenvironment of chronic lymphocytic leukemia." Journal of Immunology 196, no. 1_Supplement (2016): 73.12. http://dx.doi.org/10.4049/jimmunol.196.supp.73.12.
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Abstract Chronic lymphocytic leukemia (CLL) is a B-cell malignancy that is stringently associated with a tumor-supportive microenvironment and defective anti-tumor immunity. T cells from CLL patients show features of exhaustion, including expression of PD-1, and are highly impaired in their activity. Our previous work showed that immune checkpoint blockade using anti-PD-L1 effectively prevents disease and restores T-cell activity in the Eμ-TCL1 mouse model of CLL. Development of disease in these mice was shown to be associated with a relative loss of naïve T cells and an enrichment of effector T cells displaying an exhausted phenotype. Our work showed that T cells in the Eμ-TCL1 mice express high levels of the chemokine receptor CXCR3 accompanied with elevated serum levels of the corresponding chemokines CXCL9 and CXCL10, suggesting a role for CXCR3 in T-cell skewing in CLL. Intracellular flow cytometric analysis showed an enrichment of CXCL9+ CD45+ hematopoietic cells in leukemic spleens which were identified as Ly6C+ monocytes. Along this line, depletion of phagocytic cells by Clodronate Liposomes resulted in significantly decreased serum levels of CXCL9, a reversal of T-cell skewing and slowed down development of CLL in mice. To evaluate the role of this receptor in CLL in vivo, adoptive transfer of murine CLL cells in CXCR3 knockout mice is currently ongoing. We further perform an in-depth phenotypical analysis of T-cell subsets in blood samples of CLL patients from different prognostic subgroups to characterize T-cell aberrations along with disease progression. Taken together, our data suggest a pathomechanistic role for CXCL9, CXCL10 and their receptor CXCR3 in CLL. Their potential as novel drug targets needs to be explored in the future.
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Schulze-Edinghausen, Lena, Claudia Dürr, Selcen Öztürk, et al. "Dissecting the Prognostic Significance and Functional Role of Progranulin in Chronic Lymphocytic Leukemia." Cancers 11, no. 6 (2019): 822. http://dx.doi.org/10.3390/cancers11060822.
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Chronic lymphocytic leukemia (CLL) is known for its strong dependency on the tumor microenvironment. We found progranulin (GRN), a protein that has been linked to inflammation and cancer, to be upregulated in the serum of CLL patients compared to healthy controls, and increased GRN levels to be associated with an increased hazard for disease progression and death. This raised the question of whether GRN is a functional driver of CLL. We observed that recombinant GRN did not directly affect viability, activation, or proliferation of primary CLL cells in vitro. However, GRN secretion was induced in co-cultures of CLL cells with stromal cells that enhanced CLL cell survival. Gene expression profiling and protein analyses revealed that primary mesenchymal stromal cells (MSCs) in co-culture with CLL cells acquire a cancer-associated fibroblast-like phenotype. Despite its upregulation in the co-cultures, GRN treatment of MSCs did not mimic this effect. To test the relevance of GRN for CLL in vivo, we made use of the Eμ-TCL1 CLL mouse model. As we detected strong GRN expression in myeloid cells, we performed adoptive transfer of Eμ-TCL1 leukemia cells to bone marrow chimeric Grn−/− mice that lack GRN in hematopoietic cells. Thereby, we observed that CLL-like disease developed comparable in Grn−/− chimeras and respective control mice. In conclusion, serum GRN is found to be strongly upregulated in CLL, which indicates potential use as a prognostic marker, but there is no evidence that elevated GRN functionally drives the disease.
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Chen, Shih-Shih, Rainer Claus, David M. Lucas, et al. "Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL." Blood 117, no. 3 (2011): 862–71. http://dx.doi.org/10.1182/blood-2010-05-284638.
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Abstract Inhibitor of DNA binding protein 4 (ID4) is a member of the dominant-negative basic helix-loop-helix transcription factor family that lacks DNA binding activity and has tumor suppressor function. ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function, and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to various degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. The crossing of ID4+/− mice with Eμ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in nontransformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.
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Lapalombella, Rosa, Virginia Goettl, Katie Williams, et al. "Significant in Vivo Efficacy of the SINE KPT-330 in Mouse Models of CLL." Blood 120, no. 21 (2012): 2452. http://dx.doi.org/10.1182/blood.v120.21.2452.2452.
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Abstract Abstract 2452 CRM1 (chromosomal regional maintenance 1) or XPO1 (exportin 1) is the major protein that mediates nuclear export. XPO1 has been shown to be over-expressed in cancer cells leading to ineffective cytoplasmic localization of multiple tumor suppressor genes such us p53, FOX03a, and Iκ-β. Chronic Lymphocytic Leukemia (CLL) is characterized by disrupted apoptosis caused both by microenviromental stimuli and aberrant activation of survival pathways including PI3K/Akt, NF-kB, and p53. We previously showed that first generations Selective Inhibitor of Nuclear Export (SINE), KPT-185 and KPT-251, specifically bind to and block XPO1 function resulting in re-activation of tumor suppressor pathways and apoptosis in CLL. Furthermore, we showed that KPT-251 at 75mg/Kg, 3 times per week (QODX3) slowed disease progression, and improved overall survival in the Eμ-TCL1-SCID mouse model of CLL (median OS = 130 and 72 days for KPT-330 and vehicle respectively; p=0.01) with minimal weight loss or other toxicities. The goal of this study was to determine the efficacy of KPT-330, a new generation SINE currently in Phase 1 human studies, with improved pharmacokinetic and oral bioavailability, in the Eμ-TCL1-SCID mouse model of CLL. We first compared the in-vitro activity of KPT-330 to KPT-251. KPT-330 induced improved levels of cytotoxicity on CLL cells compared to KPT-251 with retained selective cytotoxicity against tumor cells when compared to peripheral blood mononuclear cells and isolated B, and T cells. Similar to KPT-251, KPT-330 showed enhanced cytotoxic potency on CLL cells under coculture conditions with stromal cells compared to CLL alone (p<0.05). Interestingly KPT-330 prevented CPG induced proliferation of CLL cells (p<0.005) and migration of CLL cells driven by CXCL12 and CXCL13, homing factors for hematopoietic cells to the marrow that are constitutively secreted by bone marrow stromal cells. KPT-330 was also more effective than KPT-251 in mediating cytotoxicity of murine TCL1+ cells in-vitro. We therefore tested the ability of KPT-330 to slow disease progression in Eμ-TCL1-SCID mouse model of CLL in which CD19+ TCL1 leukemia cells were engrafted into SCID mice via tail vein injection. Treatments began 2 weeks post engraftment. 9 or 10 mice in each treatment group were treated with: a) vehicle control twice weekly (BIW), b) KPT-330 5 mg/kg BIW, c) KPT-330 15 mg/kg BIW d) vehicle control QODX3, e) KPT-330 1 mg/kg QODX3 for 9 weeks followed by 15 mg/kg QODX3, f) KPT-330 3 mg/kg QODX3, g) KPT-330 10 mg/kg QODX3 for up to a maximum of 36 weeks. In surviving mice, treatment was stopped after 36 weeks and animals were followed up for 30 additional days. Overall survival (OS) was the primary endpoint. Within the BIW groups, only KPT-330 at 15 mg/kg (median OS = undefined p=0.0002) showed a significant improvement over vehicle (median OS = 98 days) with 100% of the mice still alive at the end of the study (week 40). Within the QODX3 groups, KPT-330 10 and 15 mg/kg QODX3 (median OS = undefined, 213 days respectively) both showed a significant improvement (p<0.0001) over vehicle (median OS = 98.5). No improvement in survival compared to vehicle treated animals was induced by KPT-330 3 mg/kg. KPT-330 was well tolerated in mice, weight loss suggestive of drug toxicity was seen at the beginning of the study only in groups treated with KPT-330 at doses of >10 mg/kg QODX3 but was fully reversed by the end of the study and did not appear to adversely affect the animals. Moreover, KPT-330 at doses of >10 mg/kg BIW and QODX3 significantly prevented an increase in circulating CLL cells compared to vehicle. To further validate KPT-330 in mice with leukemic phase (ie, very high tumor burdens), 20 additional mice were engrafted with CD19+ TCL1 leukemia cells and treatment with vehicle or 15 mg/kg KPT-330 (QoDx3/wk) was initiated 70 days post engraftment. Mice treated with KPT-330 had a significant survival advantage (p=0.0008, median OS = undefined; 99 days, for KPT-330 and vehicle respectively). KPT-330 was strikingly active in prolonging survival of TCL1-SCID mice. Using CLL as a disease model we validate the clinical effectiveness of targeting XPO1 with use of SINE, with favorable therapeutic index in-vivo. These data strongly support the ongoing Phase 1 clinical studies of KPT-330 in patients with advanced hematologic malignancies including CLL. Disclosures: Sandanayaka: Karyopharm Therapeutics: Employment. Shechter:Karyopharm Therapeutics: Employment. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment.
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Maharaj, Kamira, John J. Powers, Alex Achille та ін. "The dual PI3Kδ/CK1ε inhibitor umbralisib exhibits unique immunomodulatory effects on CLL T cells". Blood Advances 4, № 13 (2020): 3072–84. http://dx.doi.org/10.1182/bloodadvances.2020001800.
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Abstract The in-clinic phosphatidylinositol 3-kinase (PI3K) inhibitors idelalisib (CAL-101) and duvelisib (IPI-145) have demonstrated high rates of response and progression-free survival in clinical trials of B-cell malignancies, such as chronic lymphocytic leukemia (CLL). However, a high incidence of adverse events has led to frequent discontinuations, limiting the clinical development of these inhibitors. By contrast, the dual PI3Kδ/casein kinase-1-ε (CK1ε) inhibitor umbralisib (TGR-1202) also shows high rates of response in clinical trials but has an improved safety profile with fewer severe adverse events. Toxicities typical of this class of PI3K inhibitors are largely thought to be immune mediated, but they are poorly characterized. Here, we report the effects of idelalisib, duvelisib, and umbralisib on regulatory T cells (Tregs) on normal human T cells, T cells from CLL patients, and T cells in an Eμ-TCL1 adoptive transfer mouse CLL model. Ex vivo studies revealed differential effects of these PI3K inhibitors; only umbralisib treatment sustained normal and CLL-associated FoxP3+ human Tregs. Further, although all 3 inhibitors exhibit antitumor efficacy in the Eμ-TCL1 CLL model, idelalisib- or duvelisib-treated mice displayed increased immune-mediated toxicities, impaired function, and reduced numbers of Tregs, whereas Treg number and function were preserved in umbralisib-treated CLL-bearing mice. Finally, our studies demonstrate that inhibition of CK1ε can improve CLL Treg number and function. Interestingly, CK1ε inhibition mitigated impairment of CLL Tregs by PI3K inhibitors in combination treatment. These results suggest that the improved safety profile of umbralisib is due to its role as a dual PI3Kδ/CK1ε inhibitor that preserves Treg number and function.
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Widhopf, George F., Bing Cui, Esther Avery, et al. "ROR1 Expression Accelerates Leukemia Development in RORxTCL1 Transgenic Mice,." Blood 118, no. 21 (2011): 3905. http://dx.doi.org/10.1182/blood.v118.21.3905.3905.
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Abstract Abstract 3905 ROR1 is a receptor tyrosine kinase-like orphan receptor and an oncofetal protein that is expressed on chronic lymphocytic leukemia (CLL) B cells, but not on normal B cells or most other adult tissues. The leukemia-associated expression of ROR1 suggests that it potentially could contribute to the development and/or progression of CLL. To investigate its functional significance in the development and/or progression of CLL, we generated B6 mice transgenic (Tg) for human ROR1 (hROR1) under the control of the murine Ig promoter/enhancer, which drives B-cell-restricted expression of hROR1. These mice developed increased numbers of splenic B lymphocytes relative to that of control littermates lacking the hROR1 transgene; a few of these animals also developed hROR1+/CD5+/B220low B cell leukemia resembling human CLL at 15–18 months of age. We crossed hROR1-Tg mice with B6 Eμ-TCL1-Tg mice (TCL1), which at 7 months of age develop CD5+B220low leukemia B cells detectable in the blood and resembling human CLL cells except lacking expression of mouse ROR1. We found the F1 animals with both transgenes (ROR1XTCL1) developed hROR1+/CD5+/B220low B-cell CLL at a significantly younger median age than did littermate-control mice having either transgene alone. Comparison of the number of CD5+B220low leukemia B cells in ROR1xTCL1 or TCL1 Tg mice at five, six, and seven months of age demonstrated that ROR1xTCL1 Tg mice have significantly higher median percentages of circulating leukemic cells in the blood compared to that of age-matched TCL1 Tg mice (Welchs's t test based on the average of all 3 measurements, p=0.038). ROR1xTCL1 leukemia B cells also expressed higher levels of hROR1 than non-leukemia B cells of ROR1 Tg mice. To confirm that ROR1 enhances leukemia-cell expansion, we adoptively transferred CD5+B220low leukemia B cells from ROR1xTCL1 Tg mice into syngeneic ROR1-Tg mice. Adoptive transfer of equal numbers (1×105) of leukemia B cells from ROR1xTCL1 or TCL1 Tg mice resulted in transfer of leukemia in both cases without requiring prior conditioning of recipient animals. However, animals engrafted with ROR1xTCL1 leukemia cells developed more aggressive disease and more marked splenomegaly than did animals engrafted with TCL1 leukemia cells. Moreover, eight weeks after adoptive transfer, animals engrafted with ROR1xTCL1 leukemia cells had ≥4-fold greater median numbers of neoplastic B cells in the spleen (e.g. 4 × 108) than did animals engrafted with TCL1 leukemia cells. In vivo labeling studies with bromodeoxyuridine (BrdU), measurement of the relative proportions of Ki-67-positive leukemia cells from either type of adoptive host animals, and use of the terminal deoxynucleotidyl transferase (dUTP) nick end-labeling (TUNEL) assay to detect cells that had undergone apoptosis. These studies revealed that animals engrafted with hROR1+ leukemia cells had significantly lower-rates of leukemia-cell turnover due to higher rates of cell proliferation and lower rates of apoptosis than that animals engrafted with TCL1 leukemia cells. These studies indicate that hROR1 can accelerate development of de novo B-cell leukemia, lower the rate of leukemia-cell turnover, and enhance disease progression in this model system. Disclosures: No relevant conflicts of interest to declare.
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Hayakawa, Kyoko, Anthony M. Formica, Joni Brill-Dashoff, et al. "Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression." Journal of Experimental Medicine 213, no. 13 (2016): 3007–24. http://dx.doi.org/10.1084/jem.20160712.
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In mice, generation of autoreactive CD5+ B cells occurs as a consequence of BCR signaling induced by (self)-ligand exposure from fetal/neonatal B-1 B cell development. A fraction of these cells self-renew and persist as a minor B1 B cell subset throughout life. Here, we show that transfer of early generated B1 B cells from Eμ-TCL1 transgenic mice resulted in chronic lymphocytic leukemia (CLL) with a biased repertoire, including stereotyped BCRs. Thus, B1 B cells bearing restricted BCRs can become CLL during aging. Increased anti-thymocyte/Thy-1 autoreactive (ATA) BCR cells in the B1 B cell subset by transgenic expression yielded spontaneous ATA B-CLL/lymphoma incidence, enhanced by TCL1 transgenesis. In contrast, ATA B-CLL did not develop from other B cell subsets, even when the identical ATA BCR was expressed on a Thy-1 low/null background. Thus, both a specific BCR and B1 B cell context were important for CLL progression. Neonatal B1 B cells and their CLL progeny in aged mice continued to express moderately up-regulated c-Myc and down-regulated proapoptotic Bmf, unlike most mature B cells in the adult. Thus, there is a genetic predisposition inherent in B-1 development generating restricted BCRs and self-renewal capacity, with both features contributing to potential for progression to CLL.
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Rattmann, Ina, David Zhu, Carlo M. Croce, et al. "The Expression of the GTPase-Deficient, Hematopoietic-Specific RhoH GTPase Is Implicated in Development of Chronic Lymphocytic Leukemia (CLL)." Blood 110, no. 11 (2007): 339. http://dx.doi.org/10.1182/blood.v110.11.339.339.
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Abstract RhoH is a hematopoietic-specific small GTPase which is constitutively active due to lack of intrinsic GTPase activity, suggesting that expression levels of RhoH regulate cellular function. Our lab has previously shown that RhoH in T-cells directly interacts with ZAP-70, a critical protein tyrosine kinase in TCR cell signaling and function (Gu et al., Nature Immunol., 2006). As in human B-CLL ZAP-70 expression has been correlated with an unmutated IgVH gene status and poor prognosis, we have investigated RhoH expression and function in B-CLL. PBMC samples from 29 B-CLL patients, all containing >90% CD19+/CD5+ human B-CLL cells, were compared to CD19+ B-cells from normal donors. Significantly higher RhoH mRNA levels were detected in CLL samples by quantitative PCR (p<0.001) with a 2–4 fold increase in the majority of samples. In addition, RhoH levels in these CLL samples were directly correlated with ZAP-70 expression (p<0.01). To further delineate the role of RhoH in the development and progression of B-CLL, we utilized a genetic mouse model of B-CLL, the Eμ-TCL1 transgenic mouse (Bichi et al. PNAS, 2002), and interbred these with Rhoh−/− mice. Starting at 2 months of age peripheral blood of these mice was analyzed every month for different blood cell lineages and in particular for the number of IGM+/CD5+/B220low cells as a readout for the progression of the CLL-like disease. In TCL1 transgenic mice with normal RhoH expression (Rhohwt; TCL1Tg) and with one Rhoh allele knocked out (Rhoh+/−; TCL1Tg) an accumulation of IgM+/CD5+ B-cells was observed which started as early as an age of 3 and 4 months, respectively, and increased dramatically thereafter. In contrast, Rhoh−/−; TCL1Tg showed no significant increase of IgM+/CD5+ B-cells until month 5 with a modest increase thereafter and values were significantly reduced in comparison to Rhohwt; TCL1Tg from month 3–5. As expected, no increase of IgM+/CD5+ B-cells was observed in the nontransgenic controls Rhoh−/+; TCL1NTg, Rhoh−/−; TCL1NTg (data not shown), and Rhohwt; TCL1NTg (Table 1). Table 1: IGM+/CD5+ cells in the peripheral blood. Age [months] Rhoh wt ; TCL1Tg Rhoh +/−;TCL1Tg Rhoh −/−;TCL1Tg Rhoh wt ;TCL1 NTg Mean ± SEM (x1000/ul); () animals analyzed; * p<0.05 in comparison to Rhohwt; TCL1NTG (data collection still in progress) 2 0.12 ± 0.02 (10) 0.28 ± 0.07 (8) 0.15 ± 0.04 (9) 0.08 ± 0.02 (9) 3 0.33 ± 0.09 (11) 0.22 ± 0.06 (8) 0.07 ± 0.03 (8)* 0.07 ± 0.01 (10) 4 0.83 ± 0.38 (11) 0.57 ± 0.20 (18) 0.11 ± 0.05 (16)* 0.08 ± 0.01 (6) 5 3.32 ± 2.27 (6) 1.35 ± 0.46 (17) 0.26 ± 0.06 (15)* 0.11 ± 0.03 (5) 6 4.38 ± 2.41 (7) 5.01 ± 3.01 (9) 0.66 ± 0.23 (8) 0.07 ± 0.02 (5) When mice from each genotype were sacrificed at 6 months of age the number of IgM+/CD5+ B-cells detected in Rhoh−/− TCL1Tg vs Rhohwt TCL1Tg mice was 6-fold reduced in the peritoneal cavity, 3-fold reduced in the bone marrow and a 2-fold reduced in the spleen. In summary our data strongly suggest that RhoH plays a role in the progression of the B-CLL-like disease in the transgenic Eμ-TCL1 mouse model. This will allow detailed molecular studies of the role of RhoH in pathways altered in TCL1-induced expansion of B-cell precursor populations in this model.
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Egle, Alexander, Josefina D. Pinon, Christoph Heyder, et al. "T Cell Dynamics during the Pretumor and Tumor Phase in the Murine Tcl1 Transgenic Chronic Lymphocytic Leukemia Model." Blood 112, no. 11 (2008): 3145. http://dx.doi.org/10.1182/blood.v112.11.3145.3145.
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Abstract Chronic lymphocytic leukaemia of B cells (B-CLL) is characterised by a clonal expansion of CD5-expressing B cells. However, the absolute number of T cells in patients with B-CLL is also increased, primarily due to an enlarged CD8+ population. Despite the enhanced number of T cells, they appear to be dysfunctional as they are largely devoid of anti-tumor activity and may even support the growth and maintenance of the malignant B-CLL clone. To further investigate the contribution this T cell dysfunction has on the establishment and progression of B-CLL, we monitored the changes in the T cell compartment in Eμ-TCL1 transgenic mice of various ages, during the progression of B-CLL-like disease. The deregulated expression of TCL1 in the B cell compartment of these mice leads to a hyperplasia of CD5+ CD19+ B cells early in life that is first detectable in the peritoneal cavity and later spleen and bone marrow. Later, this develops into a clonal disease, and onset of frank leukaemia, with infiltration of the CD5+ CD19+ cells into other organs (Bichi et al, 2003). Similar to human B-CLL, leukaemic TCL1 transgenic mice have increased absolute numbers of T cells, owing mainly to an expanded CD8+ population. Analyses of the cell surface expression of CD25, CD44, and CD62L, revealed a marked increase the number of memory and effector T cells compared to naïve T cells in the CD8+ subset that is also observed in the CD4+ subset, although to a lesser degree. Importantly, a decrease in the number of naïve T cells along with a corresponding increase in T memory and effector cells has been observed in the CD4+ T cell pool from patients with unmutated B-CLL (Tinhofer et al, unpublished), and the TCL1 mouse has been shown to be a model for the unmutated form of B-CLL. The relative shift from naïve to central memory T cells occurs alongside the CD5+ CD19+ hyperplasia in the TCL1 mice. In younger mice that exhibit the CD5+ CD19+ hyperplasia only in the peritoneal cavity, the changes in the T cell compartment are also observed solely within this organ. At later stages, when the CD5+ CD19+ hyperplasia has spread to other organs, the shift to memory T cells is also detectable in all infiltrated organs. Because memory and effector T cells represent antigen-experienced cells, we analysed the complementarity determining region 3 (CDR3) of the T cell receptor (TCR) of purified CD4+ and CD8+ populations by spectratyping in order to determine the degree of clonality (mono-, oligo-, or polyclonal) of the different T cell families within the T cell pool. More clonal T cells were observed in the CD8+ T cell subset in the infiltrated organs of leukaemic mice, though we could not observe a skewing towards any particular BV gene family. Interestingly, more mono- and oligoclonal CD4+ T cells have also been observed in the peripheral blood of patients with unmutated B-CLL. Thus, in many aspects, the TCL1 mouse recapitulates many of the T cell abnormalities observed in patients with B-CLL. It should be noted that, although driven from a B-cell specific promoter, the hTCL1 transgene is also expressed in the T cells of TCL1 mice. Interestingly, we have also found that TCL1 is overexpressed in the CD4+ and CD8+ T cells of patients with unmutated B-CLL compared to mutated CLL or healthy controls. Thus the Tcl1 transgenic murine CLL model seems suitable as a model for the immune aberrations found in human unmutated CLL.
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Ghia, Paolo, Maria TS Bertilaccio, Cristina Scielzo, et al. "Novel Mouse Models of Chronic Lymphocytic Leukemia (CLL) Unravel the Molecular Mechanisms Controlling Bone Marrow Involvement by Leukemic B Cells." Blood 114, no. 22 (2009): 360. http://dx.doi.org/10.1182/blood.v114.22.360.360.
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Abstract Abstract 360 In CLL, the bone marrow (BM) represents a typical site of involvement and relapse, suggesting a preferential homing of leukemic cells to this anatomical site compared to other lymphoid organs, though the mechanisms controlling CLL cell migration and accumulation within the BM are unclear. In order to define the rules driving in vivo CLL cell re-circulation between the blood and tissutal compartments, we specifically generated two different mouse models and investigated the role played by HS1; this molecule, other than being a putative prognostic factor in CLL, is also involved in cytoskeleton reorganization of lymphocytes, and, potentially, in the control of cellular shape, migration and homing. First, we established a novel transplantable xenograft murine model of CLL by engrafting the cell line MEC1 into RAG2-/-γc-/- mice, at a variance with previous studies in nude mice where MEC cells failed to engraft. Likely due to the lack of B, T and NK cells (while nude mice retain NK cells), RAG2-/-γc-/- animals were successfully transplanted with the CLL cell line through either subcutaneous or intravenous routes, resulting in a systemic blood and tissutal involvement. When subcutaneous MEC1 cells silenced for HS1 expression were injected in these animals, we observed a preferential localization in the tumor draining axillary and inguinal lymph nodes and especially in the BM, when compared to controls. As we have previously demonstrated that CLL cases with hyper-phosphorylated HS1 show a worse clinical outcome, we took advantage of this mouse model to investigate the in vivo homing ability of primary CLL cells from patients showing different HS1 phosphorylation patterns. Purified leukemic cells from 4 patients with hyper-phosphorylated HS1 were labeled with high concentration of the dye CSFE, and each sample was paired and admixed with purified CLL cells obtained from patients with low levels of HS1 phosphorylation and separately labeled with low CSFE concentration. Each pair of samples was injected i.v. into RAG2-/-γc-/- mice recipients. When we analyzed the different organs of the animals by flow-cytometry, the differential expression of CFSE fluorescence (CFSE-high vs CFSE-low) allowed us to distinguish between the two leukemic cell populations with opposite HS1 phosphorylation status. In 3/4 experiments, CLL cells with hyper-phosphorylated HS1 revealed a preferential homing to the BM. Based on these results and on the in vitro evidence that B lymphocytes from HS1-/- mice have an impaired spontaneous migration, we have crossed HS1-/- (H-/-) mice with the Eμ-TCL1 transgenic (Ttg) mouse, an animal model that between 13 and 18 months of age develops a disease resembling human CLL. In the H-/-/Ttg mice, monoclonal CD19+CD5+ cells became evident earlier (at 7-13 months of age) and in significantly higher proportion as compared to Eμ-TCL1 transgenic mice. Cells preferentially localized in the BM where leukemic cells are usually observed at low frequencies in the Eμ-TCL1 mouse (mean value: 28%±16 vs 5%±2, respectively, p=0,008). These findings suggest that HS1 may have a relevant role in both normal and leukemic B-cells and in particular is crucial for cell migration, through its involvement in cytoskeleton organization. Accordingly, we also provide evidence that, in the absence of HS1, cells fail to form actin-myosin complexes, leading to an instability of the cell signalling complex. Our findings suggest a relationship between the expression of HS1 and the development and progression of CLL, most notably in terms of BM involvement, indicating that specific abnormalities in the cytoskeleton organization may be pivotal in regulating leukemic migration and infiltration in selected anatomical sites. This points at HS1 as a target for development of novel cancer treatments, aiming at interfering with the lymphoid tissue infiltration and invasion which is characteristic of the disease. In addition, these animal models could become very useful for evaluating the biological basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents. Disclosures: No relevant conflicts of interest to declare.
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Papait, Andrea, Tiziana Vaisitti, Sara Serra та ін. "Targeting the Adenosinergic Axis in the Eμ-TCL1 Chronic Lymphocytic Leukemia Mouse Model Offers Novel Therapeutic Opportunities". Blood 132, Supplement 1 (2018): 240. http://dx.doi.org/10.1182/blood-2018-99-118057.
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Abstract Background. The tumor microenvironment is typically an immunosuppressive niche often characterized by low oxygen tension, representing an intrinsic limitation to the success of immunotherapeutic approaches. Several lines of evidence indicate hypoxia is a master regulator of the adenosinergic axis, up-regulating on one side expression of CD39 and CD73, the two enzymes that generate adenosine starting from ATP/ADP and on the other side the adenosine receptors, which are powerful inhibitors of immune responses. Our previous studies using primary samples indicate that CLL cells can produce extracellular adenosine and that the CLL microenvironment is rich in expression of the A2A receptor, which potently inhibits T cell responses and skews macrophages towards type 2 responses(1, 2). Aim of the work. The main aims of this work are i) to dynamically study expression and activity of the adenosinergic axis using an in vivo model of CLL, and ii) to investigate whether its targeting may restore immune responses. Results. We exploited the TCL1 mouse model of CLL by adoptively transferring different leukemias into immunocompetent wild-type C57BL/6 mice and following the re-organization of the microenvironment and the reshaping of immune responses during disease progression. The first observation is that the appearance of large leukemic nodules that subvert normal splenic architecture is accompanied by increased staining with the hypoxia marker pimonidazole and by lactic acidosis, as witnessed by progressive increase in LDH activity within the tissue. The second observation concerns the onset of progressive immunosuppression with appearance of terminally differentiated and dysfunctional T lymphocytes and skewing of classical inflammatory monocytes to anti-inflammatory patrolling monocytes. Leukemia development is also accompanied by increased adenosine bio-synthetic potential due to the up-regulation of CD39 and CD73 and to the marked and generalized up-regulation of the A2A adenosine receptor, which is evident in all cells of the microenvironment, including leukemic cells, T lymphocytes and macrophages, as documented by real-time PCR studies on purified populations and by flow cytometry and immunohistochemistry analyses. This finding suggests that A2A may be a common mediator of immune suppression and that it may be a suitable therapeutic target. To test this hypothesis, we adoptively transferred different TCL1 leukemias in naïve C57BL/6 mice, allowed engraftment for 10 days and then treated with the commercially available SCH58621 A2A inhibitor every other day for two weeks (1mg/kg, intra-peritoneally). At the end of treatment, mice were euthanized and immune features examined as above. Results very consistently showed that A2A inhibition increased the naïve component of both CD4+ and CD8+ subsets, with a concomitant partial reduction in effector T lymphocytes and in regulatory T cells. Moreover, both CD4+ and CD8+ subsets recovered their cytotoxic functions as indicated by the production of IFN-γ and IL-2. When examining the macrophage compartment, SCH58261 repolarized monocytes, by increasing the inflammatory subset at the expense of patrolling monocytes. Conclusions. Taken together, these results highlight the relevance of the adenosinergic axis in the creation and maintenance of a tumor microenvironment that favors immune escape. This could be due, at least in part, to the presence of a highly hypoxic leukemic niche. Interrupting this network using an A2A receptor antagonist restores the functions of the different immune cell subsets. Therefore, these data suggest that the adenosinergic axis may represent a good target in treatment strategies that combine anti-leukemic drugs with agents able to repolarize the CLL environment towards immune competence.Serra S, et al. (2016) Adenosine signaling mediates hypoxic responses in the chronic lymphocytic leukemia microenvironment. 1(1).doi:10.1182/bloodadvances.2016000984.The.Serra S, et al. (2011) CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death. 118(23):6141-6153. Disclosures Deaglio: iTeos therapeutics: Research Funding; Verastem: Research Funding; VelosBio inc: Research Funding.
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Zanesi, Nicola, Rami Aqeilan, Alessandra Drusco та ін. "Effect of Rapamycin on Mouse Chronic Lymphocytic Leukemia and the Development of Nonhematopoietic Malignancies in Eμ-TCL1 Transgenic Mice". Cancer Research 66, № 2 (2006): 915–20. http://dx.doi.org/10.1158/0008-5472.can-05-3426.
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Rivas, Jacqueline R., Sara S. Alhakeem, Joseph M. Eckenrode, et al. "Enhancing Anti-Tumor Immunity and Responses to Immune Checkpoint Blockade By Suppressing Interleukin-10 in Chronic Lymphocytic Leukemia." Blood 134, Supplement_1 (2019): 5486. http://dx.doi.org/10.1182/blood-2019-127178.
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B-cell Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world, accounting for nearly one third of all leukemia cases. In CLL abnormal B-cells accumulate in the blood and lymphoid organs leading to serious immune dysfunction. This immune suppression is in part due to CLL-produced mediators that downregulate T-cell responses, such as the regulatory cytokine Interleukin-10 (IL-10). We previously found that eliminating T-cell IL-10 signaling enhanced their ability to control CLL growth in vivo. Therefore, we investigated the potential for IL-10 blockade to enhance the anti-tumor activity of CD8+ T-cells. In our studies we use human CLL cells as well as the Eμ-Tcl1 mouse model of CLL, in which the oncogene Tcl1 is expressed under the immunoglobulin VH promoter and µ-enhancer. IL-10 production by CLL cells depends on the transcription factor Sp1, and we found that the Sp1 inhibitor mithramycin (MTM) suppresses CLL IL-10 production. However, MTM is not well tolerated in vivo, so we synthesized novel, less toxic analogues of MTM to test for IL-10 suppression. One of these MTM analogues similarly suppresses mouse and human CLL IL-10 with little to no effect on effector T-cell cytokines and viability. Therefore, we treated mice with this analogue in the adoptive transfer model of Eμ-Tcl1, and later combined this with anti-PD-L1 checkpoint blockade to determine its effects on anti-tumor immunity. Here we show that this MTM analogue enhances the efficacy of anti-CLL T-cells in vivo by suppressing CLL IL-10 production, allowing for increased CD8+ T-cell proliferation, effector memory cell prevalence, and CD8+ interferon-γ (IFN-γ) production. Treatment slowed the growth of Eμ-TCL1 CLL cells in the spleen and blood and reduced the spread of CLL to the bone marrow. Furthermore, suppressing IL-10 in this manner improved responses to anti-PD-L1 treatment, decreasing the burden of CLL cells and the functionality of CD8+ T-cells in comparison to anti-PD-L1 alone. The overall number and frequency of CD8+ T-cells was higher in double treated mice, with more IFN-γ+ CD8+ cells, more effector memory cells, and fewer exhausted T-cells. This paradigm shifting approach is novel as current therapies for CLL do not target IL-10 and it may increase the efficacy of T-cell-based immunotherapies in human CLL. T-cell-based immunotherapies have experienced limited success in trials with CLL, and since there is no cure for this disease, our approach may provide a new avenue for combination therapies. Moreover, IL-10 blockade could be applicable to other B-cell malignancies and even solid tumors where T-cell suppression plays a significant role. Disclosures Hildebrandt: Axim Biotechnologies: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Sangamo: Equity Ownership; Novartis: Equity Ownership; Axim Biotechnologies: Equity Ownership; Juno Therapeutics: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Novartis: Equity Ownership; Insys Therapeutics: Equity Ownership; Abbvie: Equity Ownership; GW Pharmaceuticals: Equity Ownership; Cardinal Health: Equity Ownership; Immunomedics: Equity Ownership; Endocyte: Equity Ownership; Clovis Oncology: Equity Ownership; Cellectis: Equity Ownership; Aetna: Equity Ownership; CVS Health: Equity Ownership; Celgene: Equity Ownership; Bluebird Bio: Equity Ownership; Bristol-Myers-Squibb: Equity Ownership; crispr therapeutics: Equity Ownership; IDEXX laboratories: Equity Ownership; Johnson & Johnson: Equity Ownership; Pfizer: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Procter & Gamble: Equity Ownership; Vertex: Equity Ownership; Bayer: Equity Ownership; Scotts-Miracle: Equity Ownership; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Research Funding; Pharmacyclics: Research Funding; Astellas: Other: Travel.
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Rassenti, Laura, L. Huynh, G. W. Basak, et al. "TCL1 Expression in Chronic Lymphocytic Leukemia Correlates with the Intensity of 11q Deletions and ZAP-70." Blood 110, no. 11 (2007): 2068. http://dx.doi.org/10.1182/blood.v110.11.2068.2068.
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Abstract The TCL1 (T-cell leukemia/lymphoma1) oncogene is a coactivator of the AKT oncoprotein, an essential molecule in the transduction of antiapoptotic signals in T and B cells. Eμ-TCL transgenic mice with B cells with high TCL1 expression develop the aggressive phenotype of chronic lymphocytic leukemia (CLL). Studies in human CLL have found that expression of TCL1 correlates with high expression of ZAP-70 and use of unmutated IgVH genes. The expression of TCL1 may be regulated in part by microRNA, miR-29 and miR-181, which map to chromosome 11(11q). Because aberrations at 11q have been associated with poor prognosis in CLL, we interrogated the relationship between deletions at 11q and expression of TCL1 and ZAP-70. We used a direct immunophenotyping method to investigate the relative co-expression levels of TCL1 and ZAP-70 within CLL cells and examined the relationship between such levels and the proportion of leukemia cells within the CLL population bearing 11q deletions, as detected by FISH analysis. Direct staining of intracellular TCL1 protein was performed by using the monoclonal anti-TCL1 antibody (clone 1–21) labeled with Alexa647 in combination with the established ZAP-70 protocol (NEJM2004; 351:893) together with mAb directed against CD5 and B cell surface antigens. Negative staining levels were set using isotype control antibodies. FISH was performed on interphase nuclei by using uniform and cross-validated procedures at all CRC sites using the CLL-panel from Vysis. Chromosomal abnormalities were detected in 76% (520) of the 680 CLL samples analyzed. Sixteen percent of the patients had leukemia cells with monoalleleic deletions at 11q. We performed flow cytometry for intracellular co-expression TCL1 and ZAP-70 on cryopreserved samples obtained from 25 CLL patient samples with varying proportions of cells with the 11q deletion (10% to 98% abnormal cells with 11q deletion, mean 70%) and 30 CLL samples lacking any chromosomal abnormalities. We detected significantly higher levels of TCL1 in CLL cells that expressed ZAP-70 and/or unmutated IgVH genes. The ZAP-70pos cases (39/55) had a median percent of TCL1pos cells of 34% compared to 15% for the ZAP-70neg cases. The cases using unmutated IgVH genes (43/55) had a median percent of TCL1pos cells of 31%, which was greater than the 19% median observed for cases that used mutated IgVH genes. Multiparameter analyses revealed that the ZAP-70 positive fraction of each CLL clone had significantly higher levels of TCL1 than did the ZAP-70 negative cells (mean=45% versus 29%, respectively, p=0.002). We observed a significant difference between the expression levels of TCL1 for CLL cells that had deletions in 11q relative to that of CLL cells lacking any chromosomal abnormalities (mean=41% versus 18%, respectively p=0.0002). In addition, we observed a relationship between the levels of TCL1 expressed in leukemia cell populations and the relative proportion of leukemia cells with deletions at 11q. This study reveals a relationship between the levels of TCL1 expression in CLL leukemia-cell expression of ZAP-70, and the relative proportions of leukemia cells having deletions at 11q. Studies are in progress to define whether these relationships can be explained by altered expression of microRNA that map to 11q, which might also account for the noted adverse prognosis of CLL that has deletions at 11q.
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Wu, Qing-Li, Claudia Zierold, and Erik A. Ranheim. "Dysregulation of Frizzled 6 is a critical component of B-cell leukemogenesis in a mouse model of chronic lymphocytic leukemia." Blood 113, no. 13 (2009): 3031–39. http://dx.doi.org/10.1182/blood-2008-06-163303.
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Abstract Wnt/Fzd signaling is known to play a key role in development, tissue-specific stem-cell maintenance, and tumorigenesis, particularly through the canonical pathway involving stabilization of β-catenin. We have previously shown that Fzd9−/− mice have a deficiency in pre-B cells at a stage when self-renewing division is occurring in preference to further differentiation, before light chain immunoglobulin recombination. To determine whether pathologic usurpation of this pathway plays a role in B-cell leukemogenesis, we examined the expression of Wnt/Fzd pathway genes in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. We find that, in the course of leukemogenesis, the expression of Wnt16, Wnt10α, Fzd1, and most dramatically, Fzd6, is progressively up-regulated in the transformed CD5+ B cells of these mice, as are β-catenin protein levels. Elimination of Fzd6 expression by crossing into Fzd6−/− mice significantly delays development of chronic lymphocytic leukemia in this model. Our findings suggest that the self-renewal signals mediated by Wnt/Fzd that are enlisted during B-cell development may be pathologically reactivated in the neoplastic transformation of mature B cells.
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Yan, Xiao-Jie, Pablo Morande, Ezra B. Kolitz, et al. "Overexpression of Activation-Induced Deaminase in TCL1 Mice Leads to the Development of IGHV -Mutated and -Unmutated CLL Clones That Resemble Unique Subsets of Human CLL." Blood 126, no. 23 (2015): 1710. http://dx.doi.org/10.1182/blood.v126.23.1710.1710.
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Abstract Introduction. Somatic hypermutation (SHM) and class-switch recombination (CSR) are critical physiologic events in an effective normal B-cell immune response. Notably, both SHM and CSR are initiated by activation-induced cytidine deaminase (AID). In CLL, IGHV clonal mutations correlate strongly with better clinical outcomes. Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However because SHM and CSR occur rarely in the leukemic clones from these animals, TCL1 Tg mice mimic only IGHV -unmutated CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address these issues, we developed two new TCL1 strains by interbreeding mice over-expressing AID in all cells (Em-TCL1xActin-AID) or only in B lymphocytes (Em-TCL1xVκ-AID). Methods. B-cell clonal expansions were identified in spleen cells from 22 TCL1 and 33 TCL1xAID Tg (10 Em-TCL1xActin-AID plus 23 Em-TCL1xVκ-AID) mice at 10-20 months of age. This was done by amplifying cDNAs by PCR using consensus IGHV FR and IgM-, IgG-, and IgA-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. DNA sequences of these amplicons were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Because there were no major differences in the parameters listed below for the two TCL1xAID Tg mouse strains, data were combined. Results. Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 31 of 33 TCL1xAID mice; each animal bore an IgMκ+ clone and 7 also exhibited an IgGκ+ clone. IGHV gene use in IgMκ+ clones: IGHV use did not differ significantly between IgMκ+ TCL1 and IgMκ+ TCL1xAID clones. Approximately 50% used VH1-55, VH11-2, or VH12-3, some of which encoded stereotyped anti-phosphatidylcholine antibodies. SHM: Among the IgM+ TCL1 clones, there was a mutation frequency of 0.05% for IGHV and 0.09% IGKV. In contrast, the IGHV and IGKV mutation frequencies were considerably higher in TCL1xAID mice. Specifically, for IGHV, the TCL1xAID animals displayed a frequency of 0.47% in IgM+ and 3.0% in IgG+ clones; for IGKV, the frequency was 0.9% for IgM+ and IgG+ combined. Consistent with these mutations being mediated by AID, mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1. However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%, range: 0-1.9%) than the TCL1 level (0.05%), it was considerably less than that found in the TCL1xAID clones using other IGHV s (0.80%, range: 0-3.8%). In addition, no mutations were detected in VH1-55 clones. Consistent with a reduced AID mutational activity in clones expressing these specific genes, none of the genes were found in the isotype-switched, IgG-expressing clones. IGHV gene use and SHM in clones that underwent CSR: Notably, in only 2 of 9 instances was the same IGHV-D-J rearrangement found in IgMκ+ and IgGκ+ clones from the same mice; these IgM+ and IgG+ shared clones used VH5 genes. For the remaining 7, only the IgG+ version was detected; all but one of these used a VH1 gene. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%). Stereotyped IGHV-D-J and IGκ V-Jκ rearrangements: Among 43 IgM clones from TCL1xAID mice, we found 8 clones expressing VH11-2 and Vκ14-126 and 10 clones expressing VH12-3 and Vκ4-91; these IGHV-D-J and IGκV-Jκ rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones. Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes since despite AID over-expression certain IGHVs and IGKVs appeared less sensitive to major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, the two new TCL1xAID mouse strains described provide new models to study IGHV -mutated and IGHV -unmutated CLL and represent novel tools to evaluate the role of AID in leukemic progression. Disclosures No relevant conflicts of interest to declare.
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Yan, Xiao J., Emilia Albesiano, Gloria Telusma, Nicola Zanesi, Carlo M. Croce, and Nicholas Chiorazzi. "The BCRs Expressed by Leukemia Cells from TCL1 Transgenic Mice Resemble Those of Unmutated B-CLL." Blood 106, no. 11 (2005): 49. http://dx.doi.org/10.1182/blood.v106.11.49.49.
Full textAbstract:
Abstract Introduction: Eμ-T cell leukemia-1 (TCL-1) transgenic (Tg) mice serve as models of human B-cell chronic lymphocytic leukemia (B-CLL). These animals develop oligoclonal expansions of CD5+ B cells, one of which transforms into a B-CLL-like cell at ~13 - 18 months of age. A major unanswered question is whether the IgV gene restrictions seen in the TCL1 Tg model resemble those identified in human B-CLL. Therefore we analyzed the DNA sequences of the expressed, rearranged VHDJH and VLJL from 11 TCL1 Tg mice for V gene use, association with specific D and J segments, and shared H and L CDR3 motifs. Methods: Total RNA was isolated from spleens and lymph nodes of mice with obvious leukemia and reverse-transcribed to cDNA. To determine the Ig VH genes used by B cell clonal expansions, consensus FR1 primers and consensus JH primers were used for PCR. For Ig VL genes, Vκ consensus primers and Cκ primers were used. PCR products were either sequenced directly or cloned into vectors and then analyzed. DNA sequences were compared to the mouse Ig V gene germline genes deposited in NCBI GenBank and IMGT V-Quest. To confirm that nucleotide differences were actual point somatic mutations and not polymorphisms of known VH germline genes or heretofore unrecognized germline genes, PCR was performed on DNA from splenocytes of non-Tg mice using primers specific for the intron upstream of FR1 and the recombination signal sequences 3′ of the gene. PCR products were cloned and up to 60 colonies were sequenced. Using this approach, three new germline genes were identified and reported to GenBank. HCDR3 motifs were used to search both nucleotide and protein databases to identify similar sequences of known antigen specificity or B-cell subset origin. Results: DNA sequences of the VHDJH and VLJL from all (n=11) TCL1 Tg mice studied were &lt;2% different from the most similar germline counterpart. Eight of the 11 clones used VH 1 family genes and the other three used VH 3, 5 and 12 family genes. HCDR3 and LCDR3 of these sequences frequently contained charged amino acids at the V-(D)-J junctions. Database searches for sequences similar to those of the TCL1 clones revealed groups of non-B-CLL sequences with identical or very similar HCDR3 motifs; some of these groups used the same VH gene and others used different VH genes. These structurally similar antibodies were either autoantibodies or antibodies produced by B-1 cells. One anti-bacterial antibody also was included. Conclusions: The clones that eventually become leukemic in TCL1 mice resemble those of human B-CLL cases with the worst clinical outcome in that they do not exhibit significant levels of Ig V gene mutations and they are structurally similar to autoantibodies and anti-microbial antibodies. Therefore, this model will be valuable in analyzing the development and progression of B-CLL cells from normal CD5+ B cells and the role that antigen-receptor engagement by autoantigens and microbial antigens plays in this process.