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1
Tao, Jiangchuan, Tint Lwin, Xiaohong Zhao, Ling Zhang, Lynn Moscinski, Eduardo M. Sotomayor, William S. Dalton, and Jianguo Tao. "B Cell Receptor (BCR) Signal Pathways Confer Microenvironment-Mediated Drug Resistance and Are Promising Therapeutic Targets for B Cell Lymphomas." Blood 120, no. 21 (November 16, 2012): 4918. http://dx.doi.org/10.1182/blood.v120.21.4918.4918.
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Abstract Abstract 4918 Despite intensive efforts in developing new therapies, B-cell lymphoma remains essentially incurable with standard therapeutic approaches. Dynamic interactions between the lymphoma cell and its microenvironment play a critical role in lymphoma development and response to therapy. There is accumulating evidence pointing to an essential role of BCR signaling in B-cell lymphomas. Recently, inhibitors of BCR signaling have become an area of substantial clinical interest. We therefore studied the role of BCR signal pathways in stroma-mediated lymphoma cell survival. We demonstrated that adhesion of the various B-cell lymphoma cell lines to lymph node stromal cells enhanced activation of BCR signaling pathways: activation of phosphatidylinositol 3-kinase (PI3K), Bruton's tyrosine kinase (Btk) and extracellular signal-regulated kinase (ERK) pathways. Inhibition of Btk by PCI 32765 or PI3K by CAL101 significantly blocked BCR signal pathways, triggered lymphoma cell apoptosis and overcame stroma-mediated drug resistance. Furthermore, targeting BCR signal pathway disrupted microenvironmental stroma-lymphoma interaction through regulation of CXCR4 expression. Collectively, these data support that BCR activation not only controls intrinsic survival pathway related to B lymphoma cell but also regulates stroma-mediated extrinsic lymphoma cell survival as well as lymphoma cell homing and interplay with its microenvironment. As a result, this study suggests that targeting BCR signal pathway molecules is a promising therapeutic strategy to lymphoma therapy. Disclosures: No relevant conflicts of interest to declare.
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Pham, Lan, Juan Chen, Archie Tamayo, Jerry Bryant, David Yang, and Richard J. Ford. "Cannabinoid Receptor Signaling As a Target for Personalized Therapy in Aggressive B Cell Lymphomas." Blood 128, no. 22 (December 2, 2016): 4181. http://dx.doi.org/10.1182/blood.v128.22.4181.4181.
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Abstract Non-Hodgkin Lymphoma (NHL) is the most common hematological malignancy, with B-cell lymphoma (NHL-B) accounting for 85% of all lymphomas. In the United States, there are ~500,000 lymphoma patients currently living with this disease and ~20,000 lymphoma-related deaths occur annually. The current overall cure rate for B-cell lymphoma is estimated at ~30%, indicating that new innovative therapeutic approaches are needed to significantly reduce the high mortality rate, particularly of relapsed/refractory (r/r) NHL-B. The poor quality of life in patients suffering from chronic diseases like cancer has forced many patients to pursue alternative treatment options, including medicinal cannabinoids (CB), in order to improve their clinical prospect/outcomes. Medicinal cannabinoids have been legalized in 23 states and DC for several medical conditions such as cachexia, chronic pain, epilepsy and other similar disorders characterized by seizures, glaucoma, HIV- AIDS, Multiple Sclerosis, muscle spasticity and GI enteritis. Lately however, cannabis has been shown to have a broader biologic activity spectrum with various cannabis compounds functioning as ligands binding the two principle cannabinoid-specific G protein-coupled receptors (GPCR) CB1 (in neural cells), and CB2, in immune lymphoid, particularly B cells, but have also been identified, showing aberrant expression in a wide variety of important human cancers. This suggests not only a wider spectrum of cellular usage of cannabinoids and their cognate receptors, but also their potential utility as novel therapeutic targets. Gene expression profiling data has demonstrated, however, that B-cell lymphoma is one of the top three cancers (glioma and gastric are the other two) showing high expression of CB1 and CB2 receptors. Our studies showed that CB1 receptor is highly expressed in aggressive NHL-B, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cells in comparison to normal unstimulated (G0) B cells, and that targeting CB1 using an siRNA approach leads to cell growth inhibition. Furthermore, pharmacological approaches targeting CB1 with small molecule antagonists (Rimonabant and Otenabant) inhibited lymphoma cell viability, leading to the induction of apoptosis and G2M cell cycle arrest. Using proteomic approach via reverse-phase protein array (RPPA), we have demonstrated that lymphoma cells treated with the CB1 antagonist Rimonabant showed a robust effect on apoptosis (increases in caspase 3 and 7, Bad, and bak), cell cycle (increases in p27 and cyclin D1), DNA damage (increases in gH2AX), and autophagy (increases in LC3A) associated proteins. In addition, Rimonabant treatment also inhibited several growth and survival pathways, including STAT3, SRC, and b-catenin, while enhancing the PI3K/ATK pathway. Of note, Rimonabant treatment also activated the DNA damage response (DDR) pathway through stimulating two checkpoint kinases (Chk1 and Chk2). Blocking Rimonabant-induced Chk1 and Chk2 with a selective ATP-competitive inhibitor of Chk1 and Chk2 leads to a robust synergistic effect on cell growth inhibition and apoptotic induction, suggesting that blocking the DDR pathway with Chk kinase inhibitors prevents cells recovering from rimonabant-induced DNA damage. These findings suggest that targeting the cannabinoid receptors and the DDR pathway represents a new therapeutic strategy against resistant r/r NHL-B cells. Disclosures Pham: Vyripharm Biopharmaceuticals: Research Funding. Bryant:Vyripharm Biopharmaceuticals: Equity Ownership. Yang:Vyripharm Biopharmaceuticals: Employment.
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Byrd, John C. "Therapeutic Targeting of B-Cell Receptor Signaling Pathways." Blood 120, no. 21 (November 16, 2012): SCI—27—SCI—27. http://dx.doi.org/10.1182/blood.v120.21.sci-27.sci-27.
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Abstract Abstract SCI-27 Targeted therapy in hematologic malignancies has achieved significant therapeutic success when relatively selective inhibition is attainable to a target dispensable to the majority of normal cells. The best appreciated example of this is imatinib in chronic myeloid leukemia (CML), in which a single translocation forms a fusion protein involving the tyrosine kinase ABL that molecularly defines the disease. Kinase inhibition of ABL in this setting by imatinib (or other second- and third-generation kinase inhibitors) promotes durable, long-term remission in CML patients. The great majority of B-cell malignancies lack a characteristic translocation, activating mutation, or other aberration that facilitates such targeted therapy as employed in CML. Nonetheless, a common global signaling pathway involving the B-cell receptor (BCR) pathway has emerged as one that might be important to the control of these diseases. Furthermore, mouse models of select kinase targets (phosphoinositide-3 kinase p110 isoform-delta and Bruton's tyrosine kinase [BTK]) have shown that these are dispensable relative to long-term survival. Indeed, knockout or mutation of BTK and p110 delta each have a modest phenotype outside of diminished B-cell development and function. Based upon these preclinical observations, several molecules targeting BCR signaling have come forward to the clinic with exciting results across a wide range of B-cell malignancies. GS-1101 is a selective phosphoinositide-3 kinase p100 delta inhibitor with a very favorable toxicity profile that has shown promising clinical activity in low-grade lymphoma and chronic lymphocytic leukemia (CLL). The toxicity of this orally administered agent is quite modest in the majority of patients and allows sustained continuous dosing. Similarly, ibrutinib is an irreversible inhibitor of BTK and has shown promising clinical potential in an even broader range of B-cell malignancies, including diffuse large B-cell lymphoma, mantle cell lymphoma, low-grade lymphoma, and CLL. Toxicity with ibrutinib has also been modest, allowing long-term continuous dosing. Notably, each of these agents also produces an atypical mobilization of malignant lymphocytes into the blood soon after treatment. This treatment lymphocytosis is BCR-target-related due to diminished CXCR4/SDF-1 interface between tumor cells and stromal cells in the bone marrow, with egress of these cells to the blood. GS-1101 and ibrutinib are now entering phase III studies for regulatory approval and offer great potential to change the treatment paradigm of both CLL and B-cell non-Hodgkin lymphoma (NHL). Questions moving forward with these agents will include molecular predictors of response, feasibility and efficacy of combining with other effective therapies, and mechanisms of resistance. The scientific session presentation will provide an overview of the most promising BCR signaling agents in CLL and NHL clinical trials. Disclosures: No relevant conflicts of interest to declare.
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Witzig, Thomas E., and Mamta Gupta. "Signal Transduction Inhibitor Therapy for Lymphoma." Hematology 2010, no. 1 (December 4, 2010): 265–70. http://dx.doi.org/10.1182/asheducation-2010.1.265.
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Abstract Current research in lymphoma is focused on two areas of lymphoma biology—the signal transduction pathways used to maintain the growth of malignant lymphocytes and the role of the tumor microenvironment in lymphoma growth and survival. This review focuses on three signaling pathways: the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway, the B-cell receptor/spleen tyrosine kinase (BCR/Syk) pathway, and the protein kinase C-beta (PKC-β) pathway, known to be important to lymphoma cells. The mTOR inhibitors temsirolimus and everolimus have demonstrated antitumor activity in all types of lymphoma, the Syk inhibitor fostamatinib has activity in diffuse large B-cell lymphoma and chronic lymphocytic leukemia, and the PKC-β inhibitor enzastaurin is being used as consolidation therapy after remission in diffuse large B-cell lymphoma. This review discusses the biology behind the development of each new agent and the results of initial clinical trials. The goal is to provide the hematologist/oncologist background information on these new agents and understand their current and potential role in the management of patients.
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Staudt, Louis M. "Chronic Active B-Cell Receptor Signaling in Lymphoma." Blood 120, no. 21 (November 16, 2012): SCI—26—SCI—26. http://dx.doi.org/10.1182/blood.v120.21.sci-26.sci-26.
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Abstract Abstract SCI-26 We have developed loss-of-function, RNA interference-based screens to reveal genes essential for cancer cell proliferation and survival. In parallel, we are using high-throughput RNA resequencing (RNA-seq) to identify somatic mutations and other structural abnormalities in cancer. The intersection of these two data sets has helped us to discover novel pathogenetic pathways in lymphoma that are amenable to therapeutic attack. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) has constitutive activation of the NF-κB pathway, which we traced to the signaling adapter CARD11. In some ABC DLBCL biopsies (∼10%), somatic mutations produce CARD11 isoforms that spontaneously activate NF-κB signaling. In ABC DLBCL tumors with wild-type CARD11, we defined a “chronic active” form of B-cell receptor (BCR) signaling that activates NF-κB. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as Bruton's tyrosine kinase (BTK), or components of the BCR itself. Over one-fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. In 18 percent of cases, mutations occur in a single tyrosine residue in the critical “ITAM” signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Based on these findings, we are conducting clinical trials of ibrutinib in relapsed/refractory DLBCL. Ibrutinib is an irreversible and highly selective small-molecule inhibitor of BTK. Thus far, ibrutinib monotherapy has induced many complete and partial responses in patients with ABC DLBCL, including those with “primary refractory” tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 19 months, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. More recently, we have uncovered a “tonic” form of BCR signaling in Burkitt lymphoma that engages the prosurvival PI(3) kinase pathway. Two-thirds of Burkitt lymphoma cell lines die upon knockdown of BCR subunits or the proximal kinase SYK, due to loss of PI(3) kinase signaling. Moreover, a gene expression signature of PI(3) kinase activity is more highly expressed in Burkitt lymphoma biopsies than in biopsies of other aggressive lymphomas. Tonic BCR signaling in Burkitt lymphoma is mechanistically distinct from chronic active BCR signaling in ABC DLBCL, since it does not engage BTK, CARD11, or NF-κB. RNA-seq revealed that 70 percent of sporadic Burkitt lymphoma cases harbor somatic mutations that potentiate the action of the transcription factor TCF3 by preventing its inhibitory heterodimerization with the DNA-binding inhibitor ID3. TCF3 promotes tonic BCR signaling and PI(3) kinase activity in Burkitt lymphoma by transactivating the immunoglobulin heavy- and light-chain genes, thereby increasing surface BCR expression, and by repressing the phosphatase SHP-1, a potent negative regulator of BCR signaling. Hence, inhibitors of proximal BCR signaling and the PI(3) kinase pathway should be evaluated in Burkitt lymphoma, especially in patients for whom high-dose chemotherapy is infeasible, such as older individuals and those with the endemic form of this lymphoma. Disclosures: Off Label Use: I will be discussing clinical trials of ibrutinib (PCI-32765) in lymphoma. Ibrutinib is an investigational drug that has not yet received FDA approval for any indication.
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Blum, Kristie A. "B-cell receptor pathway modulators in NHL." Hematology 2015, no. 1 (December 5, 2015): 82–91. http://dx.doi.org/10.1182/asheducation-2015.1.82.
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AbstractWith the recent success of the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, and the phosphoinositide-3-kinase (PI3K) inhibitor, idelalisib, in the treatment of patients with relapsed or refractory non-Hodgkin's lymphoma (NHL), a number of new agents targeting the B-cell receptor (BCR) pathway are in clinical development. In addition, multiple trials combining these agents with conventional cytotoxic chemotherapy, immunomodulatory agents, monoclonal antibodies, or other kinase inhibitors are underway. This review will summarize the current data with the use of single agent and combination therapy with BCR inhibitors in NHL. In addition, commonly encountered as well as serious toxicities and hypothesized resistance mechanisms will be discussed. Lastly, this review will examine the future of these agents and opportunities to maneuver them into the front-line setting in selected NHL subtypes.
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Guidoboni, Massimo, Paola Zancai, Roberta Cariati, Silvana Rizzo, Jessica Dal Col, Alessandro Pavan, Annunziata Gloghini, et al. "Retinoic Acid Inhibits the Proliferative Response Induced by CD40 Activation and Interleukin-4 in Mantle Cell Lymphoma." Cancer Research 65, no. 2 (January 15, 2005): 587–95. http://dx.doi.org/10.1158/0008-5472.587.65.2.
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Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavorable prognosis. Here, we show that retinoic acid (RA) isomers significantly inhibit the proliferation of both primary MCL cultures (n = 7) and established cell lines (Granta 519 and SP-53) as shown by [3H]thymidine uptake and carboxyfluorescein diacetate succinimidyl ester labeling coupled with cyclin D1 staining. RA induces cell accumulation in G0-G1 together with a marked up-regulation of p27Kip1 by inhibiting ubiquitination and proteasome-dependent degradation of the protein. The p21Cip1 inhibitor was also up-regulated by RA in Granta 519 cells, whereas the expression of cyclin D1 is unaffected. Most of RA-induced p27Kip1 was bound to cyclin D1/cyclin-dependent kinase 4 complexes, probably contributing to the decreased cyclin-dependent kinase 4 kinase activity and pRb hypophosphorylation observed in RA-treated cells. Experiments with receptor-selective ligands indicate that RA receptor α cooperates with retinoid X receptors in mediating RA-dependent MCL cell growth inhibition. Notably, RA isomers, and particularly 9-cis-RA, also inhibited the growth-promoting effect induced in primary MCL cells by CD40 activation alone or in combination with interleukin-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results therefore further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds, our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting.
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Mondello, Patrizia, Enrico Derenzini, Zahra Asgari, John Philip, Elliot J. Brea, Venkatraman E. Seshan, Ronald Hendrickson, Elisa de Stanchina, David A. Scheinberg, and Anas Younes. "Dual Inhibition of Histone Deacetylases and Phosphoinositide 3-Kinase Enhances Therapeutic Activity Against B Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 293. http://dx.doi.org/10.1182/blood.v128.22.293.293.
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Abstract Oncogenic co-operation between c-Myc and activated phosphoinositide 3-kinase (PI3K) signaling pathways is crucial in lymphomagenesis, providing an opportunity for developing mechanism-based therapy to disrupt this co-operative survival mechanism. Combining constitutive c-Myc expression with constitutive PI3K activity in mouse germinal center B (GCB) cells resulted in Burkitt lymphoma-like tumors. Furthermore, analysis of primary human Burkitt lymphoma (BL) tissue sections revealed that two-thirds of the cases expressed high levels of phosphorylated AKT and S6 proteins, indicative of PI3K and mTORC1 activation. Prior attempts to develop small molecule inhibitors that specifically and directly target c-Myc protein have been unsuccessful. However, c-Myc cellular protein abundance can be decreased by using epigenetic modifying drugs (HDAC inhibitors or bromodomain/BET inhibitors) that are known to inhibit c-Myc transcription. Several investigators attempted disrupting c-Myc and PI3K cooperation by combining HDAC inhibitors and PI3K pathway inhibitors and synergic activity was demonstrated in DLBCL irrespective of subtype. In this study, we assessed the efficacy of CUDC-907, a new rationally designed dual inhibitor of PI3K and HDACs, in a panel of lymphoma cell lines. CUDC-907 treatment resulted in a dose- and time-dependent growth inhibition and cell death of DLBCL cell lines, irrespective of the cell of origin. CUDC-907 treatment down-regulated the phosphorylation of PI3K downstream targets, including AKT, PRAS40, S6, and 4EBP1, increased histone 3 acetylation, and decreased Myc protein levels. SILAC-based quantitative mass spectrometry demonstrated that CUDC-907 treatment decreased the protein levels of several components of the B cell receptor (BCR) and Toll like receptor (TLR) pathways, including BTK, SYK, and MyD88 proteins. These cellular changes were associated with an inhibition of NF-kB activation. CUDC-907 demonstrated in vivo efficacy with no significant toxicity in a human DLBCL xenograft mouse model. Collectively, these data provide a mechanistic rationale for evaluating CUDC-907 for the treatment of patients with c-Myc and PI3K-dependent lymphomas. Disclosures No relevant conflicts of interest to declare.
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Wu, Xiaosheng, Mary Stenson, Jithma Abeykoon, Kevin Nowakowski, Lianwen Zhang, Joshua Lawson, Linda Wellik, et al. "Targeting glycogen synthase kinase 3 for therapeutic benefit in lymphoma." Blood 134, no. 4 (July 25, 2019): 363–73. http://dx.doi.org/10.1182/blood.2018874560.
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Abstract Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.
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Huynh, Minh Quang, Ulrich Kaiser, Jennifer Goßmann, and Andreas Neubauer. "Expression of Phospholipse C Gamma 2 in Diffuse Large B-Cell Lymphomas,." Blood 118, no. 21 (November 18, 2011): 3667. http://dx.doi.org/10.1182/blood.v118.21.3667.3667.
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Abstract Abstract 3667 Cell survival and proliferation of diffuse large B-cell lymphomas (DLBCL) are dependent on the NF-κB signaling pathway. Antigen binding of the B-cell receptor (BCR) leads to phosphorylation of Src tyrosine kinases, resulting in PLCg2 activation, which is important for regulation of NF-κB through the protein kinase C (PKC). The PLC-PKC cascade also activates the Ras signaling pathway. There are several clinical studies showing that inhibition of PKCβ with the specific inhibitor Enzastaurin could be a promising new therapy target for DLBCL. To our knowledge, there are little data about the role of PLCg2 in DLBCL. It has been shown that the activity of Syk and PLCg2 are well correlated with sensitivity of DLBCL to dasatinib. Our study performed immunohistological staining of PLCg2 in 86 primary DLBCL. PLCg2 was strongly expressed in 54 out of 86 cases (63%) and weakly expressed in 28 cases (32%). Only 4 cases (5%) were negative for PLCg2. In three DLBCL cell lines (SuDHL-4, SuDHL-6 and U2932) we could show an inhibitory effect of the PLC-inhibitor U73122 on cell proliferation. Treatment in combination with Enzastaurin or the Src-inhibitor pp2 had an additive effect on cell proliferation compared to U73122 alone. In conclusion, PLCg2 is strongly expressed in a great subset of DLBCL. Inhibition of PLCg2 could be, at least in these cases, a new target for lymphoma treatment. Disclosures: No relevant conflicts of interest to declare.
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Kloo, Bernhard, Daniel Nagel, Matthias Pfeifer, Michael Grau, Michael Düwel, Michelle Vincendeau, Bernd Dörken, Peter Lenz, Georg Lenz, and Daniel Krappmann. "Critical role of PI3K signaling for NF-κB–dependent survival in a subset of activated B-cell–like diffuse large B-cell lymphoma cells." Proceedings of the National Academy of Sciences 108, no. 1 (December 20, 2010): 272–77. http://dx.doi.org/10.1073/pnas.1008969108.
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The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy.
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Arita, Adriana, Katherine Hanlon, Halina Chkourko, Brian Joseph Lannutti, David Michael Johnson, Janice Lynn Gabrilove, Eileen Scigliano, Samir S. Parekh, and Joshua Brody. "Effect of phosphotidylinositol 3-kinase-delta inhibitor idelalisib (GS-1101) on signaling in primary non-Hodgkin lymphoma cells: Correlative studies from NCT01306643." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 8579. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.8579.
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8579 Background: Studies of GS-1101, an oral phosphatidylinositol 3-kinase (PI3K)d-specific inhibitor, in heavily pre-treated patients with indolent-non-Hodgkin's lymphomas (iNHL) have shown marked clinical activity [Kahl BS, et al., Blood 2010]. We have shown that GS-1101 blocks both constitutive and inducible signaling events proximally downstream of PI3K [Lannutti BJ, et al., Blood 2011]. We hypothesized that flow-cytometric interrogation of PI3Kd-inhibition in iNHL samples might demonstrate signaling differences between patients that can be correlated with clinical outcomes. Methods: Single-cell suspensions of biopsy specimens from patients were incubated with or without GS-1101, B-cell receptor stimulated and stained with lineage markers and phospho-specific antibodies targeting signaling nodes proximally (e.g. pAkt S473) or distally (e.g. pS6 S235/6) downstream, or "parallel" to (e.g. pErk1/2) described PI3K pathways. Results: Healthy and iNHL B cells demonstrated complete or near-complete inhibition of PI3K proximal downstream signaling by GS-1101. PI3K distal downstream signaling was completely inhibited by GS-1101 in healthy B cells. The degree inhibition of distal downstream signaling was variable between iNHL patients. iNHL showed marked variability in the degree of PI3K distal downstream signaling amongst tumor cells within the same sample, suggesting an admixture of "PI3K-dependent“ and "PI3K-independent" tumor cells. We also observe variable PI3K-independence amongst other NHL histologies such as mantle cell lymphoma. Additionally, combining inhibition of PI3K with that of other signaling nodes shows additive distal downstream effects. Conclusions: We demonstrate that GS-1101 blocked both constitutive and invoked signaling proximally downstream of PI3K in primary iNHL cells. By correlating clinical outcomes in the ongoing study with signaling readouts between patients we may develop predictive rules for response. Ongoing studies will focus on studying the change in intratumoral cell subsets which develop in vivo as patients progress on PI3Kd inhibitor therapy.
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Weng, Jing-Ru, Chang-Fang Chiu, Jing-Lan Hu, Chia-Hsien Feng, Chiung-Yao Huang, Li-Yuan Bai, and Jyh-Horng Sheu. "A Sterol from Soft Coral Induces Apoptosis and Autophagy in MCF-7 Breast Cancer Cells." Marine Drugs 16, no. 7 (July 17, 2018): 238. http://dx.doi.org/10.3390/md16070238.
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The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that plays a key role in regulating cellular metabolism, and is a therapeutic target for cancer therapy. To search for potential PPARγ activators, a compound library comprising 11 marine compounds was examined. Among them, a sterol, 3β,11-dihydroxy-9,11-secogorgost-5-en-9-one (compound 1), showed the highest PPARγ activity with an IC50 value of 8.3 μM for inhibiting human breast adenocarcinoma cell (MCF-7) growth. Western blotting experiments showed that compound 1 induces caspase activation and PARP cleavage. In addition, compound 1 modulated the expression of various PPARγ-regulated downstream biomarkers including cyclin D1, cyclin-dependent kinase (CDK)6, B-cell lymphoma 2 (Bcl-2), p38, and extracellular-signal-regulated kinase (ERK). Moreover, compound 1 increased reactive oxygen species (ROS) generation, upregulated the phosphorylation and expression of H2AX, and induced autophagy. Interestingly, pre-treatment with the autophagy inhibitor 3-methyladenine rescued cells from compound 1-induced growth inhibition, which indicates that the cytotoxic effect of compound 1 is, in part, attributable to its ability to induce autophagy. In conclusion, these findings suggest the translational potential of compound 1 in breast cancer therapy.
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Jain, Neeraj, Satishkumar Singh, Georgios Laliotis, Amber Hart, Elizabeth Muhowski, Kristyna Kupcova, Tereza Chrbolkova, et al. "Targeting phosphatidylinositol 3 kinase-β and -δ for Bruton tyrosine kinase resistance in diffuse large B-cell lymphoma." Blood Advances 4, no. 18 (September 14, 2020): 4382–92. http://dx.doi.org/10.1182/bloodadvances.2020001685.
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Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-β expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-β/δ. Treatment with the selective PI3K-β/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.
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Lim, Kian-Huat, Donna L. Romero, Divya Chaudhary, Shaughnessy D. Robinson, and Louis M. Staudt. "IRAK4 Kinase As A Novel Therapeutic Target in the ABC Subtype of Diffuse Large B Cell Lymphoma." Blood 120, no. 21 (November 16, 2012): 62. http://dx.doi.org/10.1182/blood.v120.21.62.62.
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Abstract Abstract 62 Activating mutations of MyD88, particularly L265P, occur in about 30% of activated B cell-like diffuse large B cell lymphomas (ABC DLBCLs), the more malignant molecular subtype of DLBCL that responds poorly to standard chemotherapy. We found that the oncogenic signal of mutant MyD88 is transduced predominantly through the Interleukin-1 Receptor-Associated Kinase (IRAK) cascade consisting of IRAK4 and IRAK1, resulting in constitutive activation of classical NF-κB signaling. Importantly, using knockdown-rescue experiments, we found that the kinase activity of IRAK4, but not IRAK1, is required for the oncogenic effect of mutant MyD88 in the survival of ABC DLBCL cell lines. As such, we have proposed that inhibitors of IRAK4 kinase activity could have a therapeutic impact in lymphomas with MyD88 mutations. We have identified two potent small molecule IRAK4 inhibitors, ND-2158 and ND-2110, that are highly selective across a panel of more than 300 kinases. When administered to rodents, both compounds lead to >90% suppression of LPS-induced TNF-α release in serum. Both inhibitors are universally toxic towards ABC DLBCL but not GCB DLBCL cell lines, consistent with the highly specific mechanism of action. The molecules demonstrate good pharmacologic drug-like properties and are expected to have a suitable safety profile for clinical evaluation. Mechanistic studies indicate that both inhibitors potently abrogate IRAK4-mediated phosphorylation of IRAK1 and NF-κB activity resulting from the MyD88 (L265P) mutation in ABC DLBCL. Additionally, these agents suppress secretion of the pro-inflammatory cytokines IL-6 and IL-10 by ABC DLBCL cells. A second, parallel survival pathway in ABC DLBCL is engaged by “chronic active” B cell receptor signaling, which can be blocked by inhibiting Bruton's tyrosine kinase (BTK) either genetically or pharmacologically. Notably, the IRAK4 inhibitors strongly synergized with BTK knockdown in killing multiple ABC DLBCL cell lines. Our results provide a solid rationale for the further development of IRAK4 inhibitors for the therapy of ABC DLBCL and suggest that simultaneous inhibition of B cell receptor signaling may provide superior clinical responses. Disclosures: Romero: NIMBUS Discovery: Employment. Chaudhary:NIMBUS Discovery: Employment. Robinson:NIMBUS Discovery: Consultancy, Employment.
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Zhao, Xiaoxian, Andrew E. Schade, and Eric Hsi. "Distinct Role of Src Family Kinase Inhibitors in Burkitt Lymphoma Cells Vs. Diffuse Large B-Cell Lymphoma Cells." Blood 112, no. 11 (November 16, 2008): 3765. http://dx.doi.org/10.1182/blood.v112.11.3765.3765.
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Abstract Introduction: The Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of malignancies, with approximately 85% of NHL belonging to the B-cell lineage. Src family kinases (SFKs) are non-receptor intracellular tyrosine kinases which are important in the regulation of multiple signaling pathways including cell proliferation, tumor invasiveness, angiogenesis and apoptosis. Syk is another predominant tyrosine kinase expressed in B-cell lines in addition to SFKs. We attempted to correlate SFK and Syk inhibitor efficacy with the presence of phospho-SFK or phospho-Syk in lymphoma cell lines and tissues. Methods: Cell proliferation was measured with WST-1 reagent. Apoptotic assay was performed with Annexin-V and 7-AAD by flow cytometry (FC, FACSCalibur, BD Bioscience). Phospho-Src (Y416) antibody (cell signaling Technology, CSL) was used for immunoblotting and immunohistochemistry. (IHC, Discovery, Ventana Medical Systems). Phospho-Syk (Y525/526) antibody (CSL) was used for FC and immunoblotting. Results: In a screening for the effects of different kinases’ inhibitors on B-cell lymphoma lines, we observed that SFK inhibitors, PP2 and dasatinib (Sprycel, Bristol Myers Squibb), inhibited proliferation and caused dose-dependent apoptosis induction at 24 h (PP2: 31% at 10 mM; dasatinib: 39% at 100 nM) in Burkitt’s lymphoma cell line Raji. The apoptotic induction was associated with cleavage of caspase-3 and caspase-8. The ability of SFK inhibitors to induce apoptosis in Raji cells paralleled high level expression of constitutive phospho- SFK (Y416). In contrast to this Burkitt’s line, diffuse large B-cell lymphoma (DLBCL) lines (Sud-HL4, Sud-HL-6 and OCI-LY3, OCI-LY10) were less-sensitive to these SFK inhibitors but showed apoptosis induction upon exposure to the Syk inhibitor (piceatannol & syk inhibitor IV). Interestingly, the DLBCL lines that were resistant to SFK inhibitors had undectable or low levels of phospho-SFK (Y416); while their susceptibility to the Syk inhibitor-induced apoptosis paralleled detectable constitutive phospho-Syk (Y525/526). Immunohistochemical staining of burkitt’s lymphoma tissues and a tissue microarray panel of NHL indicated 13/20 (65%) of Burkitt’s lymphoma, 3/5 of small lymphocytic lymphoma, 2/5 of mantle cell lymphoma, 3/10 of follicular lymphoma, 2/5 of DLBCL, 2/5 of marginal zone lymphoma, 1/5 of lymphoblastic lymphoma are positive for phospho-Src (Y416). Staining of normal tonsil tissue showed germinal center cells are strong positive for phospho-Src (Y416), while marginal zone cells are weak positive and plasma cells are negative. We are currently testing the correlation of phospho-Src (Y416) expression in fresh NHL tissues and their sensitivity to Src family kinase inhibitors. Conclusion: These data suggest that rational application of molecularly targeted therapy for aggressive NHL is possible by directly examining key signaling nodes promoting survival and proliferation. For instance, the clinical SFK inhibitor dasatinib is currently being examined in a clinical trial for NHL (NCT00550615). Our results suggest that profiling patients’ lymphoma cells for phospho-SFK could optimize therapeutic efficacy and minimize unnecessary treatment-related side effects.
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Ponader, Sabine, and Jan A. Burger. "Bruton's Tyrosine Kinase: From X-Linked Agammaglobulinemia Toward Targeted Therapy for B-Cell Malignancies." Journal of Clinical Oncology 32, no. 17 (June 10, 2014): 1830–39. http://dx.doi.org/10.1200/jco.2013.53.1046.
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Discovery of Bruton's tyrosine kinase (BTK) mutations as the cause for X-linked agammaglobulinemia was a milestone in understanding the genetic basis of primary immunodeficiencies. Since then, studies have highlighted the critical role of this enzyme in B-cell development and function, and particularly in B-cell receptor signaling. Because its deletion affects mostly B cells, BTK has become an attractive therapeutic target in autoimmune disorders and B-cell malignancies. Ibrutinib (PCI-32765) is the most advanced BTK inhibitor in clinical testing, with ongoing phase III clinical trials in patients with chronic lymphocytic leukemia and mantle-cell lymphoma. In this article, we discuss key discoveries related to BTK and clinically relevant aspects of BTK inhibitors, and we provide an outlook into clinical development and open questions regarding BTK inhibitor therapy.
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Xia, Bing, Xiaowu Li, Le Zhang, Qing Guo, Xin Jin, Shanqi Guo, Eduardo M. Sotomayor, and Yizhuo Zhang. "BTK inhibitor PCI-32765 targets endogenous and microenvironment-mediated B-cell receptor signaling to suppress lymphoma cell growth and is highly synergistic with Bortezmib against non-Hodgkin B-cell lymphomas." Blood 120, no. 21 (November 16, 2012): 4721. http://dx.doi.org/10.1182/blood.v120.21.4721.4721.
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Abstract Abstract 4721 Background: B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of B-cell malignancies and Bruton tyrosine kinase(Btk) is essential for BCR signaling and function. PCI-32765, a specific and irreversible small-molecule Btk inhibitor, has recently been reported to display a significant clinical activity against non-Hodgkin B-cell lymphomas (NHL) especially chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL). In this study we set to explore 1) the role of Btk in NHL cell apoptosis and proliferation, 2) the role of BtK in bone marrow strom-mediated lymphoma cell survival and 3) to test if PCI-32765 as a therapeutic agent in single or in combination with Bortezomib for NHL therapy. Methods: B-cell lymphoma cell lines including mantle cell lymphoma lines (Jeko-1 and HBL-2), Burkitt lymphoma cell line (Raji) and transformed large cell lymphoma cell line (SUDHL-10) as well as primary lymphoma cells from various NHL samples were used for the experiments. These cells were cultured in the presence or absence of bone marrow mesenchymal stromal cells (MSC). The endogenous and MSC-induced Btk and its signaling activation such as BtK, ERK1/2 and AKT expression and phosphorylation status as well as its inhibition by were examined PCI-32765 by Western blot. The effects of PCI-32765 on lymphoma cell growth and appotosis were analyzed by using MTT, DAPI stain and flow cytometric annexin V/PI staining. Furthermore, the combined effect of PCI-32765 and Bortezomib on lymphoma cell growth and apoptosis was analyzed using the CalcuSyn software program in search for a synergistic or additive effect. Results: We found constitutive expression and activation of Btk and its downstream signaling in most of these cell lines and primary lymphoma cells. Furthermore, co-culture with MSC cells further enhanced the phosphoration of Btk and AKT in these cells. Incubation of Jeko-1, Raji, HBL-2 and SUDHL-10 cell lines with PCI-32765 induced cell growth inhibitory effects. We found that PCI-32765 exhibited a significant dose-dependent induction of cytotoxicity in these cells at various time points as measured by MTT. We also found significant apoptosis in these cells treated with PCI-32765. In addition, PCI-32765 significantly inhibited phpsphorylation of AKT and Btk, confirming the block of BCK signal pathways in these cells. Finally, MTT assays indicated that combined PCI-32765 with Bortezomib induced a synergistic cytotoxicity against these NHL cells (CI<1). Discussion: Our studies therefore highlight the biological significance of Btk in B-cell lymphoma cell growth and survival. PCI-32765 effectively antagonizes B-cell survival provided by bone marrow stromal cells and synergistically in combination with Bortezomib eliminates lymphoma cells. This study provide rational for targeting BCR and Btk as a novel therapeutic approach for NHL. Disclosures: No relevant conflicts of interest to declare.
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Roué, Gaël, Patricia Pérez-Galán, Mónica López-Guerra, Neus Villamor, Elias Campo, and Dolors Colomer. "Selective Inhibition of I kappaB Kinase Sensitizes Mantle Cell Lymphoma B Cells to TRAIL by Decreasing c-FLIP Level." Blood 108, no. 11 (November 16, 2006): 258. http://dx.doi.org/10.1182/blood.v108.11.258.258.
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Abstract Mantle cell lymphoma (MCL) is an aggressive B lymphoid neoplasm with a mature B-cell phenotype and genetically characterized by the t(11;14)(q13;q32) leading to cyclin D1 overexpression with the consequent deregulation of cell cycle at the G1-S checkpoint. MCL cells also present a constitutive activation of the NF-kappaB pathway which leads to the overexpression of several anti-apoptotic regulators. We have analyzed sensitivity to the extrinsic apoptotic signal triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on six human MCL cell lines and primary cells from 10 MCL patients, which differ in their p53-dependent pathway status, growth characteristics and sensitivity to cytotoxic drugs. TRAIL has been shown to exert in vivo a selective anti-tumor activity with minimal toxicity on normal cells. We observed that TRAIL was able to trigger apoptosis in a majority of MCL cell lines and primary MCL tumor cells, while sparing normal peripheral B cells. TRAIL-induced cell death was characterized by a time- and dose-dependent loss of membrane potential, Bax and Bak activation, caspase activation and phophatidylserine exposure. MCL sensitivity to TRAIL was irrespective of TRAIL-R1 and TRAIL-R2 receptor levels, Bcl-2 family members or caspase regulators expression, but was closely linked to the activity of the NF-kappaB p50 factor and to the expression of c-FLIP, a NF-kappaB-regulated factor. C-FLIP accumulated into the TRAIL-dependent complex in resistant cells and its transient knockdown overcame MCL resistance to TRAIL. In parallel, NF-kappaB inhibitors differentially modulated TRAIL cytotoxicity. Indeed, sub-toxic doses of bortezomib increased TRAIL cytotoxic effects by up-regulating TRAIL-R2 receptor expression, but also led to the intracellular accumulation of c-FLIP, impeding full synergistic interaction in cells with highest c-FLIP basal level. In contrast, the IkappaB kinase (IKK) inhibitor BMS-354451 allowed to consistent reduction of NF-kappaB activity, decreased total and DISC-associated c-FLIP levels, and sensitized all MCL cells to TRAIL cytotoxic effects. These results indicate that pharmacological enhancement of MCL cells sensitivity to TRAIL does not depend on TRAIL receptors level but is rather regulated by NF-kappaB-regulated c-FLIP expression. Considering that both TRAIL and BMS-345541 have already demonstrated selective cytotoxicity against malignant cells, combining TRAIL, with pharmacological inhibitors of IkappaB kinase signaling may represent an attractive model for the design of a new and rational combination therapy for MCL.
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Yin, Wu, Nie Zhe, Andrew Placzek, Michael Trzoss, Goran Krilov, Shulu Feng, Morgan Lawrenz, et al. "Identification of Potent Paracaspase MALT1 Inhibitors for Hematological Malignancies." Blood 136, Supplement 1 (November 5, 2020): 30. http://dx.doi.org/10.1182/blood-2020-141146.
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Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), was identified as a translocation protein fused with cIAP2 in mucosa-associated lymphoid tissue (MALT) B cell lymphomas. MALT1, a key mediator of NF-κB signaling and the main driver of a subset of B-cell lymphomas, functions via formation of a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 has been considered as a potential therapeutic target for several non-Hodgkin B cell lymphomas as well as chronic lymphocytic leukemia (CLL). Here, we describe the discovery of novel, potent MALT1 inhibitors that result in antiproliferative effects in non-Hodgkin B-cell lymphoma cells. Results: We have identified novel small molecule MALT1 inhibitors using our proprietary physics-based Free Energy Perturbation (FEP+) modeling technology. Our compounds show potent (sub nM) inhibition of MALT1 enzymatic activity, as well as high binding affinity (sub nM) to MALT1 protein measured by Surface Plasmon Resonance (SPR). BCL10 is a binding partner of MALT1 that is cleaved by MALT1 at the C-terminus. Our inhibitors were efficacious in a target engagement assay showing prevention of BCL10 cleavage in Activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines OCI-LY3 and OCI-LY10, which are Bruton tyrosine kinase (BTK) inhibitor ibrutinib-resistant and -responsive respectively. Our compounds are potent inhibitors of IL10 secretion in both OCI-LY3 and OCI-LY10 cells, which is consistent with the inhibition of NF-κB signaling. We also examined the effect of our MALT1 inhibitors on ABC-DLBCL cell proliferation. Our inhibitors demonstrated potent anti-proliferative effects in both OCI-LY3 and OCI-LY10 cell lines, as well as synergistic effects with ibrutinib in a BTKi sensitive ABC-DLBCL cell panel. Examinations of a protease panel and off-target safety screening panel, as well as in vivo high dose tolerability study showed our compound had excellent selectivity and significant safety margin. Plasma IL10 and tumor BCL10 have been identified as robust PD markers in PK/PD studies in both OCI-LY3 and OCI-LY10 tumor bearing mice. Dose-dependent tumor growth inhibition was observed after 3 weeks of treatment in OCI-LY3 xenograft model, with efficacy also observed in combination with venetoclax. Ongoing work: We are continuing to explore the synergistic effects of our compounds with BTK inhibitors in B-cell lymphoma mouse models. Preliminary data showed potent inhibition of IL-2 secretion in Jurkat cells from our compound treatment. Additional studies are ongoing to elucidate the role of MALT1 inhibition in Treg as well as Teffector cells in vitro and in vivo. Refinement of the current inhibitor series, using co-crystal structures, is in progress in preparation for further development of optimized molecules. Conclusion and Future Plans: We have identified novel potent MALT1 protease small molecule inhibitors that are efficacious in the in vitro B-cell lymphoma cell proliferation assays and in the in vivo B-cell lymphoma xenograft model. Our data suggest that targeting MALT1 may expand therapy options for patients with selected B-cell lymphomas, such as ABC-DLBCL. Our work provided insight into the anti-tumor efficacy of our inhibitors in B-cell lymphomas as single agent, and ongoing work will continue to assess the potential combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphoma. Disclosures Yin: Schrodinger: Current Employment, Current equity holder in publicly-traded company. Zhe:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Placzek:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Trzoss:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Krilov:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feng:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lawrenz:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Pelletier:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lai:Triplet Therapeutics: Current Employment, Current equity holder in private company. Bell:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Calkins:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Grimes:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Tang:Schrodinger: Current Employment, Current equity holder in publicly-traded company. McRobb:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Gerasyuto:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feher:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Mondal:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Jensen:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Wright:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Akinsanya:Schrodinger: Current Employment, Current equity holder in publicly-traded company.
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Wiestner, Adrian. "BCR pathway inhibition as therapy for chronic lymphocytic leukemia and lymphoplasmacytic lymphoma." Hematology 2014, no. 1 (December 5, 2014): 125–34. http://dx.doi.org/10.1182/asheducation-2014.1.125.
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Abstract Chronic lymphocytic leukemia (CLL) and lymphoplasmacytic lymphoma (LPL) are malignancies of mature B cells. In LPL, mutations of the adaptor protein MYD88 (L265P) in the Toll-like receptor pathway have been recognized recently as being a hallmark of the disease and indicate a dependence of the tumor on this pathway. In CLL, functional studies have implicated BCR activation in the tissue microenvironment as a pivotal pathway in the pathogenesis. Bruton's tyrosine kinase (BTK) and the PI3Kδ isoform are essential for BCR signaling and also seem to be required for signal transduction in LPL cells, even if the role of BCR signaling in this disease remains less well defined. Ibrutinib, a covalent inhibitor of BTK approved by the Food and Drug Administration as a second-line treatment for CLL, and idelalisib, a selective inhibitor of PI3Kδ, achieve excellent clinical responses in both diseases irrespective of classic markers indicating high-risk disease. Several additional inhibitors targeting BTK and PI3Kδ, as well as the spleen tyrosine kinase, have entered clinical trials. This review discusses the biologic basis for kinase inhibitors as targeted therapy for CLL and LPL and summarizes the clinical experience with these agents.
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Ghosh, Asish K., Debabrata Mukhopadhyay, and Neil E. Kay. "Bosutinib, a Src/Abl Kinase Inhibitor Induces Apoptosis in CLL B Cells by Targeting Multiple Tyrosine Kinases and Overcomes Stroma Protection." Blood 114, no. 22 (November 20, 2009): 2368. http://dx.doi.org/10.1182/blood.v114.22.2368.2368.
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Abstract Abstract 2368 Poster Board II-345 Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. A novel therapeutic agent, Bosutinib, was initially developed as an inhibitor of Src and Abl kinases and is currently in phase II clinical trials for the treatment of several human malignancies. Recently, Bosutinib has been shown to inhibit phosphorylation of a novel receptor tyrosine kinase, Axl which has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Previously, Dasatinib, another Src/Abl kinase inhibitor, showed cytotoxic effects on CLL B cells by decreasing levels of activated, phosphorylated forms of Akt, Erk1/2 and p38 and reducing expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in CLL B cells. Here, we wished to examine receptor or non-receptor tyrosine kinases active in primary CLL B-cells and determine their status after exposure to Bosutinib as well as the latter drug's effect on CLL B-cell viability. Methods: We used freshly isolated CLL B cells after obtaining written consent from patients. Bosutinib was used at various doses (2.5, 5.0, 10.0 and 20.0 μM) to treat CLL B cells in vitro for 24/48 hrs. Induction of apoptosis was assessed by annexin/propidium staining. To examine the impact of stroma on Bosutinib induced CLL B-cell death, primary CLL-bone marrow stromal cells (BMSC) were cocultured with CLL B cells at a cell density ratio of 1:20 and treated with various doses of Bosutinib for 24 hrs. Expression status of various kinases and downstream targets were analyzed in CLL B cell lysates with or without Bosutinib-treatment by Western blot using specific antibodies. Results: Treatment of CLL B-cells with Bosutinib induces a massive apoptotic cell death in a dose- and time-dependent manner (IC50 for 24 h; ∼10 μM and IC50 for 48 h: 5-10 μM) which involves PARP cleavage as demonstrated by Western blot analysis. Moreover, Bosutinib-treatment reduced expression of several key anti-apoptotic proteins, Mcl-1, XIAP and Bcl-2 reported to be overexpressed in CLL B cells. Interestingly, we detected that the majority of CLL B-cells express constitutively active Axl. Importantly, Bosutinib treatment inhibited phosphorylation of Axl in CLL B cells resulting in inhibition of AKT-activation, one of its downstream signaling pathways. Previous studies have suggested a possible physical association between Axl and Src kinase. We observed that expression of constitutively active Axl was associated with the presence of highly phosphorylated Src kinase when compared with that in CLL B-cells with low or unphosphorylated Axl. These observations suggest that phosphorylation of Axl may be an upstream event for Src activation in CLL. We found inhibition of constitutively active Axl resulted in subsequent inhibition of Src kinase activation in CLL B-cells following Bosutinib-treatment. We also detected inhibition of ZAP70/Syk-phosphorylation in CLL B cells upon Bosutinib-treatment. Finally, we found Bosutinib was able to overcome stomal protection of CLL B cells at a dose of 10 μM in an in vitro coculture system suggesting its potential as a therapeutic agent against CLL. Conclusion: Together, these observations suggest that Bosutinib induces apoptosis in CLL B-cells, even in the presence of stromal cells, in association with the down regulation of multiple kinases including the novel receptor-tyrosine kinase, Axl, and reduces expression of the anti-apoptotic proteins critical to CLL B-cell survival. In total, these findings for the first time indicate that Bosutinib has the potential to be a very potent therapeutic agent for CLL patients. Disclosures: Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.
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Faisal, Muhammad Salman, Hira Shaikh, Ahmed Khattab, Mary Albrethsen, and Salman Fazal. "Cerebral aspergillosis in a patient on ibrutinib therapy—A predisposition not to overlook." Journal of Oncology Pharmacy Practice 25, no. 6 (July 25, 2018): 1486–90. http://dx.doi.org/10.1177/1078155218788717.
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Ibrutinib has revolutionized the treatment of B-cell malignancies since its approval for chronic lymphocytic leukemia. It is also used in mantle cell lymphoma, diffuse large B-cell lymphoma, Waldenstrom’s macroglobulinemia, among others. It is a Bruton’s tyrosine kinase inhibitor that acts on B-cell receptor signaling pathway and predisposes to various infections due to its effects on neutrophils, monocytes and T cells. We present a case of cerebral invasive aspergillosis in a patient being treated with ibrutinib for relapsed chronic lymphocytic leukemia. It was hard to associate the condition to ibrutinib versus the chronic lymphocytic leukemia. The patient was successfully treated with a combination of voriconazole and micafungin, resulting in complete recovery and no residual deficits. This highlights the importance of recognizing the rare complication in those on ibrutinib and initiating the treatment immediately with appropriate antifungal agents to improve prognosis of this potentially fatal condition.
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Ghosh, Subrata K., Charles Wood, Lawrence H. Boise, Abdul M. Mian, Vadim V. Deyev, Gerold Feuer, Ngoc L. Toomey, et al. "Potentiation of TRAIL-induced apoptosis in primary effusion lymphoma through azidothymidine-mediated inhibition of NF-κB." Blood 101, no. 6 (March 15, 2003): 2321–27. http://dx.doi.org/10.1182/blood-2002-08-2525.
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The survival of viral mediated lymphomas depends upon constitutive nuclear factor kappa B (NF-κB) activity. AIDS-related human herpesvirus type 8–associated primary effusion lymphoma (PEL) responds poorly to chemotherapy and is almost invariably fatal. We have previously demonstrated that the antiviral combination of interferon alpha (IFN-α) and azidothymidine (AZT) induces apoptosis in PEL cell lines. We therefore used these agents as therapy for an AIDS patient with PEL. The patient had a dramatic response, with complete resolution of his malignant effusion in 5 days. In PEL cells, the death receptor ligand known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is markedly up-regulated by IFN-α; however, signals transduced by death receptors may also activate an antiapoptotic response mediated by NF-κB. In both the primary tumor cells from our patient and PEL cell lines, AZT selectively blocked nuclear entry of the NF-κB heterodimer p50 and p65, an effect not seen with other nonthymidine antiviral nucleosides. AZT monophosphate, the principal intracellular metabolite, inhibited phosphorylation and degradation of IκB by the IκB kinase complex. AZT- and IFN-α-mediated apoptosis was blocked by expression and nuclear localization of an IκB-resistant form of NF-κB (the p50 subunit linked to the transactivation domain of herpes simplex virus VP16). The proapoptotic effect of AZT and IFN-α in PEL occurs through the concomitant activation of TRAIL and blockade of NF-κB and represents a novel antiviral therapy for a virally mediated tumor.
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Du, Wei, Rebecca Goldstein, Yanwen Jiang, Omar Aly, Leandro Cerchietti, Ari M. Melnick, and Olivier Elemento. "A Virtual B Cell Lymphoma Model to Predict Effective Combination Therapy." Blood 124, no. 21 (December 6, 2014): 928. http://dx.doi.org/10.1182/blood.v124.21.928.928.
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Abstract Aberrant activation of the B cell receptor (BCR) signaling network drives survival and proliferation of many B cell malignancies, such as activated B cell like diffuse large B cell lymphoma (ABC-DLBCL). A number of small molecule inhibitors targeting various kinases in the BCR signaling network have been developed. However, clinical application of these targeted agents is facing several challenges such as low response rate and acquired drug resistance. The limited efficacy of single agent targeted therapy is at least partially due to pathway reactivation through crosstalks and compensatory circuits. By simultaneously repressing multiple nodes in the signaling network, combination therapy has the potential to completely extinguish signaling and induce more potent and durable response. The complexity of BCR signaling network makes it difficult to infer which combinations will be effective and synergistic. Given the large number of possible drug combinations, comprehensive experimental screening – including exploration of multiple dosages – is not practically feasible. Computational models of signaling networks that can accurately reconstruct signaling dynamics in silico may represent a useful alternative to experimental screening and trial-and-error experimental investigation. Here, we developed a computational model that integrates signal transduction, tumor growth, and drug kinetics to accurately simulate BCR signaling dynamics and the effect of drug-induced perturbation on signaling output and cell viability. We used this model to predict effective combinatorial therapy in silicoand validated some of these predictions using in vitro experiments. Based on experimentally verified protein-protein interactions, we constructed the first detailed kinetic model of the BCR signaling network covering three major signaling pathways downstream of BCR, namely NFkB, PI3K/AKT and MAPK. The model captured complex crosstalk between these three pathways and multiple feedback loops. Simulated kinase activation time courses under temporal antigen stimulus successfully recapitulated normal BCR signaling dynamics as reported in literature. Using published drug response data in the BCR signaling-dependent ABC-DLBCL cell line TMD8, we trained a tumor growth model which in combination with the kinetic model enabled reliable prediction of viability response of many drug combinations at various dosages. For example, predicted viability response of BTK inhibitor ibrutinib in combination with inhibitors targeting other kinases in the network, e.g. BKM-120 against PI3K, sotrastaurin against PKC-beta closely matched previously published experimental data in TMD8 (r>0.86,p<1e-11). We then sought to identify synergistic drug combinations by simulating viability response at 10x10 virtual dosages for each drug combination and estimating synergism using Bliss independence model. Computational screening predicted dual blockage of LYN and SYK as the most synergistic combination, which we confirmed experimentally by treating TMD8 cells with LYN inhibitor Dasatinib and SYK inhibitor R406 at multiple doses. Finally we sought to use our model to predict biomarkers of sensitivity and resistance to specific treatment strategies. By integrating expression levels of BCR signaling network components assessed by published primary DLBCL RNA-seq data, we simulated patient-specific drug responses and computed the correlation between expression level of specific components with viability response under specific treatments. We found that overexpression of PTP1B, which dephosphorylates BTK substrate PLCg2, predicts relative sensitivity to BTK inhibition. Supporting this prediction, we observed increased PTP1B expression in DLBCL cell lines sensitive to ibrutinib treatment, suggesting PTP1B as potential biomarker for ibrutinib sensitivity. In summary, this study provides a novel approach to computationally optimize combinatorial targeted therapy against aberrant BCR signaling and paves the way for the discovery of effective patient-specific drug combinations. Disclosures Melnick: Bioreference: Scientific Advisory Board, Scientific Advisory Board Other; Calgene: Consultancy; Janssen: Research Funding; Genentech: Speakers Bureau.
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Lannutti, Brian J., Sarah A. Meadows, Adam Kashishian, Bart Steiner, Galina Pogosov, Olga Sala-Torra, Amy J. Johnson, John C. Byrd, Jerald Radich, and Neill A. Giese. "CAL-101, An Oral p110δ Selective Phosphatidylinositol-3-Kinase (PI3K) Inhibitor for the Treatment of B Cell Malignancies Inhibits PI3K Signaling, Cellular Viability and Protective Signals of the Microenvironment." Blood 114, no. 22 (November 20, 2009): 286. http://dx.doi.org/10.1182/blood.v114.22.286.286.
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Abstract Abstract 286 It is well established that deregulation of the PI3K signaling pathway plays an important role in the etiology of human malignancies including those of hematologic origin. In 30–50% of solid tumors, oncogenic activation of the PI3K pathway is the result of gain-of-function mutations in the PI3K p110α isoform or due to the loss-of-function of the PTEN phosphatase that is responsible for PI3K downregulation. In B cell malignancies these mutations are rarely observed in spite of the fact that PI3K pathway activation is commonly observed and often essential for tumor cell growth and survival. In this case, PI3K pathway activation has been shown to result from constitutive B cell receptor (BCR) activation and/or from exposure to survival factors present in the microenvironment. The activation of the PI3K pathway by cell surface receptors is directly mediated by the Class I isoforms (α, β, δ, and γ), however, their relative contribution in B cell tumors is unknown. Interestingly, genetic and pharmacological approaches that specifically inactivate the p110δ isoform have demonstrated its important role in normal B cell signaling in response to multiple factors including antigen, CD40L, BAFF, SDF-1 and CXCR13 making it an attractive target for therapeutic intervention in B cell malignancies. CAL-101 is an oral p110δ specific inhibitor which is currently being evaluated in a phase I clinical trial for the treatment of patients with select hematologic malignancies. This compound is a novel potent p110δ inhibitor with an IC50 of 2.5 nM against purified p110δ and EC50 of 65 nM in p110δ-mediated basophil activation in whole blood. CAL-101 demonstrates 300-, 200-, and 40-fold selectivity over the other class I family members (α, β, and, γ respectively) and no activity against class II and III PI3K family members or other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a kinome-wide screen failed to detect activity against any additional kinases. To investigate the potential role of p110δ in B cell hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for constitutive PI3K pathway activation. The expression of p110δ was observed in >90% of these cell lines and in many cases was accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-S6, and GSK-3β in cells that represent a range of tumor types including follicular lymphoma, acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, and mantle cell lymphoma (MCL). Furthermore, treatment with CAL-101 resulted in growth suppression and induction of apoptosis which was accompanied by PARP and caspase-3 cleavage. Growing evidence suggests that signals from the microenviroment are essential for the expansion, homing, and survival of malignant B cells, in addition to promoting resistance to conventional drug therapy. To investigate the potential role p110δ plays during B cell signaling via interactions with the microenvironment, we examined invoked stimulation of leukemia and lymphoma cell lines with CXCL12, CXCL13, BAFF, or BCR crosslinking in the presence or absence of CAL-101. Stimulation with either chemokines or growth factors resulted in the phosphorylation of Akt which was inhibited by CAL-101 in a dose dependent manner. Moreover, p110δ inhibition with CAL-101 inhibits the chemotaxis of ALL and MCL cell lines to CXCL12. These studies have now been extended to the analysis of primary patient B-ALL and MCL cells to further establish preclinical proof of concept for therapeutic application of CAL-101. In summary, CAL-101 is a potent and selective p110δ kinase inhibitor with broad anti-tumor activity against cancer cells of hematologic origin. Our results demonstrate that selective inhibition of p110δ with CAL-101 inhibits malignant B cell growth, survival, and migration. Furthermore, p110δ inhibition may enhance the effect of cytotoxic drugs or overcome cell mediated drug resistance by inhibiting the protective signals of the microenviroment, providing a rational for combination therapy. These data suggest that p110δ may play an important role in regulating signals between malignant B cells and their microenvironment thereby providing the preclinical rationale for clinical evaluation of CAL-101 alone or in combination with cytotoxics or biologics in patients with hematologic malignancies. Disclosures: Lannutti: Calistoga Pharmaceuticals: Employment. Meadows:Calistoga Pharmaceuticals: Employment. Kashishian:Calistoga Pharmaceuticals: Employment. Steiner:Calistoga Pharmaceuticals: Employment. Johnson:Calistoga Pharmaceuticals: Research Funding. Giese:Calistoga Pharmaceuticals: Employment.
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Miles, Rodney R., Zhaosheng Lin, Megan S. Lim, and Kojo S. J. Elenitoba-Johnson. "Deregulation of the Insulin-Like Growth Factor Type 1 Receptor (IGF-1R) in Transformed Follicular Lymphomas: Implications for Novel Therapy." Blood 108, no. 11 (November 1, 2006): 2407. http://dx.doi.org/10.1182/blood.v108.11.2407.2407.
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Abstract Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin lymphoma in the western hemisphere. The vast majority of cases are incurable and transformation to diffuse large B-cell lymphoma (DLBCL) is an important cause of death. The molecular and biologic mechanisms underlying FL transformation are largely uncharacterized. In this study, we utilized a global quantitative proteomics approach for the identification of differentially expressed proteins associated with follicular lymphoma transformation. Five matched pairs of clonally identical cases of follicular lymphoma and their transformed counterparts (DLBCL) arising in the same individual were utilized. Quantitative analysis of differentially expressed proteins was performed by isotope-coded affinity tagging (ICAT™) followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Equivalent quantities of total cell lysates obtained from the FLs and the DLBCLs were ICAT™ labeled, and subjected to avidin affinity chromatography. Offline fractions were collected, digested with trypsin, and analyzed by automated reverse phase nanospray LC-MS/MS. Our proteomic studies revealed upregulation of the IGF-1R (3–5 fold) in the transformed lymphomas. Western blot analysis using an antibody to the a-subunit of IGF-1R revealed overexpression in the transformed lymphoma samples as compared to their preceding FL counterparts (discovery set). Similarly, IGF-1R upregulation was demonstrated in an additional independent set of 6/7 DLBCL samples as compared to their preceding FL counterparts. Immunohistochemical studies were performed on formalin-fixed paraffin-embedded tissue sections of 15 matched pairs of FL and their transformed DLBCL counterparts. The neoplastic cells of FL demonstrated negligible levels of IGF-1R whereas in 5/15 cases, the neoplastic cells of DLBCL demonstrated strong cytoplasmic and membranous expression of IGF-1R. We carried out studies to determine the functional role of IGF-1R in the survival of lymphoma cells in vitro. Blocking antibodies to IGF-1R caused a significant reduction of cell viability in all three transformed FL cell lines (SUDHL-4, OCI-LY1, Karpas 4224) as determined by MTT assays. In contrast, antibodies against EGFR, EphA and Frizzled 8 protein did not affect the cell viability of any of the transformed FL cell lines, indicating specificity. Furthermore, knockdown of IGF-1R expression in SUDHL-4 cells by RNA interference resulted in significant reduction in cell viability whereas the control “scramble” siRNA or EGFR siRNA did not have an effect. Cell cycle analysis of the IGF-1R siRNA transfected cells indicated an increase in cells undergoing apoptosis relative to control cells. We utilized a synthetic tyrphostin compound (AG1024) which selectively inhibits the IGF-1R tyrosine kinase activity to determine the effects of pharmacologic inhibition of IGF-1R on the viability of transformed FL cells. Inhibition of IGF-1R resulted in inhibition of cell viability with IC50 of 22mM. This study, for the first time, reveals the role of deregulated expression of IGF-1R in transformed FL and provides a rational basis for the use of IGF-1R blocking agents in the therapy of these neoplasms.
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Sakemura, Reona, Nan Yang, Michelle J. Cox, Sutapa Sinha, Mehrdad Hefazi, Michael J. Hansen, Kendall J. Schick, et al. "Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies." Blood 132, Supplement 1 (November 29, 2018): 728. http://dx.doi.org/10.1182/blood-2018-99-112209.
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Abstract Despite the remarkable outcomes and recent FDA approval of CD19 directed chimeric antigen receptor T (CART19) cell therapy in B cell malignancies, the durable responses in diffuse large B cell lymphoma are less than 40% and CART activity in chronic lymphocytic leukemia (CLL) is further limited. This is thought to be related to loss of CART persistence, poor trafficking to lymph nodes and inhibition by the leukemic microenvironment. Therefore, strategies to enhance CART cell function to overcome these limitations are needed. Recent studies have shown that abnormal expression of the receptor tyrosine kinase (RTK) AXL is associated with poor prognosis in human cancers. AXL signaling is associated with tumor proliferation, survival, metastasis, and drug resistance. Inhibition of AXL RTK with TP-0903, a high affinity AXL inhibitor has been found to induce robust apoptosis of CLL B cells. Based on the significant modulation of T cell functions observed with BTK inhibitor, we examined the role of AXL RTK inhibition with TP-0903 on T cell function in CLL and other B cell malignancies. First, we investigated the effect of AXL inhibition on T cell phenotype in normal donors. When naïve T cells were stimulated with PMA/Ionomycin and cultured with low dose TP0903, cytokine production was favorably altered through the promotion of Th1 and reduction of Th2 cytokines. This was associated with a significant reduction of inhibitory receptors (Fig 1a). Western blot of T cell lysates suggests low dose TP-0903 results in inhibition of LCK. When effector T cells and regulatory T cells (Treg) were treated with TP-0903 for 3 days, there was a preferential reduction of Treg (Fig 1b). Next, we investigated the influence of TP-0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. Similar to our findings on naïve T cells, TP-0903 treatment led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP-0903 treatment also significantly downregulated inhibitory receptors on activated CART cells, including a reduction of canonical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1c). The combination of CART19 cells and TP-0903 yielded a synergistic antitumor activity against JeKo in vitro, at low E:T ratios (Fig 1d). Western blot of T cell lysates revealed phosphorylation of LCK was remarkably reduced in the presence of TP-0903, suggesting a mechanism for the observed Th1 polarization. We compared the transcriptome of activated CART cells treated with TP-0903 and more than 100 genes were differentially expressed compared to non-treated cells. Among these genes, immune synapse related genes such as cell junction and cell migration related genes were significantly increased in activated CART cells treated with TP-0903. To investigate the effect of AXL RTK inhibition of CART cells with TP-0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of JeKo into NSG mice. A week after the injection of JeKo, mice were treated with either vehicle alone, TP-0903 (20mg/kg/day) alone, 0.5x106 of CART19 alone, or TP-0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of JeKo. Mice treated with CART19 and TP-0903 rejected the JeKo tumor challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1e). Finally, we validated our preclinical findings in a correlative analyses of Phase I clinical trial of TP-0903 for patients with solid tumors (NCT02729298). Blood T cells from 3 patients were isolated and analyzed before and a week after treatment with TP-0903. Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned Phase I clinical trial of TP-0903 in relapsed/refractory CLL (NCT03572634). In summary, we demonstrated for the first time that AXL inhibitior is capable of polarizing T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes with CART cells in B cell malignancies. These findings encourage further study of TP-0903 as an enhancer of T cell immunotherapies. Disclosures Mouritsen: Tolero Pharmaceuticals: Employment. Foulks:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria. Ding:Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.
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Rudd, Christopher E., David Rothstein, and Alison Taylor. "Small molecule drug inhibition of PD-1 transcription is as effective as anti-PD-1 biologic blockade in cancer therapy." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 56.11. http://dx.doi.org/10.4049/jimmunol.198.supp.56.11.
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Abstract Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway(s) that regulate PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoform α and β) is a serine-threonine kinase implicated in cellular processes. We have shown that GSK-3 is a key upstream kinase that regulates PD-1 expression in CD8+ T cells. GSK-3 inactivation increased Tbx21 transcription for enhanced Tbet expression, and its suppression of Pdcd1 transcription in CD8+ cytolytic T-cells (CTLs) (Taylor et al 2016 Immunity 44, 274–86). Here, we show that GSK-3 inhibitor blockade of pcdc1 (PD-1) transcription with a small molecule inhibitor (i.e. SB415286) is as effective as anti-PD-1 and PDL-1 blocking antibodies in the control of pulmonary metastasis of B16 melanoma, intra-dermally injected B16 and EL4 lymphoma solid tumors, and in the ex vivo pre-treatment of T-cells before adoptive transfer into mice carrying tumors. In an analysis of knock out mice, GSK-3α/β−/− mice, which had greatly reduced PD-1 expression on T-cells, showed the same reduction in B16 pulmonary metastasis as Pdcd1−/− mice. Further, the ex vivo treatment of T-cells with SB415286, anti-PD-1, or in combination followed by adoptive transfer, had identical effects in inhibiting EL4 lymphoma growth. In all tumor models, GSK-3 inactivation markedly inhibited Pdcd1 transcription and PD-1 expression on tumor infiltrating T-cells (TILs), while increasing Tbx21 (Tbet) transcription and the presence of CD8+ TILs expressing CD107a+ (LAMP1) and granzyme B (GZMB). Our findings define for the first time that a next generation approach using small molecule inhibition of PD-1 expression is as effective as anti-PD-1/PL1 biologics in cancer therapy.
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Xu, Xin. "BTK Inhibitors Induce ABC-DLBCL Cell Apoptosis By Inhibiting CYLD Phosphorylation." Blood 134, Supplement_1 (November 13, 2019): 5046. http://dx.doi.org/10.1182/blood-2019-126763.
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BTK inhibitors induce ABC-DLBCL cell apoptosis by inhibiting CYLD phosphorylation Xin Xu1, Zhigang Zhu1, Feng Liu1 , Weijie Zhong1,Huabao Xiong2, Tiefeng Feng3, and Qingshan Li3 1Geriatrics Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. 2Precision Immunology Institute, Mount Sinai School of Medicine, New York, NY 10029, U.S.A. 3Hemotology Department, GuangZhou First People's Hospital, GuangDong, 510180, People's Republic of China. Activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) is a common aggressive diffuse large B cell lymphoma (DLBCL). Even though CD20 antibody (Rituximab) combination therapy improved the total prognosis of DLBCL, there are still many relapsed and refractory ABC-DLBCL cases. The new treatment options are urgently needed. Pathogenesis of ABC-DLBCL is different from germinal center B cell-like diffuse large B cell lymphoma (GCB-DLBCL), which is related to activation of B cell antigen receptor(BCR) signaling. The Bruton tyrosine kinase(BTK) plays a central role in BCR signaling. Inhibition of BTK function will affect BCR signaling activity, which is involved in maintaining the malignant phenotype in B-cell lymphoma. Now in clinic, BTK inhibitors like Ibrutinib or Acalabrutinib are used extensively in chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma(MCL) as first line therapy or second line therapy. But in DLBCL its role is unclear now.Some DLBCL clinic trials got the better PFS and OS in BTK inhibitor combination therapy group(combined with Rituximab or RCHOP) but some got the negative results. Whether BTK inhibitors can definitely improve ABC-DLBCL survival and the mechanism how BTK inhibitors inducing cell death are needed further investigations. CYLD (cylindromatosis) is a tumor suppressor, its activity is inhibited by phosphorylation, which is related to apoptosis regulation pathway NF-kB in many kinds of tumors.In this study, we found that CYLD to be constitutively phosphorylated in ABC-DLBCL cell line models (HBL-1 and OCI-Ly10) as well as primary samples from patients with non-GCB DLBCL. We subsequently examined the effect of BTK inhibitors Ibrutinib (PCI-32765) and Acalabrutinib (ACP-196) on CYLD phosphorylation. We found that BTK inhibitors Ibrutinib and Acalabrutinib both induced ABC-DLBCL apoptosis by down-regulating CYLD phosphorylation in vivo and in vitro, even in the Rituximab resistant ABC-DLBCL cell line.Combination of BTK inhibitors and Rituximab would enhance this effect. Knockdown of CYLD in ABC-DLBCL cells reduced the level of cell death induced by BTK inhibitors, which showed that this cell death induced by BTK inhibitors was CYLD dependent. Through this study, we conclude that BTK inhibitors induce CYLD inactivation by regulating CYLD phosphorylation is clinical beneficial in ABC-DLBCL therapy, especially the relapsed or refractory ABC-DLBCL cases after Rituximab therapy. Disclosures No relevant conflicts of interest to declare.
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Carlo-Stella, Carmelo, Cristiana Lavazza, Arianna Giacomini, Loredana Cleris, Daniela Sia, Paolo Longoni, Marco Milanesi, et al. "Preclinical Rationale for the Use of the Multikinase Inhibitor Sorafenib in the Treatment of Human Lymphomas." Blood 112, no. 11 (November 16, 2008): 2605. http://dx.doi.org/10.1182/blood.v112.11.2605.2605.
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Abstract Introduction: The multikinase inhibitor Sorafenib (Nexavar, Bayer) exerts a remarkable activity against a variety of nonhematological tumors by blocking tumor cell proliferation and angiogenesis through the inhibition of the RAF/MEK/ERK pathway, as well as the receptor tyrosine kinases vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptor (PDGFR), c-KIT, Flt3, and RET. Several lines of evidence suggest that sorafenib might have a relevant clinical impact in the therapy of malignant lymphomas by overcoming the cytoprotective effects of ERK, Mcl-1, and Bcl-XL. However, preclinical data establishing a rationale for the clinical use of sorafenib in lymphomas are still lacking. The present studies aimed to investigate the activity and the mechanism(s) of action of sorafenib in human lymphomas. Methods: The effects of sorafenib were evaluated in vitro using a panel of six human cell lines of different phenotypes, including JVM-2 (B-Chronic Lymphocytic Leukemia), Granta-519 (Mantle Cell Lymphoma), DOHH2 (Follicular Lymphoma), SU-DHL-4V (Diffuse Large B-Cell Lymphoma), HD-MY-Z (Hodgkin Lymphoma), and KMS-11 (Multiple Myeloma) cell lines. Additionally, the antitumor efficacy and mechanism of action of sorafenib were investigated in vivo by means of five lymphoma xenograft models in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results: In vitro, the response of cell lines to sorafenib (1–10 μM for 24–48 hours) was evaluated by detecting apoptotic cell death with the annexin-V/propidium iodide double staining assay, and viable cell countings with the Trypan blue dye exclusion test. All 6 cell lines responded to sorafenib with values of 50% inhibitory concentrations ranging from 1 to 7.5 μM. In contrast, normal CD34+ cells remain insensitive to the drug up to 15 μM. Despite significant rates of sorafenib-induced apoptosis were seen in all cell lines, activation of caspase-3 analyzed by fluorescent-activated cell sorter was only detected in DOHH-2 and JVM-2 cell lines. The phosphorylation status of mitogen-activated protein kinase (MAPK) was investigated using the human phospho-MAPK Array kit (R&D systems), analyzed with the open source imaging software ImageJ (http://rsb.info.nih.gov/ij/), and then validated by Western blotting. Sorafenib induced a significant reduction of pAkt1, pAkt2, and pAkt3 in SU-DHL-4V, Granta-519, and JVM-2 cell lines, whereas p38 phosphorylation levels were significantly reduced in all but one cell line (KMS-11). Reduced levels of pMEK, pERK1 and pERK2 were detected in SU-DHL-4V, KMS-11, Granta-519, and HD-MY-Z cell lines. Down-regulation of MCL-1 was seen in HD-MY-Z, JVM-2, and DOHH-2 cell lines. In vivo, the activity of sorafenib was evaluated in NOD/SCID mice bearing subcutaneous tumor nodules. Animals with tumors averaging from 140 to 160 mg were randomly grouped to receive sorafenib (90 mg/kg body weight, IP, once daily for 15 days) or control vehicle. Sorafenib significantly (P ≤0.001) reduced the growth of subcutaneous HD-MY-Z, KMS-11, Granta-519, SU-DHL-4V, and JVM-2 nodules, with values of tumor growth inhibition of 70%, 52%, 40%, 37%, and 24%, respectively. In control mice, TUNEL staining of tumor sections showed large areas of viable cells without significant necrosis, whereas a 2- to 5-fold increase of necrotic areas was detected in sorafenib-treated mice bearing the different lymphoma xenografts. Analysis of tumor vasculature by means of in vivo biotinylation of endothelial cells with sulfo-NHS-LC-biotin showed a 30% to 60% reduction of vessel density in sorafenib-treated mice bearing the different lymphoma xenografts. Conclusions: Sorafenib efficiently targets a variety of human lymphomas representative of different phenotypes by inhibiting tumor angiogenesis and directly affecting tumor cell survival. Our preclinical data establish a rationale for exploring the clinical activity of sorafenib in human lymphomas.
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Saba, Nakhle S., Hana F. Safah, and Laura S. Levy. "Effect of Protein Kinase C β Specific Inhibition On Acute Lymphoblastic Leukemia Cell Lines." Blood 114, no. 22 (November 20, 2009): 4817. http://dx.doi.org/10.1182/blood.v114.22.4817.4817.
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Abstract Abstract 4817 Acute lymphoblastic leukemia (ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction–consolidation–maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is basically due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, PKCβ targeting arose as a new, promising, and well tolerated treatment strategy in a variety of neoplasms, especially in B-cell malignancies. It showed encouraging results in preclinical and clinical studies involving chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma. PKCβ plays a major role in B-cell receptor signaling, but studies describing the role of PKCβ in B-cell ALL are lacking. In the present study, we measured the sensitivity of a variety of B-cell ALL cell lines to PKCβ specific inhibition. Three cell lines were studied: RS4;11 (characterized by the t(4;11) chromosomal abnormality), TOM-1 (characterized by the t(9;22) chromosomal abnormality), and REH (characterized by the t(12;21) chromosomal abnormality). Cells were tested for PKCβ1 and PKCβ2 expression by immunoblot. Cell viability was measured when PKCβ-specific inhibitor at concentrations of 1, 2.5, 5, 10, 20 and 30 μM was added for 48 hours in the presence of 10% fetal bovine serum (FBS). MTS assay was performed to quantify cell viability. Results showed that all three cell lines express PKCβ1 and PKCβ2. Treatment with PKCβ-specific inhibitor resulted in a dose-dependent inhibition of cell proliferation; Sensitivity was evident at 1 μM for RS4;11 cell line, and at 2.5 μM for TOM-1 and REH cell lines, with 10% cell growth inhibition; Growth inhibition increased to 90% for all cell lines at an inhibitor concentration of 30 μM. These results indicate that PKCβ plays an important role in the malignant process in B-cell ALL, and suggest that PKCβ targeting should be considered as a potential treatment, whether in combination with the current regimens used or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCβ and adverse cytogenetics like t(4;11) and t(9;22). Disclosures: No relevant conflicts of interest to declare.
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Denker, Sophy, Aitomi Bittner, Il-Kang Na, Julia Kase, Mareike Frick, Ioannis Anagnostopoulos, Michael Hummel, and Clemens A. Schmitt. "A Phase I/II first-line study of R-CHOP plus B-cell receptor/NF-κB-double-targeting to molecularly assess therapy response." International Journal of Hematologic Oncology 8, no. 4 (December 1, 2019): IJH20. http://dx.doi.org/10.2217/ijh-2019-0010.
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The ImbruVeRCHOP trial is an investigator-initiated, multicenter, single-arm, open label Phase I/II study for patients 61–80 years of age with newly diagnosed CD20+ diffuse large B-cell lymphoma and a higher risk profile (International Prognostic Index ≥2). Patients receive standard chemotherapy (CHOP) plus immunotherapy (Rituximab), a biological agent (the proteasome inhibitor Bortezomib) and a signaling inhibitor (the Bruton's Tyrosine Kinase-targeting therapeutic Ibrutinib). Using an all-comers approach, but subjecting patients to another lymphoma biopsy acutely under first-cycle immune-chemo drug exposure, ImbruVeRCHOP seeks to identify an unbiased molecular responder signature that marks diffuse large B-cell lymphoma patients at risk and likely to benefit from this regimen as a double, proximal and distal B-cell receptor/NF-κB-co-targeting extension of the current R-CHOP standard of care. EudraCT-Number: 2015-003429-32; ClinicalTrials.gov identifier: NCT03129828.
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Dreyling, Martin, Armando Santoro, Luigina Mollica, Sirpa Leppä, George A. Follows, Georg Lenz, Won Seog Kim, et al. "Phosphatidylinositol 3-Kinase Inhibition by Copanlisib in Relapsed or Refractory Indolent Lymphoma." Journal of Clinical Oncology 35, no. 35 (December 10, 2017): 3898–905. http://dx.doi.org/10.1200/jco.2017.75.4648.
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Purpose Phosphatidylinositol 3-kinase (PI3K) signaling is critical for the proliferation and survival of malignant B cells. Copanlisib, a pan-class I PI3K inhibitor with predominant activity against PI3K-α and -δ isoforms, has demonstrated efficacy and a manageable safety profile in patients with indolent lymphoma. Patients and Methods In this phase II study, 142 patients with relapsed or refractory indolent lymphoma after two or more lines of therapy were enrolled to receive copanlisib 60 mg intravenously on days 1, 8, and 15 of a 28-day cycle. The primary end point was objective response rate; secondary end points included duration of response, progression-free survival, and overall survival. In addition, safety and gene expression were evaluated. Results Median age was 63 years (range, 25 to 82 years), and patients had received a median of three (range, two to nine) prior regimens. The objective response rate was 59% (84 of 142 patients); 12% of patients achieved a complete response. Median time to response was 53 days. Median duration of response was 22.6 months, median progression-free survival was 11.2 months, and median overall survival had not yet been reached. The most frequent treatment-emergent adverse events were transient hyperglycemia (all grades, 50%; grade 3 or 4, 41%) and transient hypertension (all grades, 30%; grade 3, 24%). Other grade ≥3 events included decreased neutrophil count (24%) and lung infection (15%). High response rates to copanlisib were associated with high expression of PI3K/B-cell receptor signaling pathway genes. Conclusion PI3K-α and -δ inhibition by copanlisib demonstrated significant efficacy and a manageable safety profile in heavily pretreated patients with relapsed or refractory indolent lymphoma.
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Hu, Jia, Jianhan Wang, Xiaoming Dai, Jianlin He, Junqing Liang, Ying Yu, Juntao Yu, et al. "Abstract 5454: Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma models." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5454. http://dx.doi.org/10.1158/1538-7445.am2022-5454.
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Abstract Background: The delta isoform of PI3K (PI3Kδ) plays an important role in B-cell development and function by mediating the signaling of key receptors on B cells. Aberrant activation of PI3Kδ signaling pathway promotes the survival and proliferation of malignant B cells, making selective inhibition of this isoform a promising therapeutic approach for the treatment of B cell malignances. Amdizalisib (HMPL-689, Amdiz), discovered and being currently developed in a pivotal phase II clinical trial (NCT04849351) by HUTCHMED, is a highly potent and selective PI3Kδ inhibitor. Based on encouraging clinical data from the phase Ib study (2021 ESMO, abstract# 8330), Amdiz is a designated breakthrough therapy in China for treatment of relapsed and refractory follicular lymphoma. Herein, we report the pre-clinical anti-tumor activity of Amdiz. Methods: Kinase activity was measured by Transcreener™ Fluorescence Polarization assay. The kinome selectivity profile of Amdiz was evaluated in Eurofins KinaseProfiler™ panel containing 323 kinases. Cell based phosphorylation of AKT was determined by using Acumen Explorer system. CD63 expression in basophils was evaluated by FACS assay. Inhibitory effects of Amdiz alone or in combination with other agents on cell viability were investigated in a panel of B cell lymphoma cell lines by CellTiter-Glo luminescent or CCK-8 assay. The ability of Amdiz to inhibit the activation of B cells in animals was evaluated in naïve rats using an ex vivo assay, and B cell activation was determined by evaluating CD86 expression using FACS analysis. Human B cell lymphoma cell line derived xenograft models were used to determine the anti-tumor activity of Amdiz in combination with other agents such as rituximab, BTK inhibitor, and venetoclax. Results: Amdiz is a highly selective inhibitor of PI3Kδ, showing more than 250-fold selectivity over other PI3K isoforms and no significant inhibition over other 319 protein kinases at the concentration of 1 µM. Amdiz potently inhibited PI3Kδ in biochemical, cellular and human whole blood assays with IC50s ranging from 0.8-3 nM. Its inhibitory effects on cell viability were also evaluated in a panel of B -cell lymphoma cell lines. Results showed that Amdiz potently inhibited cell survival with IC50s from 0.005 to 5 μM. Amdiz showed a long-lasting and strong inhibition on B cell activation in a rat pharmacodynamics (PD) study at a dose as low as 0.1 mg/kg. Moreover, Amdiz significantly improved anti-tumor activity of standard-of-care agents as well as targeted agents in multiple B-cell lymphoma models both in vitro and in vivo. Conclusion: Amdiz is a highly potent and selective PI3Kδ inhibitor with strong activity against B-cell lymphoma in pre-clinical studies, supporting clinical evaluation as either a single agent or in combination with other therapeutic agents for the treatment of B-cell malignancies. Citation Format: Jia Hu, Jianhan Wang, Xiaoming Dai, Jianlin He, Junqing Liang, Ying Yu, Juntao Yu, Na Yang, Linfang Wang, Yu Cai, Xiong Li, Weiguo Qing, Yongxin Ren, Weiguo Su. Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma models [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 5454.
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Chen, Jing, Yong Zhang, Michael N. Petrus, Wenming Xiao, Alina Nicolae, Mark Raffeld, Stefania Pittaluga, et al. "Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations." Proceedings of the National Academy of Sciences 114, no. 15 (March 29, 2017): 3975–80. http://dx.doi.org/10.1073/pnas.1700682114.
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Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)− anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK− ALCL, we treated ALK− cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK− ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK− ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK− ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK− ALCL who are phosphorylated STAT3+.
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Kampa, Kerstin M., Tanja Grandl, Sandra Mueller, Emmanuel Normant, Michael Walter, Wolfgang Schuetz, Lothar Kanz, Michael C. Heinrich, and Marcus M. Schittenhelm. "KIT Inhibition Affects Heat Shock Protein (HSP) Activity: A Potential Rescue Mechanism towards KIT Tyrosine Kinase Inhibition and a Rationale for Dual KIT/HSP Inhibition." Blood 114, no. 22 (November 20, 2009): 2753. http://dx.doi.org/10.1182/blood.v114.22.2753.2753.
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Abstract Abstract 2753 Poster Board II-729 Activating mutations of the KIT class III receptor tyrosine kinases are associated with core binding factor leukemias (CBF AML), systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanomas, seminoma/dysgerminoma and sinonasal natural killer/T-cell non-Hodgkin lymphoma. Despite the encouraging therapeutic potential of KIT-tyrosine kinase inhibitors (TKI), resistance leading to disease progression occurs in many patients, specifically after TKI monotherapy. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways which override TKI inhibition. To explore the downstream signaling pathways of class III receptor tyrosine kinases, we performed unbiased phoshoproteomic analyses of mutant FLT3 or KIT leukemia and mastocytosis cell lines before and after TKI treatment. Tantalizingly, immunoaffinity purification of phosphopeptides followed by tandem mass spectrometry following KIT-inhibition with Imatinib at IC90 (100nM) revealed a significant upregulation of phosphorylation levels of peptides identified as members of the heat shock protein (HSP) family. Of interest, mRNA GeneChip® Array analysis of hematopoietic Ba/F3 cells transfected with either a mutant KIT isoform (D816V) or a mutant FLT3 isoform (ITD) and treated with TKI revealed significant downregulation of HSP family members in the FLT3 model – but stable mRNA levels in the KIT model. Taken together, our phosphoproteome and mRNA data suggest a protective function of HSP in mutant-KIT tumors treated with TKI. Next we studied the antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504, a 17-AGG-derivative, in mutant-KIT cell models. IPI-504 potently inhibited proliferation and induced apoptosis with an IC50 of 0.5 up to 5μM depending on the KIT isoform. Importantly, combination of IPI-504 with TKIs resulted in potentiation of the antiproliferative and proapoptotic effects achieved by either drug alone. Antitumor efficacy in combination therapy was observed even at HSP90 inhibitor concentrations that did not display antitumor activity if administered alone. In conclusion, our model suggests that inhibition of KIT affects heat shock protein activity serving to stabilize the functionality of targeted autoactivated receptor tyrosine kinases, which provides a potential mechanism for resistance to TKI therapy. Importantly, we provide a rationale to combine TKI with (low-dose) HSP-inhibitors such as IPI-504 to optimize TKI therapy. Disclosures: Normant: Infinity: Employment.
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Kaptein, Allard, Gerjan de Bruin, Maaike Emmelot-van Hoek, Bas van de Kar, Anouk de Jong, Michael Gulrajani, Dennis Demont, Todd Covey, Diana Mittag, and Tjeerd Barf. "Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies." Blood 132, Supplement 1 (November 29, 2018): 1871. http://dx.doi.org/10.1182/blood-2018-99-109973.
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Abstract Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity. Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z'-LYTE) with IC50 determination at a fixed time point. On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB). Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells. Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells. Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were <10 nM for ibrutinib, acalabrutinib and zanubrutinib. Spebrutinib had an EC50 of 140 nM in hWB, showing the greatest loss of potency versus biochemical assays and hPBMCs, going to the physiologically most relevant cellular experimental conditions (Table 2). Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates. Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested. Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively. No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values <1 µM in Jurkat T cells. Similar results were observed in primary human peripheral T cells. Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. Disclosures Kaptein: Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Meadows, Sarah, Anella Yahiaoui, Rick Sorensen, Zhi-Hua Cui, Robert Brockett, Kathleen S. Keegan, and Stacey Tannheimer. "Evaluation of Idelalisib with B-Cell Receptor or Orthogonal Pathway Inhibitors in Diffuse Large B-Cell Lymphoma Cell Lines in Vitro and In Vivo." Blood 128, no. 22 (December 2, 2016): 1845. http://dx.doi.org/10.1182/blood.v128.22.1845.1845.
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Abstract Background: Idelalisib, a selective oral inhibitor of PI3Kd, is approved for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with follicular lymphoma who have received at least 2 prior therapies. Despite remarkable clinical efficacy, complete responses are rare, highlighting the need to identify more effective therapies, including combinations of novel agents. GS-4059 (ONO-4059) is an investigational next generation Bruton's tyrosine kinase (BTK) inhibitor with improved selectivity compared with ibrutinib. We report here the results of the combination of a PI3Kd inhibitor and GS-4059 in a diffuse large B-cell lymphoma (DLBCL) xenograft model, demonstrating supportive data for our ongoing combination trial in B-cell malignancies (NCT02457598). Additionally, we investigated preclinical orthogonal combination approaches for DLBCL. Methods: Growth inhibition was assessed using CellTiter-Glo Assay after 96 h incubation with idelalisib and GS-4059. CB17-SCID mice were irradiated, implanted subcutaneously with TMD8, and treated BID PO with the PI3Kd inhibitor GS-649443, GS-4059, or coformulated combination when tumors reached 200 mm3. Lysates from tumors or cell cultures were analyzed by Simple Western (Protein Simple). Synergy for antiproliferative effects was assessed using Chalice software (Horizon Discovery, Inc., Lehar et al., Nature Biotech, 2009). Results: Idelalisib and GS-4059 potently inhibited the ABC subtype DLBCL cell line TMD8, which is a B-cell receptor (BCR)-dependent line that exhibits chronic activated B-cell signaling due to mutations in CD79A/CD79B and MYD88 (Kim Y. et al., Hum Pathol, 2014). When a clinically relevant single concentration of idelalisib or GS-4059 was added in combination to a dose responsive effect of the other, a shift in EC50 on cell viability was seen. GS-4059 (50 nM) shifted the EC50 of idelalisib from 141 nM to 5 nM, a 28-fold shift. Idelalisib (1 µM) shifted the EC50 of GS-4059 from 27 nM to 2 nM, a 14-fold shift. Evaluation of downstream signaling pathways implicated in malignant B-cell survival and proliferation showed enhanced inhibition of pAkt S437, pBTK Y223, pErk1/2 T202/Y204, and MYC with a combination of idelalisib and GS-4059, more than either single agent alone. When TMD8 xenografts were treated with a PI3Kd tool compound, GS-649443, GS-4059 or a combination of the 2 inhibitors, a statistically significant decrease in tumor volume was seen as well as tumor regression, when compared with single agent effects (Figure 1A). Evaluation of TMD8 tumor lysates showed strong suppression of pAkt S437, pBTK Y223, pS6RP S235/236, and MYC in tumors treated with both GS-649443 and GS-4059 (Figure 1B). pS6RP S235/236 and MYC, in formalin-fixed paraffin-embedded (FFPE) TMD8 tumors, were profoundly inhibited in tumors treated with combination therapy compared to the monotherapies (Figure 1C). Since the combination of a PI3Kd inhibitor and GS-4059 led to TMD8 tumor regression, an effect correlated to strong down-modulation of MYC, the combination of idelalisib with a bromodomain and extra-terminal (BET) family inhibitor was explored as a potential new orthogonal combination approach for DLBCL. A panel of DLBCL cell lines was evaluated for inhibition of cell viability by idelalisib in combination with GS-5829, a BET inhibitor currently being evaluated in a phase 1 clinical trial. At clinically relevant concentrations, the combination of idelalisib and GS-5829 showed synergistic effects on cell viability in 2 of 6 ABC subtype, 4 of 5 GCB subtype, and 2 of 2 double-hit DLBCL cell. As compared with combination with other agents that inhibit the BCR pathway (GS-4059) or the Bcl-2 pathway (ABT-199), the broadest activity across cell lines was seen with the combination of idelalisib and GS-5829. Conclusion: Idelalisib and GS-4059 demonstrated synergistic inhibition of the TMD8 xenograft with concomitant inhibition of MYC. Screening of other targeted agent combinations in a panel of DLBCL lines revealed broad preclinical activity for the BET inhibitor GS-5829 in combination with idelalisib. This represents a potential orthogonal approach for a new therapeutic strategy for the treatment of B-cell malignancies. Figure 1A Figure 1A. Figure 1B Figure 1B. Figure 1C Figure 1C. Disclosures Meadows: Gilead Sciences: Employment. Yahiaoui:Gilead Sciences: Employment. Sorensen:Gilead Sciences: Employment. Cui:Gilead Sciences: Employment. Brockett:Gilead Sciences: Employment. Keegan:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment.
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Van den Eynden, Jimmy, Ganesh Umapathy, Arghavan Ashouri, Diana Cervantes-Madrid, Joanna Szydzik, Kristina Ruuth, Jan Koster, et al. "Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways." Science Signaling 11, no. 557 (November 20, 2018): eaar5680. http://dx.doi.org/10.1126/scisignal.aar5680.
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Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements inALKtrigger the activation of the encoded receptor and its downstream signaling pathways.ALKmutations have been identified in both familial and sporadic neuroblastoma cases as well as in 30 to 40% of relapses, which makes ALK a bona fide target in neuroblastoma therapy. Tyrosine kinase inhibitors (TKIs) that target ALK are currently in clinical use for the treatment of patients with ALK-positive non–small cell lung cancer. However, monotherapy with the ALK inhibitor crizotinib has been less encouraging in neuroblastoma patients withALKalterations, raising the question of whether combinatorial therapy would be more effective. In this study, we established both phosphoproteomic and gene expression profiles of ALK activity in neuroblastoma cells exposed to first- and third-generation ALK TKIs, to identify the underlying molecular mechanisms and identify relevant biomarkers, signaling networks, and new therapeutic targets. This analysis has unveiled various important leads for novel combinatorial treatment strategies for patients with neuroblastoma and an increased understanding of ALK signaling involved in this disease.
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Ghosh, Asish K., Charla Secreto, Justin Boysen, Traci Sassoon, Sacha Holland, Tait Shanafelt, Debabrata Mukhopadhyay, and Neil E. Kay. "A Novel Receptor Tyrosine Kinase Axl is Constitutively Active in B-Cell Chronic Lymphocytic Leukemia and Acts as a Docking Site of Non-Receptor Kinases: Implications for Therapy." Blood 116, no. 21 (November 19, 2010): 54. http://dx.doi.org/10.1182/blood.v116.21.54.54.
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Abstract Abstract 54 B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B-lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. Deregulated expression of protein kinases by gene deletion, mutation, or amplification has been found to be important for tumor initiation and progression involving cancer cell proliferation, survival, motility, and invasiveness as well as tumor angiogenesis and chemotherapy resistance. Because of their critical functions in oncogenesis, protein kinases have been at the forefront of targeted cancer therapy development since the 1980s. A novel receptor tyrosine kinase (RTK) Axl has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Indeed, our recent investigation (ASH 2009, Abstract #2368) identified Axl to be constitutively phosphorylated in CLL B-cells obtained from a majority of CLL patients. We wished to examine whether the constitutively phosphorylated Axl plays a pivotal role in regulating leukemic CLL B-cell survival and could be a potential target to treat this disease. We used freshly isolated purified CLL B-cells after obtaining informed written consent from the patients. Expression status of various kinases in relation to Axl expression and their functional involvement with this novel RTK to drive downstream cell survival signaling pathway(s) were analyzed in CLL B-cell lysates by immunoprecipitation/Western blot analyses using specific antibodies. Finally, we evaluated the functional importance of Axl in CLL by employing two RTK inhibitory agents (Bosutinib [SKI-606] and R428) of different specificity and affinity for receptor tyrosine kinases in order to target Axl and determined the impact on apoptotic/cytotoxic effects on CLL B-cells by flow cytometric analysis. In some experiments, we used bone marrow stromal cells to evaluate if they could diminish the impact of the RTK agent R428. To further our initial examination of Axl status in CLL, we assessed the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B-cells by Western blot analysis. Indeed, we detected differential levels of P-Axl in CLL B-cells (n=10). Further analysis revealed that the expression of P-Axl was associated with co-expression of other constitutively phosphorylated kinases/enzymes including Src family kinases (SFKs), PI3K, SyK/ZAP70 and PLC-g2 in CLL B-cells. Importantly, we found by immunoprecipitation of Axl and subsequent Western blotting that these intracellular signaling molecules were complexed with P-Axl in primary CLL B-cells. Further analysis using a siRNA approach targeting Axl suggests that phosphorylation of Axl is an upstream event of SFK-activation in CLL B-cells indicating a pivotal role of Axl in regulating CLL B-cell survival. Finally, when Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, SKI-606 (dose range 2.5–20mM) or the specific-inhibitor of Axl, R428 (dose range 0.5–10 mM) robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner (Figure). Although both RTK inhibitory agents showed clear induction of apoptosis in CLL B-cells, R428 was found to be the most potent in inducing apoptosis exhibiting an IC50 ∼4-fold lower than SKI-606. Furthermore, we have observed that R428 could target P-Axl resulting in inhibition of SFK-activation further suggesting that Axl regulates SFK-activity in CLL B-cells. Interestingly, R428-induced apoptosis in CLL B-cells (doses of 1.0 mM and 2.5 mM) was significantly inhibited when these leukemic cells were co-cultured with bone marrow stromal cells, but when we tested a higher dose (5 mM) of R428 this stroma-mediated protection was overcome. Further studies are underway to test R428 in combination other chemotherapeutic agents. Thus, we have identified a novel RTK, Axl, in CLL B-cells which appears to work as a docking site for multiple non-RTKs and drives cell survival signals. These findings indicate a unique therapeutic target for CLL treatment. Importantly, there are currently several Axl inhibitory agents that are available to be tested in CLL patients. Disclosures: Holland: Rigel: Employment.
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Hagiwara, Kazumi, Hiroatsu Iida, Yasuhiko Miyata, Tomoki Naoe, and Hirokazu Nagai. "Combination of the Histone Deacetylase Inhibitor Vorinostat with a B-Cell Receptor Signaling Inhibitor Markedly Decreases Cyclin D1 Expression in a Mantle Cell Lymphoma Cell Line." Blood 124, no. 21 (December 6, 2014): 3631. http://dx.doi.org/10.1182/blood.v124.21.3631.3631.
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Abstract Objective: Exploration of the pathogenesis of B-cell lymphomas, including mantle cell lymphoma (MCL), has led to the development of new therapeutic strategies. We focused on the aberrant activation of histone deacetylase (HDAC) and the B-cell receptor (BCR) signaling pathway, which are both novel targets of lymphoma therapy. We evaluated the effects of combined treatment with an HDAC inhibitor and a BCR signaling inhibitor on MCL. Methods: Using the MTT assay, we investigated the cytotoxic effects of BCR signaling pathway inhibitors (fostamatinib, ibrutinib, enzastaurin and idelalisib) alone or in combination with vorinostat, a potent HDAC inhibitor, using MCL cell lines. Apoptosis was determined with flow cytometry using annexin V/propidium iodide staining and detection of cleaved caspase-3 and its substrate poly-(ADP-ribose) polymerase (PARP). Gene expression profiling was analyzed with a cDNA microarray, and the lists of differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The expression of mRNA and protein was validated with quantitative real-time PCR and western blotting, respectively. Results: Among the BCR signaling inhibitors we examined, fostamatinib, a spleen tyrosine kinase inhibitor, and ibrutinib, a Bruton’s tyrosine kinase inhibitor, significantly inhibited cell growth of Jeko-1 cells when combined with vorinostat. Single treatment with fostamatinib and ibrutinib induced 25.1 ± 3.2% and 27.3 ± 2.1% apoptosis, respectively. When combined with vorinostat, the apoptotic fraction was significantly increased to 63.1 ± 5.0% and 50.8 ± 4.9%, respectively. The expression of activated caspase-3 and PARP cleavage were increased, suggesting that the combination of vorinostat and these BCR signaling inhibitors induced caspase-dependent apoptosis. Cyclin D1, which is overexpressed in MCL, was markedly down-regulated by both combination treatments. To elucidate the underlying mechanism, we performed gene expression profiling and found that numerous genes involved in multiple cellular pathways were differentially expressed following combination treatment compared to each agent alone. KEGG pathway analysis identified the NF-κB signaling pathway, which regulates cyclin D1, as a significantly enriched pathway among the down-regulated sets of genes. We validated the expression of genes involved in this pathway and found that the expression of NF-κB1/p105 was strongly down-regulated by the combination treatment. Conclusion: The combination of vorinostat and a BCR signaling inhibitor enhanced apoptosis and induced synergistic growth inhibition of MCL cells, probably by down-regulating the NF-κB signaling pathway and suppressing cyclin D1 expression. Disclosures Naoe: Otsuka Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis Pharma: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding. Nagai:Chugai Pharmaceutical Co.LTD: Speakers Bureau; Symbio Pharmaceutical Co. LTD: Research Funding; Mundi Pharma: Research Funding.
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Rule, Simon, David Tucker, Anup Kalapur, Barbara Sarholz, Jürgen Scheele, and Pier Luigi Zinzani. "Phase I/II, first in human trial of the Bruton’s tyrosine kinase inhibitor (BTKi) M7583 in patients with B cell malignancies." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e14101-e14101. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e14101.
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e14101 Background: BTK is a cytoplasmic tyrosine kinase expressed in cells of hematopoietic origin that is involved in innate and adaptive immunity. BTK inhibition blocks B-cell functions including proliferation, antigen presentation, antibody production, and cell migration. The B-cell receptor pathway is implicated in the pathogenesis of B-cell malignancies such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and B-cell chronic lymphocytic leukemia (CLL). Small molecule BTKis are a new class of agent against B-cell malignancies; ibrutinib has recently been approved for the treatment of resistant/refractory CLL, MCL, and Waldenström’s macroglobulinemia (WM). This ongoing phase I/II clinical trial (NCT02825836) is designed to determine the safety, tolerability, PK, BTK occupancy, and preliminary antitumor activity of the highly selective BTKi M7583 in patients (pts) with refractory/resistant B cell malignancies. Methods: The trial is in two parts: a dose-escalation part for which pts with refractory/resistant B cell malignancies are eligible; and a dose-expansion part for which pts with DLBCL (ABC subtype) or MCL who have failed 1-3 prior lines of therapy are eligible. Pts in the dose-escalation part will be enrolled to sequential dose escalation, with ascending once-daily M7583 doses starting at 80 mg (3 initial days)/160 mg (full 28-day cycle) and increasing according to an adaptive Bayesian design. Results: Three pts have been enrolled to treatment with M7583 80/160 mg (Table). All 3 patients have had an objective response or stable disease and relevant clinical benefit.Conclusions: M7583 appears to have a favorable benefit:risk profile, with an efficacy signal at a dose of 80 mg (3 initial days)/160 mg (full cycle) with objective response or clinical benefit. Clinical trial information: NCT02825836. [Table: see text]
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El-Gamal, Dalia, Zachary A. Hing, Shaneice Mitchell, Taylor D. LaFollette, Paul J. Brennan, Joseph M. Flynn, Jeffrey A. Jones, et al. "A Novel Inhibitor of BET Family Bromodomains Demonstrates In Vivo and I n Vi tro Potency in B-Cell Malignancies." Blood 126, no. 23 (December 3, 2015): 318. http://dx.doi.org/10.1182/blood.v126.23.318.318.
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Abstract Chronic Lymphocytic Leukemia (CLL) is a B-cell malignancy with aberrant activation of the B-cell receptor (BCR) pathway. Despite durable remissions with targeted therapies (e.g., ibrutinib) in CLL, it remains an incurable disease. Epigenetic modifications, including DNA methylation and dysregulation of chromatin regulators have been shown to contribute to the neoplastic phenotype and the differential biologic behavior of tumor cells, including leukemia. An additional layer of epigenetic complexity in cancer cells is the acquisition of super-enhancer regions enriched at genes with known oncogenic function including MYC and BCL2. Super-enhancers in multiple myeloma cells and other tumors have been found strongly enriched for binding of BRD4, a member of the human bromodomain and extraterminal (BET) domain family of proteins which includes BRD2, BRD3, BRD4, and the testis-specific member BRDT. BRD4 binds to acetylated lysines on histones and regulates the expression of important oncogenes (e.g., MYC and BCL2). We investigated the therapeutic benefit of BET inhibition in cell culture and in vivo disease models of leukemia/lymphoma using PLX51107, a novel BRD4 inhibitor with unique binding mode. Results: We report that BRD4 is significantly overexpressed in CLL patient-derived B-cells compared to B-cells from healthy donors on both transcript and protein level (p < .001). RNA-seq analysis of 55 CLL patients revealed expression of various BRD4 isoforms with marked abundance of BRD4-long and BRD4-short. Next we sought to investigate the anti-tumor activity of PLX51107 in multiple malignant B-cell lines and patient-derived CLL cells. PLX51107 inhibited cell growth in MEC1, OCI-Ly2 and OCI-Ly6 (p < .001) dose-dependently with IC50 of 1.0 ± 0.09, 1.2 ± 0.05, 1.8 ± 0.05 μM, respectively. Notably, PLX51107 antagonized CpG-induced increase in cell proliferation of primary CLL cells (p < .01) which was consistent with the downmodulation of MYC and MCL1 along with the accumulation of the cyclin-dependent kinase inhibitor p21 and IκBα (p < .005). Furthermore, the efficacy of PLX51107 to disrupt survival signaling from the microenvironment was investigated under co-culture conditions with two different bone marrow stroma cell lines, wherein PLX51107 treatment significantly induced cytotoxicity in B-CLL cells (p < .01) without affecting stromal cell viability. By employing microarray analysis we identified possible novel targets of BRD4 in CLL. Validation of those targets is currently ongoing. Particularly, Bruton's tyrosine kinase (BTK) and phospholipase C gamma 2 (PLCG2) were markedly decreased with PLX51107 treatment (p < .005), thereby signifying potential therapeutic effect(s) for dual targeting of BRD4 and BCR-associated kinases to achieve deeper and durable responses in relapsed/refractory B-cell malignancies. Lastly, anti-tumor effects of BRD4 inhibition were evaluated in vivo using Eμ-TCL1 and cMYC/TCL1 adoptive transfer models of leukemia and lymphoma, respectively. In the Eμ-TCL1 engraftment model of aggressive CLL, PLX51107 treatment resulted in prolonged survival (p < .001) accompanied with decreased disease burden, lymphocyte infiltration and proliferation when compared to vehicle-treated mice. Next, the cMYC/TCL1 adoptive transfer mouse model was used to evaluate BRD4 inhibition in a highly penetrant, malignant leukemia/lymphoma phenotype analogous to high grade lymphoma wherein PLX51107 prolonged survival (p < .0001), decreased peripheral lymphocyte counts and neoplastic cell infiltration and proliferation in both spleen and lymph nodes. Conclusion: Collectively our findings reveal BRD4 as a valid and novel target for epigenetic therapy directed against core transcriptional programs in malignant/proliferating B-cells and provide support for use of PLX51107 as an effective treatment in clinical trials for relapsed/refractory CLL patients and related aggressive forms of B-cell malignancies, with the ultimate goal of improving the outcome of these patients. Disclosures Byrd: Acerta Pharma BV: Research Funding.
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Jóri, Balázs, Markus Falk, Iris Hövel, Peggy Weist, Markus Tiemann, Lukas C. Heukamp, and Frank Griesinger. "Acquired G2032R Resistance Mutation in ROS1 to Lorlatinib Therapy Detected with Liquid Biopsy." Current Oncology 29, no. 9 (September 16, 2022): 6628–34. http://dx.doi.org/10.3390/curroncol29090520.
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Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK)/receptor tyrosine kinase inhibitor (ROS1), demonstrated efficacy in ROS1 positive (ROS1+) non-small cell lung cancer (NSCLC), although approval is currently limited to the treatment of ALK+ patients. However, lorlatinib-induced resistance mechanisms, and its efficacy against the resistance mutation G2032R in ROS1, respectively, have not yet been fully understood. Furthermore, concomitant tumor suppressor gene p53 (TP53) mutations occur in driver alteration positive NSCLC, but their prognostic contribution in the context of ROS1 inhibition remains unclear. Here we report a ROS1+ NSCLC patient who developed an on target G2032R resistance mutation during second-line lorlatinib treatment, indicating the lack of activity of lorlatinib against ROS1 G2032R. The resistance mutation was detected in plasma-derived ctDNA, signifying the clinical utility of liquid biopsies.
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Burger, Jan A., Myriam Krome, Andrea Bürkle, and Tanja N. Hartmann. "B-Cell Receptor Signaling Enhances Migration of B-Cell Chronic Lymphocytic Leukemia Cells in Response to the Chemokine Stromal Cell-Derived Factor-1 (SDF-1/CXCL12)." Blood 106, no. 11 (November 16, 2005): 1187. http://dx.doi.org/10.1182/blood.v106.11.1187.1187.
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Abstract There is growing evidence that the microenvironment confers survival signals to Chronic Lymphocytic Leukemia (CLL) B-cells that may result in disease progression and resistance to therapy. In the marrow or secondary lymphoid tissues, CLL cells are in close contact with non-tumoral accessory cells, such as mesenchymal stromal cells or nurselike cells. We previously characterized SDF-1 (CXCL12) as a central mediator for CLL cell migration and interaction with the protective microenvironment. Constitutive secretion of CXCL12 attracts CLL cells to stroma or NLC through its cognate receptor, CXCR4. These accessory cells protect CLL cells from spontaneous or drug-induced apoptosis, which is contact-dependent and partially mediated by CXCL12. B-cell receptor (BCR) signaling has been considered another important regulator of CLL cell survival. Typically, CLL cell that lack somatic mutations in the immunoglobulin (Ig) variable region (V) genes and display high levels of the tyrosine kinase ZAP-70 strongly responds to anti-IgM stimulation. Because both, CXCL12 stimulation and BCR signaling may represent important mechanism for maintenance of CLL cell within the microenvironment, we examined whether anti-IgM stimulation affects CXCL12 responses in correlation with the ZAP-70 status. BCR signaling was modulated either by crosslinking the BCR with IgM or by blocking the tyrosine kinase Syk. Effective BCR cross-linking with anti-IgM antibodies was demonstrated by phosphorylation of Syk and p44/42 MAP kinase. In ZAP-70 positive cells, BCR crosslinking resulted in a robust activation of Syk, p44/42 MAP kinases, and protein kinase B (Akt). ZAP-70 negative CLL cells displayed a weaker activation of p44/42 upon IgM crosslinking. Pretreatment of CLL cells with anti-IgM resulted in an enhanced calcium mobilization upon CXCL12 stimulation. This was not due to changes in surface expression of CXCR4. Accordingly, Syk inhibition by piceatannol resulted in a loss of calcium response upon CXCL12 stimulation. Furthermore, anti-IgM stimulation significantly increased CLL cell chemotaxis towards CXCL12 1.4 ± 1.2fold (n=9, p=0.027), and Syk inhibition by piceatannol decreased chemotaxis to 0.6 ± 0.2fold of controls (n=8). In these experiments, we could not detect differences between ZAP-70 positive or negative cells. However, there was a strong difference regarding the spontaneous, CXCL12-dependent migration of CLL cells beneath marrow stromal cells (pseudoemperipolesis). BCR crosslinking significantly increased pseudoemperipolesis of ZAP-70 expressing CLL cells 13.4 ± 21.0fold (n=7, p=0.043), whereas there was no significant increase in pseudoemperipolesis of ZAP-70 negative cells (1.4 ± 0.2fold increase, n=8). Syk inhibition by piceatannol significantly decreased the pseudoemperipolesis of ZAP-70 positive as well as ZAP-70 negative CLL cells to 0.4 ± 0.07 of controls (n=5, p=0.043). Interestingly, spontaneous migration of CLL cells beneath follicular dendritic cells (HK cells) was also significantly enhanced by anti-IgM stimulation, in particular in ZAP-70 positive cases. In summary, BCR signaling enhances calcium mobilization, CLL cell migration to CXCL12, and pseudoemperipolesis beneath marrow stroma or follicular dendritic cells. These data suggest that BCR stimulation co-operates with CXCL12 for localization and/or maintenance of CLL cells within distinct tissue microenvironments.
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Fuhr, Viktoria, Ehsan Vafadarnejad, Oliver Dietrich, Panagiota Arampatzi, Angela Riedel, Antoine-Emmanuel Saliba, Andreas Rosenwald, and Hilka Rauert-Wunderlich. "Time-Resolved scRNA-Seq Tracks the Adaptation of a Sensitive MCL Cell Line to Ibrutinib Treatment." International Journal of Molecular Sciences 22, no. 5 (February 25, 2021): 2276. http://dx.doi.org/10.3390/ijms22052276.
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Since the approval of ibrutinib for relapsed/refractory mantle cell lymphoma (MCL), the treatment of this rare mature B-cell neoplasm has taken a great leap forward. Despite promising efficacy of the Bruton tyrosine kinase inhibitor, resistance arises inevitably and the underlying mechanisms remain to be elucidated. Here, we aimed to decipher the response of a sensitive MCL cell line treated with ibrutinib using time-resolved single-cell RNA sequencing. The analysis uncovered five subpopulations and their individual responses to the treatment. The effects on the B cell receptor pathway, cell cycle, surface antigen expression, and metabolism were revealed by the computational analysis and were validated by molecular biological methods. The observed upregulation of B cell receptor signaling, crosstalk with the microenvironment, upregulation of CD52, and metabolic reprogramming towards dependence on oxidative phosphorylation favor resistance to ibrutinib treatment. Targeting these cellular responses provide new therapy options in MCL.
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Xia, Zhizhou, Xiuli Zhang, Ping Liu, Bo Jiao, and Ruibao Ren. "Palmitoylation of GNA13 Is Essential for Its Tumor Suppressor Function and Its Inhibition Confers Hypersensitivity of B-Cell Lymphoma to BCL2 Inhibitors." Blood 134, Supplement_1 (November 13, 2019): 3787. http://dx.doi.org/10.1182/blood-2019-126368.
Full textAbstract:
GNA13, encoding G alpha subunit 13 protein, is a major tumor suppressor gene frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) and Burkitt's lymphoma (BL). GNA13 is one of the key mediators of sphingosine-1-phosphate (S1P) signaling to confine B cells in germinal center. Inactivation of GNA13 coupled signaling could release tumor B cells from germinal center of lymphoid organs to peripheral blood and promote lymphomagenesis. Due to the "undruggable" nature of GNA13 as a tumor suppressor, targeted therapy remains an unsolved clinical issue. Protein palmitoylation is a unique post-translational lipid modification in regulating protein trafficking, stability and functionality. Emerging evidence reveals that palmitoylation plays vital roles in control of G-protein-coupled receptor (GPCR) signaling transduction. In this study, we discovered that GNA13 protein relies on amino-terminal palmitoylation for its membrane association and signal transduction, which is essential for its tumor suppressor activities. By using a Isobaric iodoTMT switch labeling based mass spectrometry method, we firstly identified that palmitoylation mainly occurs on two cysteine residues, Cys14 and Cys18, one of which site was also found mutated in DLBCL patients. To validate this result, we genetically mutated the cysteine to serine residues on these two potential palmitoylation sites, respectively. We found that both single and double mutants could largely reduce the palmitoylation level of GNA13. We also found that the loss of palmitoylation dissociates GNA13 protein from cell plasma membrane. Similar results were obtained using a pan-palmitoylation inhibitor, 2-bromopalmitate (2-BP). In order to examine the role of GNA13 palmitoylation in human DLBCL cells, we reintroduced either wildtype (wt) GNA13 or palmitoylation mutant GNA13 into Su-DHL-4 lymphoma cells after knocking down the endogenous wt GNA13 expression with short hairpin RNA interference. Our data show that the loss of palmitoylation promotes GCB-DLBCL cell proliferation and tumor growth both in vitro and in vivo, indicating that palmitoylation of GNA13 is essential to its tumor suppressor function. Mechanically, inactivation of GNA13, either by knocking down GNA13 expression or mutating the palmitoylation sites of GNA13, leads to phosphoinositide 3-kinase (PI3K)/AKT activation and BCL2 overexpression. Consistent to the increased BCL2 expression, we found that BCL2 inhibitors are among the most effective drugs to kill GNA13-deficient cells in a high-throughput chemical screen. Furthermore, we show that inhibition of palmitoylation by 2-BP enhances the drug sensitivity of GNA13 wildtype GCB-DLBCL cells to BCL2 inhibitors. These results indicate that the palmitoylation of GNA13 could serve as a target for treating B-cell lymphoma in combination with a BCL2 inhibitor. Disclosures No relevant conflicts of interest to declare.
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Gobessi, Stefania, Luca Laurenti, Pablo Longo, Laura Carsetti, Simona Sica, Giuseppe Leone, and Dimitar G. Efremov. "Constitutive Activation of the Protein Tyrosine Kinase Syk in Chronic Lymphocytic Leukemia B-Cells." Blood 110, no. 11 (November 16, 2007): 1123. http://dx.doi.org/10.1182/blood.v110.11.1123.1123.
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Abstract The protein tyrosine kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation Syk is recruited to the BCR and becomes activated by sequential phosphorylation at conserved tyrosine (Tyr) residues. The first event involves phosphorylation at Tyr352 by Lyn or other Src family kinases, followed by autophosphorylation of Tyr525/Tyr526 in the activation loop. Once activated, Syk further propagates the BCR signal by associating with adaptor proteins and phosphorylating downstream signaling molecules. Recently, translocations involving Syk have been identified in MDS and T-cell lymphoma, indicating that Syk may also function as a protooncogene. In line with this possibility, expression of a constitutively active TEL-Syk fusion protein was shown to result in growth factor-independent proliferation and transformation of mouse B-cells. These findings prompted us to investigated the activation status of Syk in primary unstimulated CLL B-cells. Western blot analysis with a phospho-specific antibody revealed substantial levels of Syk phosphorylated at Tyr352 in 29 of 54 freshly isolated CLL B-cell samples. Constitutive phosphorylation of Syk at Tyr352 was confirmed by immunofluorescence and confocal microscopy, which showed punctuate staining distributed across the plasma membrane and cytoplasm of unstimulated CLL B-cells. In contrast, control experiments with BJAB lymphoma B-cells showed phosphorylation of Syk at Tyr352 only following BCR crosslinking. To investigate which downstream signaling pathways are affected by Syk activation, we produced a constitutively active Syk mutant in which Tyr352 was substituted with aspartic acid. Transfection of SykTyr352Asp in HEK293, Jurkat and BJAB cells resulted in tyrosine phosphorylation of cellular proteins and autophosphorylation of Syk at Tyr525/Tyr526, whereas no changes were observed following transfection with wild type Syk. In addition, transfection of the SykTyr352Asp mutant in the human B-CLL cell line MEC1 resulted in increased phosphorylation of ERK, Akt and GSK3, indicating that these important cellular regulatory pathways are targeted by constitutively active Syk in CLL B-cells. To determine the effect of Syk activation on CLL cell survival, we cultured leukemic B-cells in the presence of R406, a recently developed and specific Syk kinase inhibitor (kindly provided by Rigel Pharmaceutics, Inc.). Assessment of CLL cell viability after 48 hours in culture showed moderate induction of apoptosis at concentrations above 600 nM in 12 of 18 investigated cases, with a maximal cytotoxic effect at 2.5 μM (20–50% apoptotic cells after normalization for spontaneous apoptosis). Interestingly, R406 at even lower concentrations (0.16 to 0.62 μM) inhibited the proliferation of CLL B-cells that was induced by stimulation with unmethylated CpG oligonucleotides, indicating that Syk kinase activity is required for leukemic cell proliferation in this setting. In summary, these data show that Syk is frequently activated in CLL B-cells, even in the absence of BCR engagement. Expression of constitutively active Syk results in activation of pathways that regulate cellular proliferation and survival, whereas inhibition of Syk kinase activity with R406 induces apoptosis and blocks CLL cell proliferation in an in vitro model. Together, these findings suggest that Syk may be a potential candidate for targeted therapy of CLL.
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Byrd, John C., Jennifer A. Woyach, and Amy J. Johnson. "Translating PI3K-Delta Inhibitors to the Clinic in Chronic Lymphocytic Leukemia: The Story of CAL-101 (GS1101)." American Society of Clinical Oncology Educational Book, no. 32 (June 2012): 691–94. http://dx.doi.org/10.14694/edbook_am.2012.32.75.
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Overview: Targeted therapy with imatinib has transformed the treatment of chronic myeloid leukemia (CML). Unlike CML, chronic lymphocytic leukemia (CLL) lacks a common genetic aberration but does demonstrate constitutive activation of PI3-kinase (PI3K) as compared to normal B cells. This constitutively active PI3K in CLL likely relates to tonic B-cell receptor signaling that is present across a wide variety of B-cell malignancies. Although PI3K is quite proximal and represents an ideal target to pharmacologically modulate, the complexity of this pathway on which many normal functions are dependent had for many years been problematic. The p110 delta isoform of PI3K is relatively specific to hematopoietic cells, and elegant mouse studies where p110 delta was genetically inactivated demonstrated only a selective B-cell defect. Subsequent development of a potent, selective p110 delta inhibitor prompted translation into the clinic for the treatment of CLL and low-grade non-Hodgkin lymphoma (NHL). From the first patient treated where a dramatic early nodal response was noted, considerable excitement has developed for this class of drugs in CLL and NHL. We will summarize the development process of CAL-101 (now GS1101) in the treatment of chronic lymphoid malignancies such as CLL.