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1
Sharma, Vishva Mitra, Jennifer A. Calvo, Kyle M. Draheim, Leslie A. Cunningham, Nicole Hermance, Levi Beverly, Veena Krishnamoorthy, Manoj Bhasin, Anthony J. Capobianco, and Michelle A. Kelliher. "Notch1 Contributes to Mouse T-Cell Leukemia by Directly Inducing the Expression of c-myc." Molecular and Cellular Biology 26, no. 21 (September 5, 2006): 8022–31. http://dx.doi.org/10.1128/mcb.01091-06.
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ABSTRACT Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a γ-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with γ-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.
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Li, Zejuan, Miao Sun, Shuangli Mi, Roger T. Luo, Jingyue Bao, MeryBeth Neilly, Nimanthi Jayathilaka, et al. "Identification of Genes Abnormally Expressed in Both Human and Murine MLL-ELL and/or MLL-ENL Leukemia." Blood 108, no. 11 (November 1, 2006): 2249. http://dx.doi.org/10.1182/blood.v108.11.2249.2249.
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Abstract Chromosome translocations are among the most common genetic abnormalities in human leukemia. Their abnormally expressed genes identify specific markers for their clinical diagnosis. Important biological properties are often conserved across species. However, although genetically engineered mouse leukemia models are well-established, few systematic studies have validated the genes that exhibit similar abnormal expression patterns in both human and mouse leukemia models. MLL-ELL and MLL-ENL fusion genes resulting from t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3), respectively, are frequently involved in human acute leukemia, and in retrovirus-mediated mouse leukemia models. We used the SAGE technique to compare gene expression profiles between MLL-ELL or MLL-ENL myeloid leukemia progenitor cells and normal myeloid progenitor cells in both human and mouse. We analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL or MLL-ENL fusions, and a leukemia cell line with an MLL-ELL fusion. 484,303 SAGE tags were identified from the nine samples, yielding 103,899 unique tags in human and 60,993 in mouse samples. We identified 40 candidate genes that appear to be abnormally expressed in both human and murine MLL-ELL leukemias (2 up- and 38 down-regulated), and 72 in both human and murine MLL-ENL leukemias (23 up and 49 down). 25 candidate genes are down-regulated in both types of leukemias, and many of them can bind with and/or regulate other candidate genes in the candidate list. For example, LCN2 can bind directly with and positively regulate MMP9; MMP9 and TMSB4X may positively regulate FOS; FOS and JUNB can bind directly and positively regulate each other. JUNB may inhibit proliferation and promote apoptosis, and it was reported that inactivation of JunB in LT-HSC leads to MPD while its inactivation in committed myeloid progenitors also predisposes to leukemia evolution. LCN2 may also positively regulate apoptosis. Meanwhile, some important candidate genes are observed only in one type of leukemia. For example, both PXN and ARHGEF1 are down-regulated only in MLL-ELL leukemias. PXN can bind directly with ARHGEF1, and the latter may inhibit proliferation. Similarly, MYB is significantly upregulated only in MLL-ENL leukemias, which was reported to play a role in MLL-ENL-mediated transformation. Taken together, some common pathways may exist in the development of both types of leukemias, whereas each may also have their own pathway. The deregulation of the important candidate genes may contribute to leukemogenesis through inhibiting apoptosis while promoting proliferation of hematopoietic cells. We have validated the expression patterns of the candidate genes, and are studying the functions and pathways of the validated candidate genes. Our studies will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute myeloid leukemia, which may lead to more effective therapy for these leukemias.
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Draheim, Kyle M., Vishva M. Sharma, Jennifer A. Calvo, Leslie A. Cunningham, Nicole Hermance, Veena Krishnamoorthy, Levi Beverly, Manoj Bhasin, Anthony Capobianco, and Michelle A. Kelliher. "Notch1 Provides a Proliferative Signal in Mouse T Cell Leukemia by Directly Targeting c-Myc." Blood 108, no. 11 (November 16, 2006): 1434. http://dx.doi.org/10.1182/blood.v108.11.1434.1434.
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Abstract As demonstrated in mouse models and in primary human T cell leukemic samples, gain of function mutation(s) in Notch1 is a common genetic event in T cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a γ-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus where it forms part of a transcriptional activator complex. We have demonstrated that mouse leukemic growth is Notch1-dependent, since treatment with γ-secretase inhibitors (GSI) results in rapid cell cycle arrest and/or apoptosis. To specifically identify Notch1 target gene(s) in leukemia, we developed mouse T cell leukemic cell lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel and direct Notch1 target gene. Consistent with these findings, retroviral insertional mutagenesis screening of our tal1 leukemic mouse model reveal common insertions in either notch1 or c-myc. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with GSI treatment or Notch1 inhibition in 83% mouse tal1 leukemic cell lines tested. Yet in a subset of leukemic cell lines, retroviral expression of c-myc fails to rescue leukemic growth, whereas expression of intracellular Notch1 in these lines remains capable of restoring growth. These data suggest that additional Notch1 target genes other than c-myc contribute to leukemogenesis. Other Notch1 target genes in thymocyte developement and their potential role in leukemogenesis will be discussed.
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Pievani, Alice, Marta Biondi, Chiara Tomasoni, Andrea Biondi, and Marta Serafini. "Location First: Targeting Acute Myeloid Leukemia Within Its Niche." Journal of Clinical Medicine 9, no. 5 (May 18, 2020): 1513. http://dx.doi.org/10.3390/jcm9051513.
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Despite extensive research and development of new treatments, acute myeloid leukemia (AML)-backbone therapy has remained essentially unchanged over the last decades and is frequently associated with poor outcomes. Eradicating the leukemic stem cells (LSCs) is the ultimate challenge in the treatment of AML. Emerging evidence suggests that AML remodels the bone marrow (BM) niche into a leukemia-permissive microenvironment while suppressing normal hematopoiesis. The mechanism of stromal-mediated protection of leukemic cells in the BM is complex and involves many adhesion molecules, chemokines, and cytokines. Targeting these factors may represent a valuable approach to complement existing therapies and overcome microenvironment-mediated drug resistance. Some strategies for dislodging LSCs and leukemic blasts from their protective niche have already been tested in patients and are in different phases of the process of clinical development. Other strategies, such as targeting the stromal cells remodeling processes, remain at pre-clinical stages. Development of humanized xenograft mouse models, which overcome the mismatch between human leukemia cells and the mouse BM niche, is required to generate physiologically relevant, patient-specific human niches in mice that can be used to unravel the role of human AML microenvironment and to carry out preclinical studies for the development of new targeted therapies.
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Fishman, Hila, Shreyas Madiwale, Ifat Geron, Vase Bari, Wouter Van Loocke, Yael Kirschenbaum, Itamar Ganmore, et al. "ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells." Blood 139, no. 3 (January 20, 2022): 399–412. http://dx.doi.org/10.1182/blood.2020010405.
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Abstract Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.
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Sontakke, Pallavi, Richard W. J. Groen, Jennifer Jaques, Huipin Yuan, Anton Martens, Edo Vellenga, and Jan Jacob Schuringa. "Mouse Versus Human Extrinsic Cues Dictate Transformation Potential In BCR-ABL/BMI1-Induced Leukemia In Humanized Xenograft Models." Blood 122, no. 21 (November 15, 2013): 515. http://dx.doi.org/10.1182/blood.v122.21.515.515.
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Abstract The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential. In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated. Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential. Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche. In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures: No relevant conflicts of interest to declare.
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Nakamae, Ikuko, Jun-ya Kato, Takashi Yokoyama, Hidenori Ito, and Noriko Yoneda-Kato. "Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia." Blood Advances 1, no. 20 (September 1, 2017): 1682–93. http://dx.doi.org/10.1182/bloodadvances.2017007054.
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Abstract C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis.
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Serio, Justin, Wei Chen, Maria Mysliwski, Lili Chen, James Ropa, Jingya Wang, and Andrew G. Muntean. "The PAF Complex Regulation of Prmt5 Facilitates Leukemic Progression." Blood 128, no. 22 (December 2, 2016): 3914. http://dx.doi.org/10.1182/blood.v128.22.3914.3914.
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Abstract Acute myeloid leukemias have been linked with dysregulated epigenetic landscapes sometimes attributed to altered functions of epigenetic regulators. The Polymerase-Associated Factor complex (PAFc) is an epigenetic regulator involved in transcriptional initiation, elongation and termination and directly interacts with the CTD of RNA Pol II. The complex is comprised of 6 subunits in human cells, Paf1, Cdc73, Ctr9, Leo1, Rtf1 and Ski8. Many of these subunits have key roles in a variety of cancers including acute myeloid leukemia (AML). We have previously shown the relevance of the PAFc in MLL-rearranged leukemias where its interaction with MLL fusion-proteins is required for leukemic progression in vitro and in vivo (Muntean et al. 2013 Blood, Muntean et al. 2010 Cancer Cell). However, little is known about the gene programs controlled by the PAFc and how these contribute to leukemogenesis. Here we identify Prmt5, an arginine methyltransferase, as a direct downstream target gene of the PAFc. Prmt5 is upregulated in variety of cancers and has been linked to cell cycle progression and activation of known oncoproteins. In addition, Prmt5 has been implicated in AML and is essential for normal hematopoiesis where loss of Prmt5 induces bone marrow aplasia due to impaired cytokine signaling (Tarighat et al. 2015 Leukemia, Liu et al. 2015 J Clin Invest). Our work establishes a major role for the PAFc in regulating Prmt5 expression in AML. We observe that excision of the Cdc73 subunit of the PAFc results in reduced proliferation, the induction of differentiation, cell cycle arrest, and a mild increase in apoptosis. Several key epigenetic marks are reduced globally upon loss of Cdc73 including H4R3me2s, a modification catalyzed by Prmt5. RNA sequencing and bioinformatics analysis using GSEA, revealed that loss of Cdc73 led to increased expression of a gene program associated with hematopoietic differentiation, in agreement with our cellular characterization. In addition, the downregulation of a methyltransferase gene program was detected upon Cdc73 excision. Included in this signature were several members of the Prmt family. Analysis of changes in expression following loss of Cdc73 and functional relevance in MLL-AF9 leukemic cells led us to Prmt5 as a gene critically important in AML cells and modulated by the PAFc. To interrogate the function of Prmt5 in AML cells, we performed shRNA knockdown experiments which resulted in reduced proliferation, reduced cell fitness, G1 cell cycle arrest and global reduction H4R3me2s. ChIP experiments revealed that the PAFc localizes to the Prmt5 locus in mouse and human derived leukemic cells. Further, preliminary data suggests the MLL-AF9 fusion protein also localizes to the Prmt5 locus and may enhance its transcriptional output. The enzymatic activity of Prmt5 is necessary for AML cell growth as wild type PRMT5 can rescue proliferation of Prmt5 knock-down cells while a catalytic dead mutant cannot. Furthermore, we have observed that knockdown of Prmt5 increases the disease latency of Hoxa9/Meis1 induced leukemia in vivo. Utilizing a commercially available inhibitor for Prmt5, EPZ015666 (Chan-Pembre et al. 2015 Nat Chem Bio), we show pharmacologic inhibition of PRMT5 reduces the growth of a spectrum of human leukemic cell lines, suggesting PRMT5 is important for multiple subtypes of AML. Overall, our findings elucidate the PAFc as a regulator of Prmt5 expression that is necessary for the maintenance of AML. Disclosures No relevant conflicts of interest to declare.
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Chen, Jianjun, Miao Sun, Roger T. Luo, Jingyue Bao, Masha Kocherginsky, Deborah S. Johnson, Lili Wang, et al. "Genes Similarly Abnormally Expressed in Both Human and Murine MLL-Associated Leukemia." Blood 106, no. 11 (November 16, 2005): 3000. http://dx.doi.org/10.1182/blood.v106.11.3000.3000.
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Abstract Although more than 50 different loci translocate to the MLL gene at chromosome band 11q23, resulting in either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), no unifying property is shared by all partner genes. The translocations result in a functional fusion of the N-terminal part of MLL gene and the C-terminal part of each partner gene, presumably leading to changes in the expression of the normal target genes, most of which have not been identified. Although genetically engineered mouse leukemia models have been widely used, few systematic studies have evaluated whether such models are valid equivalents of human leukemia. We used serial analysis of gene expression (SAGE) to obtain genome-wide gene expression profiles in normal myeloid progenitor cells from human CD15+ and mouse Gr-1+ cells. We also analyzed four patient samples (two with each fusion) and two retrovirally-induced mouse leukemias containing either MLL-ELL [t(11;19)(q23;p13.1)] or MLL-ENL [t(11;19)(q23;p13.3)] fusions, and a cell line from a leukemia mouse transduced with an MLL-ELL fusion. MLL-ELL and MLL-ENL fusions are frequently involved in human AML, while MLL-ENL is also seen in human ALL. 484,303 SAGE tags were identified from the nine samples (40,000 to 100,000 tags per sample), yielding 103,899 unique SAGE tags in the human and 60,993 in the mouse samples. Analysis of the SAGE data identified 43 candidate genes that appear to be abnormally expressed in both human and mouse myeloid leukemia progenitor cells with either MLL-ELL or MLL-ENL fusions (9 up-regulated and 34 down-regulated; Table 1). Increasing evidence suggests that endogenous antisense RNAs may play critical roles in gene regulation and cancer. Natural antisense RNAs include cis-encoded antisense RNAs transcribed from the opposite strand of the same genomic locus as the sense target genes, and trans-encoded antisense RNAs such as microRNAs (miRNAs) transcribed from a genomic locus different from the sense target genes. 26 of the 43 candidate genes have antisense partners (with a total of 7 cis-encoded antisense RNAs and 36 trans-encoded miRNAs) and thereby might be regulated by endogenous antisense RNAs. We are currently validating the expression pattern of the 43 candidate genes in at least 30 different human and mouse leukemia and normal control samples with quantitative RT-PCR, and measuring the level of expression of all known miRNAs via microarray in these samples. Our studies on the abnormally expressed genes and their potential antisense partners will provide important insights into the complex functional pathways related to MLL rearrangements in the development of acute leukemia, which may lead to more effective therapy for these leukemias. Table 1. Genes deregulated in both human and mouse leukemiasa Total number Up-regulated genes Down-regulated genes Genes with antisense partner(s) aThe genes have at least 3 fold difference in expression with a significance P < 0.05 between each leukemia sample and the normal control sample. In MLL-ELL fusions 21 1 20 12 In MLL-ENL fusions 33 8 25 21 In both types of fusions 11 0 11 7 Total unique genes 43 9 34 26
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Korf, Katharina, Harald Wodrich, Alexander Haschke, Ron M. Evans, and Thomas M. Sternsdorf. "The Acute Promyelocytic Leukemia-Oncoprotein PML-Raralpha Blocks Senescence and Disrupts The Atrx/Daxx Chromatin Remodeling Complex To Promote Leukemia." Blood 122, no. 21 (November 15, 2013): 1267. http://dx.doi.org/10.1182/blood.v122.21.1267.1267.
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Abstract Although Acute Promyelocytic Leukemia (APL) has become a curable disease due to in-depth understanding of the underlying molecular processes, its investigation has provided unique and valuable insights into the processes involved in leukemogenesis. Therefore we use it as a model disease. 99% of APL-patients express a PML-RAR fusion protein. While involvement of RAR has proven indispensable for oncogenicity, the role of the PML domain is far less clear. In our previous study (Sternsdorf et al., Cancer Cell, 2006) we found that substitution of PML with heterologous self-interaction domains suffices to induce leukemias, but drastically decreases oncogenic potency of the resulting fusion proteins. In this study, we have chosen the inverse strategy: we have modified the PML domain to create a more active artificial model oncoprotein by adapting PR to its biological environment: As the typical model organism for APL studies is the mouse, we have replaced the human PML domain with the murine PML domain. This oncoprotein (mPR) creates APL-type leukemias in mice with higher penetrance and shorter latency than its human counterpart, hPR. We have used this system to study immediate early effects of expression of the model oncoprotein. While proliferating murine bone marrow cells go into senescence ex vivo, expression of mPR prevents this and robustly immortalizes murine bone marrow from every mouse strain tested so far. Senescence-associated upregulation of the cell-cycle regulators p21 and p19 was efficiently blocked by mPR expression. In mouse cells, mPR exhibits higher potency in disrupting the PML-associated Daxx/ATRX complex than hPR. Knockdown of ATRX, but not Daxx ameliorated ATRA-induced growth suppression and p21 upregulation in the human APL model cell line NB4. These data suggest, that PML-RAR promotes leukemogenesis by disrupting the Daxx/ATRX complex, which assembles at PML nuclear bodies during the onset of senescence. Disclosures: No relevant conflicts of interest to declare.
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Takeda, Eri, Sachiyo Tsuji-Kawahara, Mayumi Sakamoto, Marc-André Langlois, Michael S. Neuberger, Cristina Rada, and Masaaki Miyazawa. "Mouse APOBEC3 Restricts Friend Leukemia Virus Infection and Pathogenesis In Vivo." Journal of Virology 82, no. 22 (September 10, 2008): 10998–1008. http://dx.doi.org/10.1128/jvi.01311-08.
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ABSTRACT Several members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like complex 3 (APOBEC3) family in primates act as potent inhibitors of retroviral replication. However, lentiviruses have evolved mechanisms to specifically evade host APOBEC3. Likewise, murine leukemia viruses (MuLV) exclude mouse APOBEC3 from the virions and cleave virion-incorporated APOBEC3. Although the betaretrovirus mouse mammary tumor virus has been shown to be susceptible to mouse APOBEC3, it is not known if APOBEC3 has a physiological role in restricting more widely distributed and long-coevolved mouse gammaretroviruses. The pathogenicity of Friend MuLV (F-MuLV) is influenced by several host genes: some directly restrict the cell entry or integration of the virus, while others influence the host immune responses. Among the latter, the Rfv3 gene has been mapped to chromosome 15 in the vicinity of the APOBEC3 locus. Here we have shown that polymorphisms at the mouse APOBEC3 locus indeed influence F-MuLV replication and pathogenesis: the APOBEC3 alleles of F-MuLV-resistant C57BL/6 and -susceptible BALB/c mice differ in their sequences and expression levels in the hematopoietic tissues and in their abilities to restrict F-MuLV replication both in vitro and in vivo. Furthermore, upon infection with the pathogenic Friend virus complex, (BALB/c × C57BL/6)F1 mice displayed an exacerbated erythroid cell proliferation when the mice carried a targeted disruption of the C57BL/6-derived APOBEC3 allele. These results indicate, for the first time, that mouse APOBEC3 is a physiologically functioning restriction factor to mouse gammaretroviruses.
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David Chen, Chun-Wei, Amit U. Sinha, Jun Qi, Aniruddha J. Deshpande, Nan Zhu, Richard Koche, Rowena Eng, et al. "Genome-Wide RNAi Screen Identifies The Mechanistic Role For DOT1L In MLL-Rearranged Leukemia." Blood 122, no. 21 (November 15, 2013): 598. http://dx.doi.org/10.1182/blood.v122.21.598.598.
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Abstract Rearrangement of Mixed-Lineage Leukemia (MLL) gene defines a genetically distinguishable subset of aggressive leukemias with poor prognosis. Recent studies exhibit promising activity of small-molecule inhibitors of the H3K79 methyltransferase DOT1L (disruptor of telomeric silencing-1 like) against leukemias bearing MLL-translocations (Daigle et. al. 2011 Cancer Cell). However, the mechanisms underlying the epigenetic addiction of MLL-fusion oncogenic program to H3K79-methylation remain unclear. A number of labs have recently shown the expression of MLL-fusion target genes including HOXA cluster and MEIS1 is strongly dependent on DOT1L activity, whereas most of the normal genes do not require H3K79 methylation for their expression. To further investigate this unique subordination of MLL-fusion leukemic program to DOT1L/H3K79-methylation, we sought to identify genes, whose suppression would rescue the Dot1l loss-of-function phenotype in mouse MLL-AF9 cells. We conducted a suppressor screen by introducing a mouse genome-wide shRNA library (92,425 shRNA targeting 16,469 mouse genes) into Dot1lf/f-MLL-AF9 leukemic cells harboring tamoxifen-inducible Cre recombinase. Relative frequencies of integrated shRNA sequences were assessed by high throughput sequencing. Our results revealed that knockdown of sirtuin 1 (Sirt1), a histone deacetylase, significantly antagonizes the lethality cause by loss of Dot1l/H3K79me2 in MLL-AF9 leukemia. To understand the function of Sirt1 in MLL-rearranged leukemia, we performed genome-wide expression profiling and revealed that Sirt1 is essential for complete silencing of the MLL-AF9 driven oncogenic program including Hoxa7 and Meis1 upon inhibition of Dot1l. ChIP-seq analyses indicate that Sirt1 localizes to MLL-AF9 targets and mediates loss of H3K9ac upon suppression of Dot1l. Because Sirt1 is known to interact with epigenetic repressive enzymes such as polycomb repressive complex (PRC) members and Suv39h1, we them examine whether Sirt1 participates in suppression of MLL-AF9 targets by recruiting these silencing mechanisms to the loci. ChIP-seq analyses showed that suppression of Dot1l activity resulted in increased H3K9me2 and H3K27me3 levels near the transcriptional start site of MLL-AF9 target genes. Remarkably, knockdown of Sirt1 blocks the accumulation of H3K9me2 and maintains the chromatin accessibility at MLL-AF9 targets after Dot1l suppression, whereas the increase of H3K27me3 at those loci was minimally affected. Additionally, we identified that histone methyltransferase Suv39h1, a well characterized Sirt1 binding partner, is responsible for H3K9 di-methylation and gene silencing of Hoxa7 and Meis1 in response to DOT1L inhibitor. Finally, we showed that pharmacological activation of Sirt1 and inhibition of Dot1l synergistically suppresses the leukemogenetic potential of MLL-AF9 cells in a secondary leukemia drug exposure model. Our results suggest that aberrant methylation of H3K79 may render leukemic transformation by inhibition of repressive mechanisms (including SIRT1/SUV39H1-mediated heterochromatin formation and others) at MLL-fusion targets. Combination treatment model further implies that patients with leukemia bearing MLL-translocations may benefit from synergistic therapies that simultaneously target both DOT1L and SIRT1 or other epigenetic regulators. Disclosures: No relevant conflicts of interest to declare.
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Pankov, Dmitry, Tao Dao, Yiyang Wang, Andrew Scott, Tatyana Korontsvit, Victoria Zakhaleva, Nicholas Veomett, et al. "A Bi-Specific T Cell Engaging Monoclonal Antibody (mAb) Derived From a TCR-Like Mab Specific For WT1/HLA-A0201 (ESK-BiTE) Shows a Potent Activity Against Human AML and Ph+ ALL In Mouse Models." Blood 122, no. 21 (November 15, 2013): 2521. http://dx.doi.org/10.1182/blood.v122.21.2521.2521.
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Abstract The Wilms’ tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766. Disclosures: No relevant conflicts of interest to declare.
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Doubrovina, Ekaterina, Mikhail Doubrovin, Elena Kanaeva, and Richard J. O’Reilly. "In Vivo Eradication of Human Clonigenic Acute Lymphoblastic Leukemic Cells Expressing the Wilms Tumor Protein, WT-1, by HLA Restricted WT-1 Specific T-Cells in NOD/SCID Mice Monitored by Bioluminescent Imaging." Blood 110, no. 11 (November 16, 2007): 581. http://dx.doi.org/10.1182/blood.v110.11.581.581.
Full textAbstract:
Abstract WT-1 is expressed in 60–80% of acute leukemias, CML and high risk forms of MDS. Its expression has been hypothesized to be critical to the growth or survival of leukemic stem cells. Previously, alloreactive HLAA0201− T-cells recognizing a complex of WT-1 peptide and HLA A0201 were reported to prevent growth of leukemic HLA A0201+ CD34+ Ph+CML progenitor cells in NOD/ SCID mice (Transplantation, vol 75, No9, 2003). In this study, we have assessed the capacity of HLA-restricted, WT-1 peptide specific CTL (WT1-CTL) lacking alloreactivity to prevent the outgrowth of a human acute preB-lymphocytic leukemia (B-ALL)in NOD/SCID mice. This leukemia contained 65% of the blasts expressed WT-1 as determined by FACS analysis. For these studies the leukemic cells were transduced to express a luciferase reporter gene, permitting sequential monitoring of growth in vivo by bioluminescent imaging. WT-1 specific T-cells were generated from normal HLA A0201+ donor PBMC by in vitro sensitization with autologous dendritic cells loaded with the immunogenic HLA A0201 binding WT-1 peptide, RMFPNAPYL, and shown to be selectively cytotoxic against HLA A0201+WT-1+ leukemias and peptide loaded PHA blasts. T-cells from the same donor sensitized with autologous EBV BLCL and exhibiting HLA A0201 restricted EBV-specific cytotoxic activity served as controls. WT-1-CTL or EBV CTL were co-incubated in vitro with the WT-1+ HLA A0201+ BALL-LUC at a 4:1 effector target ratio for 7 hours at 37°C. Thereafter, separate groups of 5 NOD/SCID mice received intravenous infusions of cells from each of the co-cultures, at doses providing 12 × 106 WT1 CTL or EBVCTL and 3 × 106 BALL-LUC cells/mouse. A third group received 3×106 BALL-LUC alone. Leukemia growth was monitored at 2–3 day intervals from day 1–45 post infusion. In all 3 groups, BALL-LUC could be detected in the thorax by imaging at day 1. In mice treated with BALL-LUC alone or together with EBV-CTL, signal accumulation in the thorax increased steadily through 45 days of observation. By day 17, BALL-LUC were also detected throughout the head, abdomen and pelvis, and thereafter also increased until sacrifice at day 45. Autopsy confirmed presence of leukemic nodules in the lung and leukemic cells in blood, spleen and marrow as well as other organs. In contrast, in mice treated with WT1-CTL+ BALL LUC, signal intensity in lung decreased by day 4. In 4/5 of these mice, BALL-LUC could not be detected thereafter. In one mouse from this group, BALL-LUC were first detected in the head 31 days post infusion. At autopsy on day 45, this mouse had detectable BALL in the skull but in no other sites. WT-1 expression of residual leukemic cells is being analyzed. The other mice treated with WT-1 CTL had no detectable residual disease. These results suggest that clonogenic BALL cells express WT-1 and are susceptible to eradication in vivo by WT-1 peptide specific cytotoxic T-cells. The elimination of such clonogenic leukemic cells is sufficient to prevent subsequent development of leukemia.
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Mohanty, Sagarajit, and Michael Heuser. "Mouse Models of Frequently Mutated Genes in Acute Myeloid Leukemia." Cancers 13, no. 24 (December 8, 2021): 6192. http://dx.doi.org/10.3390/cancers13246192.
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Acute myeloid leukemia is a clinically and biologically heterogeneous blood cancer with variable prognosis and response to conventional therapies. Comprehensive sequencing enabled the discovery of recurrent mutations and chromosomal aberrations in AML. Mouse models are essential to study the biological function of these genes and to identify relevant drug targets. This comprehensive review describes the evidence currently available from mouse models for the leukemogenic function of mutations in seven functional gene groups: cell signaling genes, epigenetic modifier genes, nucleophosmin 1 (NPM1), transcription factors, tumor suppressors, spliceosome genes, and cohesin complex genes. Additionally, we provide a synergy map of frequently cooperating mutations in AML development and correlate prognosis of these mutations with leukemogenicity in mouse models to better understand the co-dependence of mutations in AML.
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Gough, Sheryl M., Fan Lee, Yang Jo Chung, Robert L. Walker, Fan Yang, Yuelin (Jack) Zhu, Yi Ning, Paul S. Meltzer, and Peter Aplan. "A NUP98-PHF23 Transgenic Mouse Model Develops AML and T-ALL." Blood 118, no. 21 (November 18, 2011): 2467. http://dx.doi.org/10.1182/blood.v118.21.2467.2467.
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Abstract Abstract 2467 NUP98-fusions although rare, have been associated with de novo acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, T-cell acute lymphoblastic leukemia (T-ALL) and therapy-related myeloid malignancies. The NUP98-PHF23 (NP23) gene fusion was cloned from an acute myeloid leukemia (AML) patient with a t(11;17)(p15;p13) chromosome translocation. The nucleoporin 98 protein (NUP98), normally a component of the nuclear pore complex, is known to be fused to at least 28 different fusion partners as a result of structural chromosomal rearrangements associated with hematological malignancies. PHF23 encodes the Plant homeodomain (PHD) finger 23 protein. PHF23 is largely uncharacterized, but the PHD finger motif has been shown to act as a reader of di- and tri-methylated histone 3 lysine 4 (H3K4me2/3) marks. This suggests that PHF23 may function in chromatin regulation and that the NP23 fusion protein may play a role in aberrant chromatin modification at domains of active gene transcription. To determine the oncogenic potential of NP23, we generated a transgenic mouse model that expressed the human fusion gene in hematopoietic tissues. We have characterized two founder lines (C10 and B10) expressing the NP23 fusion in hematopoietic tissue. Most of the offspring from the C10 line developed an AML that closely resembled the human disease, with increased blasts in the blood or bone marrow, widespread organ infiltration, and myeloid immunophenotype. Onset of disease was as early as 4.5 months, and 70 percent of the NP23 mice succumbed to leukemia by 12 months of age. Of note, an independent line (B10) developed a wider spectrum of leukemias but with similar age of onset and penetrance. The B10 mice predominantly developed T-ALL and AML, and four cases of B-ALL and one erythroleukemia, indicating that the NP23 protein was oncogenic in several different hematopoietic cell types. AMLs typically demonstrated an aberrant Mac-1+/B220dim phenotype, which has previously been recognized in leukemias caused by overexpression of the Hoxa cluster genes Hoxa5,7,9,10,11. Microarray gene expression analysis identified the Hoxa cluster genes to be markedly overexpressed, and validation by RQ-PCR demonstrated that Hoxa5, a7, a9 and a10 overexpression ranged from 10- to greater than 1000-fold increased in both the AML and T-ALL samples compared to wild type hematopoietic tissues; these Hoxa cluster genes were also overexpressed in hematopoietic tissues from clinically healthy NP23 transgenic mice. We also identified a novel transcript, Gm525 (homologue of H. sapiens C17orf67), that is markedly (100x) elevated specifically in the T-ALL samples. Most T-ALL samples have HD or PEST domain Notch1 mutations, and Notch1 mRNA levels, as well as its downstream target Hes1, are elevated in the T-ALLs compared to WT thymus. Immortal cell lines were established from two of the NP23 T-ALLs, and ChIP-seq was used to assay the genome wide pattern of H3K4me3 and H3K27me3 histone marks. Results show abundant levels of H3K4me3 at the Hoxa locus which tightly correlates to the increased Hoxa cluster gene expression seen in the cell lines. The NP23 model will be useful for identifying oncoproteins involved in leukemic transformation, particularly those oncoproteins which play a role in chromatin modification or are downstream targets of the HOXA genes. Disclosures: No relevant conflicts of interest to declare.
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Hu, Hsiangyu, Nirmalya Saha, Yuting Yang, Sierrah Marie Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, and Andrew G. Muntean. "Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis." Blood 136, Supplement 1 (November 5, 2020): 30–31. http://dx.doi.org/10.1182/blood-2020-138622.
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Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.
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Rodenburg, Michaela, Meike Fischer, Afra Engelmann, Stephanie O. Harbers, Marion Ziegler, Jürgen Löhler, and Carol Stocking. "Importance of Receptor Usage, Fli1 Activation, and Mouse Strain for the Stem Cell Specificity of 10A1 Murine Leukemia Virus Leukemogenicity." Journal of Virology 81, no. 2 (November 1, 2006): 732–42. http://dx.doi.org/10.1128/jvi.01430-06.
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ABSTRACT Murine leukemia viruses (MuLV) induce leukemia through a multistage process, a critical step being the activation of oncogenes through provirus integration. Transcription elements within the long terminal repeats (LTR) are prime determinants of cell lineage specificity; however, the influence of other factors, including the Env protein that modulates cell tropism through receptor recognition, has not been rigorously addressed. The ability of 10A1-MuLV to use both PiT1 and PiT2 receptors has been implicated in its induction of blast cell leukemia. Here we show that restricting receptor usage of 10A1-MuLV to PiT2 results in loss of blast cell transformation capacity. However, the pathogenicity was unaltered when the env gene is exchanged with Moloney MuLV, which uses the Cat1 receptor. Significantly, the leukemic blasts express erythroid markers and consistently contain proviral integrations in the Fli1 locus, a target of Friend MuLV (F-MuLV) during erythroleukemia induction. Furthermore, an NB-tropic variant of 10A1 was unable to induce blast cell leukemia in C57BL/6 mice, which are also resistant to F-MuLV transformation. We propose that 10A1- and F-MuLV actually induce identical (erythro)blastic leukemia by a mechanism involving Fli1 activation and cooperation with inherent genetic mutations in susceptible mouse strains. Furthermore, we demonstrate that deletion of the Icsbp tumor suppressor gene in C57BL/6 mice is sufficient to confer susceptibility to 10A1-MuLV leukemia induction but with altered specificity. In summary, we validate the significance of the env gene in leukemia specificity and underline the importance of a complex interplay of cooperating oncogenes and/or tumor suppressors in determining the pathogenicity of MuLV variants.
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Ha, Byung Hak, Mark Adam Simpson, Anthony J. Koleske, and Titus J. Boggon. "Structure of the ABL2/ARG kinase in complex with dasatinib." Acta Crystallographica Section F Structural Biology Communications 71, no. 4 (March 20, 2015): 443–48. http://dx.doi.org/10.1107/s2053230x15004793.
Full textAbstract:
ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase) family of tyrosine kinases. ARG plays important roles in cell morphogenesis, motility, growth and survival, and many of these biological roles overlap with the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is associated with constitutive ABL kinase activation resulting from fusion between parts of the breakpoint cluster region (BCR) andABL1genes. Similarly, fusion of theETV6(Tel) andARGgenes drives some forms of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Dasatinib is a tyrosine kinase inhibitor used for the treatment of CML by inhibiting ABL, and while it also inhibits ARG, there is currently no structure of ARG in complex with dasatinib. Here, the co-crystal structure of the mouse ARG catalytic domain with dasatinib at 2.5 Å resolution is reported. Dasatinib-bound ARG is found in the DFG-in conformation although it is nonphosphorylated on the activation-loop tyrosine. In this structure the glycine-rich P-loop is found in a relatively open conformation compared with other known ABL family–inhibitor complex structures.
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Fukushime-Nakase, Yoko, Mitsushige Nakao, Shigeo Horiike, Motohiro Nishimura, Masafumi Taniwaki, Tohru Sugimoto, and Tsukasa Okuda. "A Genome-Modified Mouse Line with an Insertion Mutation at the AML1/Runx1 Gene Locus Detected from a Patient with Myelodysplastic Syndrome." Blood 106, no. 11 (November 16, 2005): 1599. http://dx.doi.org/10.1182/blood.v106.11.1599.1599.
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Abstract AML1 (Acute Myeloid Leukemia 1; also known as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays important roles in initiating definitive hematopoiesis, thymocyte development, and platelet production. AML1’s DNA-binding is mediated through its Runt domain, and their further association with the non-DNA-binding subunit, CBFβ stabilizes the resultant ternary complex to be functional. AML1 is frequently involved in leukemia-associated chromosome translocations, through which AML1 fusion-proteins with strong dominant-negative effects to normal CBF are generated, thus contributing to the leukemic transformation. Recently, genomic mutations of the AML1 gene locus have been described to associate with cases of acute myeloblastic leukemia and myelodysplastic syndromes (MDS), and pedigrees of familial platelet disorder with propensity to develop acute myeloblastic leukemia (FPD/AML). Frequent sites of the point-mutations are confined to the residues within the Runt domain, such as, R139, R174, or R177, which are important for the DNA-contact of the molecule. Preliminary results from studies using knockin mice carrying mis-sense mutations for these residues so far revealed that the mutants are biologically inactive when homozygously inherited. However, the mechanisms how the mutations contribute to the onset of the leukemia remain largely to be elucidated. In order to further characterize the aberration of AML1, we generated another genome-modified mouse line which harbors an insertion mutation of this gene locus. As reported previously, we identified a case of refractory anemia with excess of blasts (RAEB) whose blast cells have a mutated allele where three base-pair, ATC, is inserted after codon 150 (I150ins) of the molecules. By this mutation, an additional isoleucine residue is inserted into the 10th β-strand of the Runt domain sequences which provides a facet to interact with CBFβ. We introduced the cDNA of I150ins into AML1/Runx1 gene locus by means of a gene-knockin approach in mouse ES cells, and transmitted the allele into mouse germline through conventional methods. Subsequent heterozygous mice were viable and fertile. In contrast, homozygous mice die in utero similarly to the mice with simple disruption of this gene. These results indicate that this insertion mutation leads to the complete loss of its biologic function as were observed for the point-mutant mice. Further careful examinations of the heterozygous mice for this mutant allele in comparison with those for point-mutation animals and simple haplo-insufficient ones will provide valuable insights into the molecular mechanisms of AML1-related leukemic transformation.
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Masselli, Marika, Serena Pillozzi, Massimo D'Amico, Luca Gasparoli, Olivia Crociani, Matteo Stefanini, Kennet Mugridge, Wolfang Tiedke, Andrea Becchetti, and Annarosa Arcangeli. "Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias." Blood 120, no. 21 (November 16, 2012): 1506. http://dx.doi.org/10.1182/blood.v120.21.1506.1506.
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Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.
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Połeć, Anna, Alexander D. Rowe, Pernille Blicher, Rajikala Suganthan, Magnar Bjørås, and Stig Ove Bøe. "PML Regulates the Epidermal Differentiation Complex and Skin Morphogenesis during Mouse Embryogenesis." Genes 11, no. 10 (September 25, 2020): 1130. http://dx.doi.org/10.3390/genes11101130.
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The promyelocytic leukemia (PML) protein is an essential component of nuclear compartments called PML bodies. This protein participates in several cellular processes, including growth control, senescence, apoptosis, and differentiation. Previous studies have suggested that PML regulates gene expression at a subset of loci through a function in chromatin remodeling. Here we have studied global gene expression patterns in mouse embryonic skin derived from Pml depleted and wild type mouse embryos. Differential gene expression analysis at different developmental stages revealed a key role of PML in regulating genes involved in epidermal stratification. In particular, we observed dysregulation of the late cornified envelope gene cluster, which is a sub-region of the epidermal differentiation complex. In agreement with these data, PML body numbers are elevated in basal keratinocytes during embryogenesis, and we observed reduced epidermal thickness and defective hair follicle development in PML depleted mouse embryos.
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Kong, Hyewon, Colleen R. Reczek, Gregory S. McElroy, Elizabeth M. Steinert, Tim Wang, David M. Sabatini, and Navdeep S. Chandel. "Metabolic determinants of cellular fitness dependent on mitochondrial reactive oxygen species." Science Advances 6, no. 45 (November 2020): eabb7272. http://dx.doi.org/10.1126/sciadv.abb7272.
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Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.
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Hu, Hsiangyu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, et al. "Abstract 2966: The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2966. http://dx.doi.org/10.1158/1538-7445.am2022-2966.
Full textAbstract:
Abstract MLL1 (KMT2A) translocations are found in ~10% of acute leukemia and give rise to an aggressive form of leukemia in infant, pediatric and adult patients. MLL1 fusion driven acute leukemia is characterized by deregulated activity of the Super Elongation Complex (SEC) and the H3K79 methyltransferase DOT1L, which alter the epigenetic landscape and transcription of pro-leukemic MLL1 fusion targets like HoxA9 and Meis1. The Eleven-Nineteen-Leukemia gene (ENL or MLLT1) is a common MLL1 fusion partner and a SEC component. The ENL protein contains a highly conserved N-terminal epigenetic reader YEATS domain that recognizes acetylated H3K9/K18/K27 (H3Kac hereinafter). Wild type ENL was recently found to be essential for leukemic cell growth, which is dependent on its YEATS domain interaction with H3Kac. While this finding highlighted the YEATS domain importance in wild type ENL function in leukemic cells, the inclusion and importance of the YEATS domain in MLL-ENL fusion protein remain to be elucidated. Here, we investigated the clinical relevance and importance of the ENL YEATS domain in MLL-ENL leukemias. We analyzed >300 t(11;19) MLL-ENL leukemia patients for the breakpoint location within the ENL gene and found that the YEATS domain is retained in the resultant MLL-ENL fusion protein in 84.1% of t(11;19) leukemia patients. We tested the importance of the YEATS domain in MLL-ENL mouse models and found that the YEATS domain and downstream sequence is required for MLL-ENL leukemogenesis in vivo. YEATS deletion decreased expression of pro-leukemic targets such as Meis1, an important factor for leukemic stem cells (LSC). To interrogate the contribution of the YEATS epigenetic reader function in MLL-ENL leukemogenesis, we introduced YEATS point mutations rendering the domain defective in interacting with H3Kac and found that this significantly increased leukemia latency in vivo. Further investigation revealed that YEATS point mutations disrupting H3Kac binding significantly decreased MLL-ENL LSC frequency while not affecting homing to the bone marrow. We attribute this LSC frequency change to altered Meis1 expression. Additionally, disruption of the YEATS epigenetic reader function in MLL-ENL leukemia cells does not induce differentiation, apoptosis nor cell cycle arrest. Therapeutically, we predicted the YEATS domain in MLL-ENL would sensitize MLL-ENL leukemia to YEATS domain inhibitors. Indeed, MLL-ENL leukemia cells are more sensitive to the ENL/AF9 YEATS domain inhibitor, SGC-iMLLT, compared to acute leukemia cells driven by other fusions. Together, our results demonstrate that YEATS-H3Kac binding plays an important role in MLL-ENL fusion mediated leukemogenesis. Our data establishes a strong rationale for future exploration of small molecules aimed at disrupting the YEATS-H3Kac interaction as a targeted therapeutics for treating t(11;19) leukemia patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia [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 2966.
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Goossens, Steven, Sofie Peirs, Geert Berx, Pieter Van Vlierberghe, and Jody J. Haigh. "Oncogenic ZEB2 Activation Drives Sensitivity Towards LSD1 Inhibition in T-Cell Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 4027. http://dx.doi.org/10.1182/blood.v128.22.4027.4027.
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Abstract T-cell acute lymphoblastic leukemias (T-ALLs) are rare aggressive hematologic tumors resulting from the malignant transformation of T-cell progenitors. The prognosis of T-ALL has gradually improved with the introduction of intensified chemotherapy. However, the outcome of T-ALL patients with primary resistant or relapsed leukemia remains extremely poor. Therefore, current research efforts are focused on the development of more effective and less toxic anti-leukemic drugs, which will likely require an improved understanding of the molecular biology of chemotherapy resistant residual tumor cells that eventually drive disease recurrence. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of an immature subtype of T-ALL with increased leukemia-initiating properties. ZEB2 is a large multidomain homeobox transcription factor that recognizes bipartite E-box motifs through its amino- and carboxyterminal Zinc finger domains. The domains outside the Zn-finger clusters have been shown to be essential for the recruitment of various tissue-specific co-activators/repressors. Identification and targeting of these interaction partners that are essential for the oncogenic properties of ZEB2 could be a feasible option for the development of novel therapeutics to treat this aggressive leukemia. In this study, we performed ZEB2 protein pull down experiments and subsequent mass spectrometry to explore the ZEB2 interactome in the context of T-ALL. We identified the Lysine-specific demethylase KDM1A/LSD1 as a novel interaction partner of ZEB2. The ZEB2-LSD1 interaction was confirmed via bi-directional co-immunoprecipitation experiments in both mouse and human T-ALL cell lines. KDM1A/LSD1 is a flavin-containing amino oxidase that specifically catalyzes the demethylation of mono- and di-methylated lysines on histone 3. LSD1 has been reported to regulate the balance between self-renewal and differentiation of pluripotent stem cells and its expression is upregulated in various cancers. Pharmacological inhibition of LSD1 has been proposed as a novel promising therapy to treat and kill cancer stem cells and novel potent inhibitors are being tested in clinical trials. We demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on LSD1 activity for their leukemic survival. In addition, doxycycline induced ZEB2 overexpression significantly increased sensitivity to LSD1 inhibition in the MLL-AFX positive T-ALL cell line KARPAS-45. We conclude that targeting the ZEB2 protein complex through direct disruption of the ZEB2-LSD1 interaction or pharmacological inhibition of the LSD1 demethylase activity itself, could serve as a novel therapeutic strategy for this aggressive subtype of T-ALL, and possibly other ZEB2-driven malignancies. Disclosures No relevant conflicts of interest to declare.
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Teitell, M., M. F. Mescher, C. A. Olson, D. R. Littman, and M. Kronenberg. "The thymus leukemia antigen binds human and mouse CD8." Journal of Experimental Medicine 174, no. 5 (November 1, 1991): 1131–38. http://dx.doi.org/10.1084/jem.174.5.1131.
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The thymus leukemia antigen (TLA) is a class Ib, or 'nonclassical' class I molecule, one of several encoded within the Tla locus of the mouse major histocompatibility complex (MHC). It structurally resembles the H-2K, D, and L class I transplantation antigens, which present processed peptides to cytotoxic T lymphocytes (CTLs). Although their function(s) are unknown, there has been recent speculation concerning the possibility that class Ib molecules may present antigens to T cells that express gamma delta T cell antigen receptors (TCRs). In this report, using both a cell-cell adhesion assay and adhesion of T lymphocyte clones to purified plate-bound TLA, we provide evidence that TLA can bind to both human and mouse CD8. We also show that a chimeric class I molecule containing the peptide antigen binding site of Ld and the alpha 3 domain, transmembrane, and cytoplasmic segments of TLA, can support a CD8-dependent immune response by CTLs. These results demonstrate for the first time binding of a class Ib molecule to CD8 with a functional outcome, as is observed for the class I transplantation antigens. The capacity to interact with CD8 has been conserved despite the extensive sequence divergence of TLA in the peptide antigen binding site, suggesting this interaction is highly significant. TLA is expressed by epithelial cells in the mouse small intestine. As these epithelial cells are in close contact with intestinal intraepithelial lymphocytes that are nearly all CD8+, and many of which express the gamma delta TCR, the data are consistent with the hypothesis that TLA is involved in antigen presentation, perhaps to gamma delta-positive lymphocytes in this site.
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Jo, Stephanie Y., Eric M. Granowicz, and Jay L. Hess. "Assessment of DOT1L as a Therapeutic Target In Acute Leukemia." Blood 116, no. 21 (November 19, 2010): 3291. http://dx.doi.org/10.1182/blood.v116.21.3291.3291.
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Abstract Abstract 3291 Histone modifying enzymes are crucial regulators of hematopoiesis that are commonly disrupted in acute leukemia. DOT1L has emerged as a particularly important methyltransferase in leukemias with Mixed Lineage Leukemia (MLL) rearrangements. Leukemogenic MLL fusion proteins transform primarily through upregulation of A-cluster HOX genes, including HOXA9 and the HOX cofactor MEIS1. Many of the most common MLL translocation partners including the AF4 family members, AF9, ENL, and AF10, form the Elongation Assisting Proteins (EAP) complex that includes DOT1L. DOT1L is the only known histone methyltransferase that methylates histone H3 on lysine 79 (H3K79). Increasing evidence suggests this histone modification, which is generally associated with transcriptional activation, is essential for MLL fusion protein mediated oncogenicity. Chromatin immunoprecipitation (ChIP) on MLL fusion protein containing cell lines shows higher levels of H3K79 methylation across the HOXA9 and MEIS1 loci compared to non-MLL fusion protein containing cell lines. Similarly, patient samples with MLL fusion proteins show elevated H3K79 methylation. Finally, knockdown of DOT1L has been shown to inhibit growth of MLL rearranged cell lines. These findings suggest that DOT1L may be an effective therapeutic target, however further development of DOT1L inhibitors will be dependent on assessing the efficacy of DOT1L disruption in a wider range of leukemic cells as well as determining the potential toxicity and effect on normal hematopoiesis. Given that constitutive Dot1l knock out is early embryonic lethal, we established conditional Dot1l knockout mouse from gene trap sperm obtained from the Knock Out Mouse Project (KOMP). Dot1l targeted animals were crossed to CreER animals so that Dot1l knock out could be induced with 4-OHT or tamoxifen. To determine the effects of Dot1l deletion in transformation ability, mouse were injected with 5-flurouracil and bone marrow cells were transduced with retrovirus expressing oncogenes in the presence or absence of 4-OHT and growth in methocult media was examined. These experiments showed that growth of cells transformed by MLL-AF9 is completely abolished by Dot1l deletion while transformations by HOXA9/MEIS1 (downstream targets of MLL fusion proteins) and E2A-HLF (which expresses very low levels of HOXA9/MEIS1 and transform through other mechanisms) were unaffected. We also examined the toxicity of Dot1l deletion in vivo by treating mice with tamoxifen and monitoring survival and assessing hematopoiesis. Immunohistochemical studies show that loss of Dot1l is associated with widespread loss of lysine 79 di- and tri-methylation in organs including liver, spleen, bone marrow, testis, muscle and gastrointestinal track with some residual methylation retained in the central nervous system. Immunophenotypic analysis of mouse bone marrow 3–4 weeks after tamoxifen treatment revealed reduction in HSCs, GMPs, MEPs, and CLPs with Dot1l excision. Bone marrow transplantations and cell cycle experiments are currently underway to further characterize the hematopoietic defects in Dot1l deficient animals. Additional experiments will be needed to determine if Dot1l activity is required in other leukemias with high level HOX expression. Together these results suggest that Dot1l is a promising therapeutic target as it is specifically required for transformation by MLL fusion leukemia, however bone marrow suppression occurs with Dot1l inhibition. Disclosures: No relevant conflicts of interest to declare.
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Jacobelli, Jordan, Eric Wigton, and Scott B. Thompson. "Myosin-IIa Is Required for Leukemia Cell Extravasation and Its Inhibition Reduces Leukemia Dissemination and Prolongs Survival in a Mouse Model of Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 967. http://dx.doi.org/10.1182/blood.v124.21.967.967.
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Abstract Background: Leukemia affects approximately 45,000 people each year in the USA with more than 20,000 fatalities. Many leukemia patients experience initial remission but often relapse, with the relapsing leukemia affecting organs such as the central nervous system (CNS). The CNS acts as a sanctuary site allowing leukemia cells to escape treatments such as tyrosine kinase inhibitors (TKIs) and chemotherapy. Leukemia dissemination is a complex process requiring leukemia cells to exit the blood circulation by extravasation and invade target tissues. To extravasate, leukemia cells cross through vascular endothelial walls in a process called trans-endothelial migration, which requires cytoskeletal remodeling. However, the specific cytoskeletal effectors of leukemia extravasation are not fully known. Notably, leukemia dissemination correlates negatively with survival rates. Goal: Our goal was to validate Myosin-IIA, a class II myosin motor protein, as a molecular target to inhibit lymphoid leukemia cell extravasation and hinder leukemia dissemination, particularly infiltration into the CNS. Myosin-IIA has been shown to play a role in cell migration. We recently showed that this myosin is more profoundly required for activated lymphocyte entry into the CNS than for homeostatic entry into secondary lymphoid organs. This suggests that Myosin-IIA may be a promising candidate to prevent leukemia infiltration into the CNS without completely inhibiting homeostatic lymphocyte trafficking. Results: For this study, we used a mouse model of Bcr-Abl driven B cell acute lymphoblastic leukemia (B-ALL), which closely recapitulates Philadelphia chromosome positive human leukemias. We used shRNA interference to inhibit Myosin-IIA protein expression in the leukemia cells. Myosin-IIA depletion did not affect baseline apoptosis of the leukemia cells but did result in a small but significant reduction in their growth rate. Myosin-IIA was key in promoting leukemia cell migration in response to the chemokine CXCL12. Expression of Myosin-IIA was also critical for leukemia cells to complete trans-endothelial migration through brain-derived endothelial cells in an in vitro model of extravasation. In addition, our data suggested that inhibition of Myosin-IIA reduces the ability of leukemia cells to disseminate in vivo, including reducing leukemia infiltration into the CNS. Finally, compared to control leukemia cells, inhibition of Myosin-IIA significantly prolonged survival in an in vivo leukemia transfer model. Conclusion: Our data suggest that Myosin-IIA regulates leukemia migration thus making this myosin a promising target to inhibit leukemia dissemination in vivo, particularly into the CNS. Given the serious side-effects of cranial irradiation and (intrathecal or systemic) chemotherapy, the ability to inhibit CNS infiltration of leukemia cells can be a valuable therapeutic tool to improve the efficacy of current therapies by reducing the relapse frequency following TKI and chemotherapy treatments. Disclosures No relevant conflicts of interest to declare.
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Bennardo, Sara, Stefano Iacovelli, Stefania Gobessi, Mirza Suljagic, Daniel Bilbao, Julia Eckl-Dorna, Hongsheng Wang, et al. "The Nature of the Antigen Determines Leukemia Development and Behavior in the Eμ-TCL1 Transgenic Mouse Model of CLL." Blood 120, no. 21 (November 16, 2012): 181. http://dx.doi.org/10.1182/blood.v120.21.181.181.
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Abstract Abstract 181 Studies conducted over the past decade have revealed a strong association between the mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes and clinical course in patients with chronic lymphocytic leukemia (CLL). In patients with aggressive CLL, the leukemic cells typically express B cell receptors (BCRs) encoded by unmutated IGHV genes, whereas these genes are most often mutated in leukemic cells from patients with indolent disease. The mutational status of the IGHV genes reflects features of the antigen, such as antigen structure, form, presentation and affinity, indicating that the difference in the clinical course between IGHV-unmutated and IGHV-mutated CLL could be due to recognition of different types of antigens. In line with this possibility, recent studies have shown that IGHV-unmutated CLL (U-CLL) cells frequently express polyreactive BCRs that bind with low affinity to both microbial antigens and autoantigens translocated or exposed on apoptotic cells, whereas such reactivity is infrequent in IGHV-mutated CLL (M-CLL). To further explore the possibility that the clinical course in CLL is determined by the availability of particular types of antigenic stimuli, we investigated the impact of different antigen/BCR interactions on leukemia development and behavior in the Eμ-TCL1 transgenic mouse model of CLL. We initially established three cohorts of Eμ-TCL1 transgenic mice that expressed transgenic BCRs with different antigen specificity. Two of these cohorts expressed low-affinity unmutated transgenic BCRs reactive with the antigens phosphatidylcholine (PtC) and Sm (IgPtC and IgSm, respectively), whereas the third cohort expressed a high-affinity mutated transgenic BCR (IgHEL) specific for the antigen hen egg lysozyme (HEL). Of note, Sm is a ribonucleoprotein complex that is translocated to the surface of apoptotic cells and has been shown to be recognized by certain human U-CLL BCRs, whereas PtC is a cell membrane component that is exposed on senescent red blood cells and gut bacteria. Because no data are currently available regarding the reactivity of the M-CLL BCRs, we subdivided the cohort of Eμ-TCL1/IgHEL double transgenic mice into four additional cohorts. These included a cohort without antigen (Eμ-TCL1/IgHEL), a cohort in which HEL was provided as a foreign antigen (Eμ-TCL1/IgHEL double transgenic mice repetitively immunized with particles coated with HEL and CpG oligonucleotides), a cohort in which HEL was provided as a soluble autoantigen (Eμ-TCL1/IgHEL/sHEL triple transgenic mice) and a cohort in which HEL was provided as a membrane-bound autoantigen exposed on apoptotic cells (Eμ-TCL1/IgHEL/mHEL-KK triple transgenic mice). Each cohort consisted of 12–14 animals, of which at least 8 have been followed for >1 year. Animals from all cohorts developed CD5-positive B cell leukemias, but only in Eμ-TCL1/IgSm and Eμ-TCL1/IgPtC mice the leukemic cells expressed a transgenic BCR. In Eμ-TCL1/IgHEL mice the leukemias were always derived from the small percentage of B cells that express an endogenous BCR, whereas B cells that express the transgenic IgHEL BCR were never transformed. Interestingly, leukemia development and progression was more rapid in Eμ-TCL1/IgPtC than Eμ-TCL1/IgSm transgenic mice (7/14 at 6 months of age and 2/10 at 8 months of age, respectively). Since PtC is expressed as both a foreign- (gut flora) and self- (senescent red blood cells) antigen, we investigated whether suppression of gut flora will affect the growth of adoptively transferred Eμ-TCL1/IgPtC leukemias. Pretreatment of syngeneic recipient mice with a three-week course of broad-spectrum antibiotics significantly delayed leukemia growth, suggesting that PtC is more potent in driving the expansion of the leukemic clone when expressed as a foreign than self antigen. To summarize, these data demonstrate that U-CLL can be induced by both microbial antigens and autoantigens exposed on apoptotic cells, including autoantigens that are recognized by human CLL cells, such as Sm. In contrast, M-CLL can not be induced by chronic or repetitive antigen stimulation, regardless whether the antigen is provided as a foreign antigen, as a soluble autoantigen, or as a membrane-bound autoantigen exposed on apoptotic cells. Collectively, these data suggest that the mechanisms that drive U-CLL and M-CLL are different and indicate that only U-CLL is an antigen-driven disease. Disclosures: No relevant conflicts of interest to declare.
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Lui, Wing Chi, Yuen Fan Chan, and Ray Ng. "PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia." Blood 124, no. 21 (December 6, 2014): 3493. http://dx.doi.org/10.1182/blood.v124.21.3493.3493.
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Abstract In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear. Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. Disclosures No relevant conflicts of interest to declare.
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Bernt, Kathrin M., Nan Zhu, Joerg Faber, Natalie Punt, Roy M. Pollock, Victoria M. Richon, Andrew L. Kung, and Scott Armstrong. "Demonstration of a Role for Dot1l In MLL-Rearranged Leukemia Using a Conditional Loss of Function Model." Blood 116, no. 21 (November 19, 2010): 62. http://dx.doi.org/10.1182/blood.v116.21.62.62.
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Abstract Abstract 62 Leukemias associated with translocations of the Mixed Lineage Leukemia (MLL) gene account for a significant percentage of both AML and ALL, and often carry a poor prognosis. The exact molecular mechanisms by which MLL-fusion proteins transform cells are incompletely understood. One proposed model involves the aberrant activation of transcriptional programs through epigenetic changes that ultimately lead to leukemogenesis. The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been shown to be recruited by the most common MLL fusion proteins, and MLL fusion protein target loci are associated with H3K79 methylation (H3K79me2/3) in mouse models and MLL-rearranged human leukemia samples. However, it is currently unclear whether H3K79 methylation is indeed a necessary step in leukemogenesis. We sought to investigate in detail the importance of Dot1l in MLL-fusion mediated leukemia, using an shRNA approach and a newly developed conditional loss of function mouse model of Dot1l (Dot1lflox/flox). shRNA mediated suppression of Dot1l in a panel of MLL-rearranged human leukemia cell lines led to a decrease in growth rate and viability, induction of apopotosis and cell cycle arrest. Bioluminescent in vivo tracking of MLL-rearranged human leukemia cell lines in xenotransplant recipients revealed that the onset of leukemia was significantly delayed after Dot1l suppression. To complement and confirm the sh-RNA results, we developed a conditional loss of function mouse model for Dot1l. In this model, deletion of the active site of Dot1l severely impaired or abrogated serial replating of Mll-Af9 transduced lineage negative (lin-) cells, and fully developed Mll-Af9 leukemia cells. Immunoblot of total cellular H3K79 and chromatin immunoprecipitation of known MLL target loci showed loss of H3K79 methylation. In addition, we observed variable induction of additional silencing mechanism such as Polycomb Repressor Complex 2 (PRC2) mediated H3K27 methylation on selected loci such as HoxA10. These epigenetic changes correlated with a reduction in expression of known MLL-fusion downstream targets. When Dot1l was inactivated in Mll-Af9 leukemia cells, in vitro colony and cell morphology demonstrated loss of blast-like phenotype and induction of differentiation. Furthermore, mice transplanted with Mll-Af9 leukemia cells from primary recipients failed to induce leukemia in secondary recipients after cre-mediated deletion of Dot1l. The role of Dot1l in normal hematopoiesis is not defined, and it is possible that deletion of Dot1l induces catastrophic collapse of the entire hematopoietic system, including leukemia cell compartments. To exclude this possibility, we are analyzing the phenotype of hematopoiesis specific deletion of Dot1l in normal mice through the use of the Vav-cre system. Initial results show that Dot1lflox/flox-Vav-Cre mice are born at mendelian ratios and display neutrophil and lymphocyte counts at the lower limit of normal despite near complete absence of H3K79 methylation in peripheral blood leukocytes. A more detailed analysis of the hematopoietic phenotype of loss of Dot1l, including hematopoietic stem cell compartments, is currently ongoing in our laboratory. These data demonstrate that Dot1l is indeed a central player in MLL-fusion mediated leukemogenesis and required for leukemia cell survival. The low-normal Dot1l-/- neutrophil and lymphocyte counts observed in the Dot1lflox/flox-Vav-Cre mice suggest that Dot1l is more critical for leukemia cells than normal hematopoietic cells. This predicts a therapeutic window for pharmacologic inhibitors of Dot1l, and highlights their potential as targeted therapy for MLL-rearranged leukemias. Disclosures: Pollock: Epizyme, Inc: Employment. Richon:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy.
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Gonder, Susanne, Anne Largeot, Ernesto Gargiulo, Sandrine Pierson, Iria Fernandez Botana, Giulia Pagano, Jerome Paggetti, and Etienne Moussay. "The Tumor Microenvironment-Dependent Transcription Factors AHR and HIF-1α Are Dispensable for Leukemogenesis in the Eµ-TCL1 Mouse Model of Chronic Lymphocytic Leukemia." Cancers 13, no. 18 (September 8, 2021): 4518. http://dx.doi.org/10.3390/cancers13184518.
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Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in the elderly and is characterized by the accumulation of mature B lymphocytes in peripheral blood and primary lymphoid organs. In order to proliferate, leukemic cells are highly dependent on complex interactions with their microenvironment in proliferative niches. Not only soluble factors and BCR stimulation are important for their survival and proliferation, but also the activation of transcription factors through different signaling pathways. The aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF)-1α are two transcription factors crucial for cancer development, whose activities are dependent on tumor microenvironment conditions, such as the presence of metabolites from the tryptophan pathway and hypoxia, respectively. In this study, we addressed the potential role of AHR and HIF-1α in chronic lymphocytic leukemia (CLL) development in vivo. To this end, we crossed the CLL mouse model Eµ-TCL1 with the corresponding transcription factor-conditional knock-out mice to delete one or both transcription factors in CD19+ B cells only. Despite AHR and HIF-1α being activated in CLL cells, deletion of either or both of them had no impact on CLL progression or survival in vivo, suggesting that these transcription factors are not crucial for leukemogenesis in CLL.
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Funk, Ryan K., Taylor J. Maxwell, Masayo Izumi, Deepa Edwin, Friederike Kreisel, Timothy J. Ley, James M. Cheverud, and Timothy A. Graubert. "Quantitative trait loci associated with susceptibility to therapy-related acute murine promyelocytic leukemia in hCG-PML/RARA transgenic mice." Blood 112, no. 4 (August 15, 2008): 1434–42. http://dx.doi.org/10.1182/blood-2008-01-132084.
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Abstract Therapy-related acute myelogenous leukemia (t-AML) is an important late adverse effect of alkylator chemotherapy. Susceptibility to t-AML has a genetic component, yet specific genetic variants that influence susceptibility are poorly understood. We analyzed an F2 intercross (n = 282 mice) between mouse strains resistant or susceptible to t-AML induced by the alkylator ethyl-N-nitrosourea (ENU) to identify genes that regulate t-AML susceptibility. Each mouse carried the hCG-PML/RARA transgene, a well-characterized initiator of myeloid leukemia. In the absence of ENU treatment, transgenic F2 mice developed leukemia with higher incidence (79.4% vs 12.5%) and at earlier time points (108 days vs 234 days) than mice in the resistant background. ENU treatment of F2 mice further increased incidence (90.4%) and shortened median survival (171 vs 254 days). We genotyped F2 mice at 384 informative single nucleotide polymorphisms across the genome and performed quantitative trait locus (QTL) analysis. Thirteen QTLs significantly associated with leukemia-free survival, spleen weight, or white blood cell count were identified on 8 chromosomes. These results suggest that susceptibility to ENU-induced leukemia in mice is a complex trait governed by genes at multiple loci. Improved understanding of genetic risk factors should lead to tailored treatment regimens that reduce risk for patients predisposed to t-AML.
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Baccelli, Irene, Yves Gareau, Bernhard Lehnertz, Gingras Stephane, Jean-Francois Spinella, Alexandre Beautrait, Sophie Corneau, et al. "Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of Mitochondrial Vulnerability in Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 910. http://dx.doi.org/10.1182/blood-2018-99-114525.
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Abstract BACKGROUND: 60% to 70% of Acute Myeloid Leukemia (AML) patients enter complete remission after induction regimen, but the majority relapse within 3 years due to the outgrowth of therapy resistant Leukemia Stem Cells (LSCs). Identification of novel treatment strategies effective against these cells thus represents an outstanding medical need. We developed a cell culture method, which transiently maintains LSC activity ex vivo (Pabst et al., Nature Methods, 2014) and enables chemical interrogation of cell types relevant for the progression of the disease. Overall, HSCs and LSCs share numerous biological traits, making specific LSC eradication challenging. However, striking differences in energy metabolism between normal and leukemic stem cells have recently been suggested. While HSCs appear to rely primarily on anaerobic glycolysis for energy production, LSCs seem to depend on mitochondrial oxidative phosphorylation for their survival. Targeting mitochondrial respiration could therefore represent an effective approach for the specific eradication of LSCs. AIM: We aimed to identify novel therapeutic targets for AMLs with poor treatment outcome. The study relied on the Leucegene approach that integrates results generated by RNA sequencing analysis of primary human AML specimens, detailed clinical and cytogenetic annotations provided by the Quebec leukemia cell bank and ex vivo responses of primary AML samples to various chemical compounds. Our study specifically focused on specimens originating from patients with poor (overall survival < 3 years) and good (overall survival ≥ 3 years) response to standard chemotherapy, and did not include cases of Acute Promyelocytic Leukemia (APL). RESULTS: We identified Mubritinib, previously described as an ERBB2 inhibitor, as a novel anti-leukemic agent, which selectively inhibits the viability of leukemic cells from therapy-resistant AML patients, but does not affect normal CD34+ cord blood cells. Exposure to Mubritinib triggered apoptotic cell death in a subset of AML samples with high mitochondrial function-related gene expression, high relapse rates, and short overall survival. Sensitivity to Mubritinib also strongly associated with the intermediate cytogenetic risk category, normal karyotype (NK), and NPM1, FLT3 (ITD) and DNMT3A mutations. Conversely, resistance to Mubritinib associated with favorable cytogenetic risk AMLs, Core Binging Factor (CBF) leukemias and KIT mutations. Mubritinib has been developed as an ERBB2 kinase inhibitor. Intriguingly, we found that ERBB2 is not expressed in Mubritinib-sensitive AML specimens, suggesting that the anti-leukemic activity of this compound is likely not mediated by ERBB2 inhibition. Using a combination of functional genomics and biochemical analyses, we demonstrated that Mubritinib directly inhibits the mitochondrial Electron Transport Chain (ETC) complex I, which leads to a decrease in oxidative phosphorylation activity and to induction of oxidative stress. The impact of Mubritinib on AML progression was explored using a syngeneic mouse model (MLL-AF9 tdTomato-positive leukemia). Recipients of MLL-AF9 cells treated with Mubritinib exhibited a 19-fold decrease in the number of tdTomato-positive cells in the bone marrow and a 42-fold decrease in the spleens compared to control mice. Short-term treatment also led to a 37% increase in the median overall survival of Mubritinib exposed recipients compared to vehicle treated mice. Importantly, and in agreement with our observation that Mubritinib treatment does not impede proliferation of normal hematopoietic CD34+ cells in vitro, Mubritinib treatment had no impact on the number of non-transduced (tdTomato negative) nucleated bone marrow cells of recipients. CONCLUSIONS: We uncovered the clinical, mutational, and transcriptional landscape of mitochondrial vulnerability in AML and identified Mubritinib as a novel ETC complex I inhibitor with therapeutic potential for approximately 30% of AML cases currently lacking effective treatment options. As Mubritinib completed a phase I clinical trial in the context of ERBB2-positive solid tumors, our work suggests an opportunity to re-purpose Mubritinib's usage for this genetically distinct subgroup of poor outcome AML patients. Disclosures No relevant conflicts of interest to declare.
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Wunderlich, Mark, Ondrej Krejci, Junping Wei, Thomas X. Lu, and James C. Mulloy. "Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability." Blood 106, no. 11 (November 16, 2005): 1379. http://dx.doi.org/10.1182/blood.v106.11.1379.1379.
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Abstract The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.
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Kim, Jun Hyun, Alexander V. Penson, Barry S. Taylor, and John H. J. Petrini. "Nbn−Mre11 interaction is required for tumor suppression and genomic integrity." Proceedings of the National Academy of Sciences 116, no. 30 (July 8, 2019): 15178–83. http://dx.doi.org/10.1073/pnas.1905305116.
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We derived a mouse model in which a mutant form of Nbn/Nbs1mid8 (hereafter Nbnmid8) exhibits severely impaired binding to the Mre11−Rad50 core of the Mre11 complex. The Nbnmid8 allele was expressed exclusively in hematopoietic lineages (in Nbn−/mid8vav mice). Unlike Nbnflox/floxvav mice with Nbn deficiency in the bone marrow, Nbn−/mid8vav mice were viable. Nbn−/mid8vav mice hematopoiesis was profoundly defective, exhibiting reduced cellularity of thymus and bone marrow, and stage-specific blockage of B cell development. Within 6 mo, Nbn−/mid8 mice developed highly penetrant T cell leukemias. Nbn−/mid8vav leukemias recapitulated mutational features of human T cell acute lymphoblastic leukemia (T-ALL), containing mutations in NOTCH1, TP53, BCL6, BCOR, and IKZF1, suggesting that Nbnmid8 mice may provide a venue to examine the relationship between the Mre11 complex and oncogene activation in the hematopoietic compartment. Genomic analysis of Nbn−/mid8vav malignancies showed focal amplification of 9qA2, causing overexpression of MRE11 and CHK1. We propose that overexpression of MRE11 compensates for the metastable Mre11−Nbnmid8 interaction, and that selective pressure for overexpression reflects the essential role of Nbn in promoting assembly and activity of the Mre11 complex.
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Sørensen, Karina Dalsgaard, Leticia Quintanilla-Martinez, Sandra Kunder, Jörg Schmidt, and Finn Skou Pedersen. "Mutation of All Runx (AML1/Core) Sites in the Enhancer of T-Lymphomagenic SL3-3 Murine Leukemia Virus Unmasks a Significant Potential for Myeloid Leukemia Induction and Favors Enhancer Evolution toward Induction of Other Disease Patterns." Journal of Virology 78, no. 23 (December 1, 2004): 13216–31. http://dx.doi.org/10.1128/jvi.78.23.13216-13231.2004.
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ABSTRACT SL3-3 murine leukemia virus is a potent inducer of T-lymphomas in mice. Using inbred NMRI mice, it was previously reported that a mutant of SL3-3 with all enhancer Runx (AML1/core) sites disrupted by 3-bp mutations (SL3-3dm) induces predominantly non-T-cell tumors with severely extended latency (S. Ethelberg, J. Lovmand, J. Schmidt, A. Luz, and F. S. Pedersen, J. Virol. 71:7273-7280, 1997). By use of three-color flow cytometry and molecular and histopathological analyses, we have now performed a detailed phenotypic characterization of SL3-3- and SL3-3dm-induced tumors in this mouse strain. All wild-type induced tumors had clonal T-cell receptor β rearrangements, and the vast majority were CD3+ CD4+ CD8− T-lymphomas. Such a consistent phenotypic pattern is unusual for murine leukemia virus-induced T-lymphomas. The mutant virus induced malignancies of four distinct hematopoietic lineages: myeloid, T lymphoid, B lymphoid, and erythroid. The most common disease was myeloid leukemia with maturation. Thus, mutation of all Runx motifs in the enhancer of SL3-3 severely impedes viral T-lymphomagenicity and thereby discloses a considerable and formerly unappreciated potential of this virus for myeloid leukemia induction. Proviral enhancers with complex structural alterations (deletions, insertions, and/or duplications) were found in most SL3-3dm-induced T-lymphoid tumors and immature myeloid leukemias but not in any cases of myeloid leukemia with maturation, mature B-lymphoma, or erythroleukemia. Altogether, our results indicate that the SL3-3dm enhancer in itself promotes induction of myeloid leukemia with maturation but that structural changes may arise in vivo and redirect viral disease specificity to induction of T-lymphoid or immature myeloid leukemias, which typically develop with moderately shorter latencies.
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Brandt, Nina, Hayley M. O'Neill, Maximilian Kleinert, Peter Schjerling, Erik Vernet, Gregory R. Steinberg, Erik A. Richter, and Sebastian B. Jørgensen. "Leukemia inhibitory factor increases glucose uptake in mouse skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 309, no. 2 (July 15, 2015): E142—E153. http://dx.doi.org/10.1152/ajpendo.00313.2014.
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Members of the IL-6 family, IL-6 and ciliary neurotrophic factor (CNTF), have been shown to increase glucose uptake and fatty acid oxidation in skeletal muscle. However, the metabolic effects of another family member, leukemia inhibitory factor (LIF), are not well characterized. Effects of LIF on skeletal muscle glucose uptake and palmitate oxidation and signaling were investigated in ex vivo incubated mouse soleus and EDL muscles from muscle-specific AMPKα2 kinase-dead, muscle-specific SOCS3 knockout, and lean and high-fat-fed mice. Inhibitors were used to investigate involvement of specific signaling pathways. LIF increased muscle glucose uptake in dose (50-5,000 pM/l) and time-dependent manners with maximal effects at the 30-min time point. LIF increased Akt Ser473 phosphorylation (P) in soleus and EDL, whereas AMPK Thr172 P was unaffected. Incubation with parthenolide abolished LIF-induced glucose uptake and STAT3 Tyr705 P, whereas incubation with LY-294002 and wortmannin suppressed both basal and LIF-induced glucose uptake and Akt Ser473 P, indicating that JAK and PI 3-kinase signaling is required for LIF-stimulated glucose uptake. Incubation with rapamycin and AZD8055 indicated that mammalian target of rapamycin complex (mTORC)2, but not mTORC1, also is required for LIF-stimulated glucose uptake. In contrast to CNTF, LIF stimulation did not alter palmitate oxidation. LIF-stimulated glucose uptake was maintained in EDL from obese insulin-resistant mice, whereas soleus developed LIF resistance. Lack of SOCS3 and AMPKα2 did not affect LIF-stimulated glucose uptake. In conclusion, LIF acutely increased muscle glucose uptake by a mechanism potentially involving the PI 3-kinase/mTORC2/Akt pathway and is not impaired in EDL muscle from obese insulin-resistant mice.
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Miyazawa, M., J. Nishio, K. Wehrly, C. S. David, and B. Chesebro. "Spontaneous recovery from Friend retrovirus-induced leukemia. Mapping of the Rfv-2 gene in the Q/TL region of mouse MHC." Journal of Immunology 148, no. 6 (March 15, 1992): 1964–67. http://dx.doi.org/10.4049/jimmunol.148.6.1964.
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Abstract The Rfv-2 gene that influences the rate of spontaneous recovery from erythroleukemia induced by a low dose of Friend retrovirus complex was mapped to the Q/TL region of mouse MHC. Rfv-2 was physically and functionally distinct from the I-A-linked Ir gene that has been shown to control the responsiveness of Th cells to the envelope glycoprotein of Friend murine leukemia helper virus. The negative effect of the Rfv-2s allele was overcome by the B10.D2-H-2dm1 mutation of the D-L genes of H-2, suggesting functional similarities between the D-L and Q/TL genes in influencing resistance against Friend murine leukemia retrovirus complex infection or possible modification of Q/TL expression by genes in the D-L region.
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Chaise, Coralie, Sarah L. Buchan, Jason Rice, Jeanine Marquet, Hélène Rouard, Mathieu Kuentz, Gisella E. Vittes, et al. "DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance." Blood 112, no. 7 (October 1, 2008): 2956–64. http://dx.doi.org/10.1182/blood-2008-02-137695.
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Abstract The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I–binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2–restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT137–45–specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT137–45, have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.
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Wang, Yiqian, Kira Hannon, Lisa Richter, Michelle Becker, Lisa Garett, Cecilia Rivas, Lemlem Alemu, Ling Zhao, Pu Paul Liu, and R. Katherine Hyde. "RUNX1 and CBFB-MYH11 Are Required for the Maintenance of Inv(16) AML." Blood 128, no. 22 (December 2, 2016): 2715. http://dx.doi.org/10.1182/blood.v128.22.2715.2715.
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Abstract The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites (CbfbflMYH11), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the CbfbflMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb+/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb+/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in Csf2rb- cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced CbfbflMYH11leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the Csf2rb-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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Richter, Lisa, Yiqian Wang, Michelle Becker, and R. Katherine Hyde. "HDAC1 Cooperates with CBFβ-SMMHC in Regulating Gene Expression in Inversion 16 Acute Myeloid Leukemia." Blood 128, no. 22 (December 2, 2016): 2690. http://dx.doi.org/10.1182/blood.v128.22.2690.2690.
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Abstract The fusion of the genes for core binding factor beta and smooth muscle myosin heavy chain (CBFB-MYH11) is the recurrent mutation found in inversion 16 (inv(16)) acute myeloid leukemia (AML). The expressed fusion protein, CBFβ-SMMHC, binds to the transcriptional regulator RUNX1, and this interaction is required for leukemogenesis. Recent data shows CBFβ-SMMHC and RUNX1 are associated with promoters of both transcribed and repressed genes, implying that the CBFβ-SMMHC:RUNX1 complex directly regulates target gene expression. However, it is not known whether other transcriptional co-factors are also required for this activity. Histone deacetylase 1 (HDAC1) removes acetyl groups from histone tails to regulate the accessibility of chromatin to transcriptional machinery. It is recruited to chromatin by transcription factors, including RUNX1. HDAC1 also colocalizes with RUNX1 and CBFβ-SMMHC to promoter regions in ME-1 cells, a human inv(16) cell line. Based on this, we hypothesized that HDAC1 could bind to the RUNX1: CBFβ-SMMHC complex and plays a role in transcriptional regulation in inv(16). To test if CBFβ-SMMHC and HDAC1 form a complex, we transfected COS-7 cells with expression plasmids for HDAC1-FLAG and CBFβ-SMMHC and performed immunoprecipitations (IP) with nuclear extracts. IP with anti-MYH11 showed an interaction between HDAC1 and CBFβ-SMMHC, as did IP with anti-FLAG. Importantly, we found that HDAC1 and CBFβ-SMMHC co-immunoprecipitate in mouse leukemia cells from our knockin model which expresses CBFβ-SMMHC from the endogenous CBFβ promoter (CBFβ-SMMHC+). Confirming the specificity of this interaction, we found that IP with anti-MYH11 in ME-1 cells shows HDAC1 interaction, but the same IP in the t(8;21) AML cell line Kasumi-1 did not show HDAC1 interaction even though similar levels of HDAC1 are expressed. We next tested whether RUNX1 mediates the interaction between CBFβ-SMMHC and HDAC1. We performed IP experiments using a CBFβ-SMMHC mutant lacking RUNX1 binding (CBFβ-SMMHCN63K,N104K,Δ179-221). This mutant was co-immunoprecipitated with HDAC1, but not RUNX1, indicating that CBFβ-SMMHC's interaction with HDAC1 does not require RUNX1. We tested a construct lacking the c-terminal 95 amino acids, CBFβ-SMMHCΔC95, the domain known to interact with the related protein, HDAC8. We found that HDAC1 immunoprecipitated with CBFβ-SMMHCΔC95, suggesting that HDAC1 binds to a unique region of CBFβ-SMMHC. To test if HDAC1 plays a role in CBFβ-SMMHC-mediated gene expression, we performed chromatin immunoprecipitations on mouse CBFβ-SMMHC+ primary leukemia cells with antibodies against HDAC1, RUNX1, and MYH11, followed by real-time PCR for the promoter regions of three CBFβ-SMMHC target genes: MPO, CSF1R, and CEBPD. We observed all three proteins enriched on the target gene promoters as compared to immunoglobulin controls. This indicates that HDAC1 localizes with CBFβ-SMMHC and RUNX1 on target gene promoters in mouse primary leukemia cells. To test if HDAC1 is required for expression of these target genes, we used shRNA to knockdown Hdac1 expression. Mouse CBFβ-SMMHC+ leukemia cells were transduced with one of 2 different shRNAs against Hdac1 or with a control construct. We found that expression of all three genes was decreased with Hdac1knockdown, implying that HDAC1 is required for CBFβ-SMMHC induced changes in gene expression. These results also suggest that HDAC1 may have a role in transcriptional activation for certain genes, which is in contrast to its traditional role as a transcriptional repressor. These findings imply that HDAC1 activity is required for the maintenance of CBFβ-SMMHC expressing leukemia cells, and that HDAC1 inhibitors may be effective against inv(16) AML. To test this possibility we performed colony-forming assays using mouse leukemia cells grown in the presence of two different HDAC inhibitors, entinostat which is specific for HDAC1, and vorinostat, a nonspecific HDAC inhibitor. Our preliminary results indicate that both entinostat and vorinostat reduce the ability of primary CBFβ-SMMHC+ mouse leukemia cells to form colonies as compared to the vehicle control, while having minimal effects on growth of normal hematopoietic cells. In summary, we demonstrated that HDAC1 forms a complex with CBFβ-SMMHC and is required for its regulation of target gene expression, and that HDAC inhibitors may be effective for the treatment of inv(16) AML patients. Disclosures No relevant conflicts of interest to declare.
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Reinart, Nina, Malgorzata Ciesla, Cornelia Rudolph, Astrid Stein, Guenter Krause, Brigitte Schlegelberger, Michael Hallek, and Guenter Fingerle-Rowson. "Macrophage Migration Inhibitory Factor (MIF) Promotes the Development of Murine Chronic Lymphocytic Leukemia (CLL)." Blood 112, no. 11 (November 16, 2008): 27. http://dx.doi.org/10.1182/blood.v112.11.27.27.
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Abstract Introduction: Tumor formation results from a complex interplay between genetic/epigenetic alterations, cell cycle dysregulation and promotion by the tumor environment. Stimulation by extracellular survival factors is important for chronic lymphocytic leukemia (CLL), since the leukemic cells undergo spontaneous apoptosis when removed from their normal milieu. Since preliminary experiments demonstrated that macrophage migration inhibitory factor (MIF), a chemokine-like proinflammatory mediator and an intracellular regulator of growth and apoptosis, is overexpressed in human CLL, we investigated whether MIF participates in the pathogenesis of murine CLL. Methods: We studied the role of MIF in CLL by crossing the Eμ-TCL1-transgenic mouse model with MIF knockout (MIF−/−) mice. B-cell-specific overexpression of T cell leukemia-1 (TCL1) leads to accumulation and proliferation of IgM+/CD5+ mature B-cells via activation of AKT. This results in a CLL-like disease with peripheral lymphocytic leukemia, lymphadenopathy, splenomegaly, BM infiltration and premature death after 8–15 months. TCL1+/wtMIF−/− and TCL1+/wtMIF+/+ mice were compared with respect to leukemia development, tumor burden, cytogenetics and survival. Results: The MIF receptors CD74/CD44 and CXCR2 are expressed on murine B-cells. TCL1+/wtMIF+/+ mice exhibited increased numbers of IgM+/CD5+ B-cells already in the preleukemic phase at month 3 and developed overt leukemia (WBC > 20G/l) 3 months earlier than their MIF−/− counterparts (p = 0.02). Leukemia load at 12 months of age as measured by hepatosplenomegaly was increased in TCL1+/wtMIF+/+ animals and lymphatic organs were densely infiltrated by small, mature lymphocytes. The accelerated disease progression in the presence of MIF translated into a median survival which was 60 days shorter than in the absence of MIF (TCL1+/wtMIF+/+ 400 days, TCL1+/wtMIF−/− 460 days, p = 0.04). SKY analysis in leukemic splenocytes yielded various complex genetic aberrations with trisomies (e.g. +15), tetraploidy, translocations and deletions. Overexpression of tp53 due to the presence of an inactivating mutation in the p53 gene was found more frequently in TCL1+/wtMIF+/+ than in TCL1+/wtMIF−/− animals. Although the rates of DNA-damage-induced apoptosis in pre-leukemic and leukemic mice ex vivo were not significantly different between the genotypes, this defect in the p53-dependent apoptosis pathway corresponded with a reduced rate of spontaneous apoptosis in spleens of leukemic TCL1+/wtMIF+/+ animals. Conclusions: Our experience with the Eμ-TCL-1-transgenic mice shows that this model is suitable for the identification of novel regulators of CLL-like disease. We provide genetic proof that MIF acts to promote the early preleukemic and the leukemic phase of TCL1-induced CLL and thereby identify MIF as a novel regulator of CLL pathogenesis. Ongoing efforts are focussing on further characterizing the differences in pathology, the activation of the AKT pathway and cell cycle control between TCL1+/wtMIF−/− and TCL1+/wtMIF+/+ mice.
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Liu, Na, Junhong Song, Yangyang Xie, Xiao-Lin Wang, Bowen Rong, Na Man, Meng-Meng Zhang, et al. "The Three E Proteins Define a Heterogeneity of the AML1-ETO-Containing Transcription Factor Complex (AETFC) and Differentially Regulate t(8;21) Leukemogenesis." Blood 132, Supplement 1 (November 29, 2018): 5247. http://dx.doi.org/10.1182/blood-2018-99-112372.
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Abstract The leukemogenic AML1-ETO fusion protein is produced by the t(8;21) translocation, which is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AETFC, that contains multiple transcription factors and cofactors. Among these AETFC components, E2A (also known as TCF3) and HEB (also known as TCF12), two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA (E-box) binding capacity to AETFC, and are functionally essential for leukemogenesis. However, we find that the third E protein, E2-2 (also known as TCF4), is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the basic helix-loop-helix (bHLH) DNA-binding domain of E2-2. Gene expression profiling and ChIP-seq analysis reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, consistent with the fact that E2-2 is a critical transcription factor in dendritic cell (DC) development, our studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with DC differentiation, and that restoration of E2-2 triggers a partial differentiation of the AML1-ETO-expressing leukemic cells into the DC lineage. Meanwhile, E2-2, but not E2A or HEB, represses MYC target genes, which may also contribute to leukemic cell differentiation and apoptosis. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates the development of leukemia. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a molecular heterogeneity of AETFC, which merits further study in different t(8;21) AML patients, as well as in its potential regulation of cellular heterogeneity of AML. These studies should improve our understanding of the precise mechanism of leukemogenesis and assist development of diagnostic and therapeutic strategies. Disclosures No relevant conflicts of interest to declare.
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Tomida, Mikio, Toshio Heike, and Takashi Yokota. "Cytoplasmic Domains of the Leukemia Inhibitory Factor Receptor Required for STAT3 Activation, Differentiation, and Growth Arrest of Myeloid Leukemic Cells." Blood 93, no. 6 (March 15, 1999): 1934–41. http://dx.doi.org/10.1182/blood.v93.6.1934.406k05_1934_1941.
Full textAbstract:
Leukemia inhibitory factor (LIF) induces growth arrest and macrophage differentiation of mouse myeloid leukemic cells through the functional LIF receptor (LIFR), which comprises a heterodimeric complex of the LIFR subunit and gp130. To identify the regions within the cytoplasmic domain of LIFR that generate the signals for growth arrest, macrophage differentiation, and STAT3 activation independently of gp130, we constructed chimeric receptors by linking the transmembrane and intracellular regions of mouse LIFR to the extracellular domains of the human granulocyte macrophage colony-stimulating factor receptor (hGM-CSFR) and βc chains. Using the full-length cytoplasmic domain and mutants with progressive C-terminal truncations or point mutations, we show that the two membrane-distal tyrosines with the YXXQ motif of LIFR are critical not only for STAT3 activation, but also for growth arrest and differentiation of WEHI-3B D+ cells. A truncated STAT3, which acts in a dominant negative manner was introduced into WEHI-3B D+ cells expressing GM-CSFR-LIFR and GM-CSFRβc-LIFR. These cells were not induced to differentiate by hGM-CSF. The results indicate that STAT3 plays essential roles in the signals for growth arrest and differentiation mediated through LIFR.
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Leslie, K. B., F. Lee, and J. W. Schrader. "Intracisternal A-type particle-mediated activations of cytokine genes in a murine myelomonocytic leukemia: generation of functional cytokine mRNAs by retroviral splicing events." Molecular and Cellular Biology 11, no. 11 (November 1991): 5562–70. http://dx.doi.org/10.1128/mcb.11.11.5562-5570.1991.
Full textAbstract:
Previously we have described the derivation of three distinct classes of leukemic cell clones from a single in vivo-passaged myelomonocytic leukemia, WEHI-274, that arose in a mouse infected with the Abelson leukemia virus/Moloney leukemia virus complex (K. B. Leslie and J. W. Schrader, Mol. Cell. Biol. 9:2414-2423, 1989). The three classes of cell clones were characterized by distinct patterns of growth in vitro, the production of cytokines, and the presence of cytokine gene rearrangements. However, all three classes of WEHI-274 clones bore a common rearrangement of the c-myb gene, suggesting that all were derived from the one ancestral cell and that at least three distinct and independent autostimulatory events were involved in the progression of a single myeloid leukemic disease. In this article, we demonstrate that the autocrine growth factor production by the WEHI-274 leukemic clones resulted from cytokine gene activations mediated by the insertion of an intracisternal A-type particle (IAP) sequence 5' to the interleukin-3 (IL-3) gene, in the case of the class I clone, or 5' to the gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), in the case of the class II clones. IAPs are defective murine retroviruses encoded by endogenous genetic elements which may undergo transpositions and act as endogenous mutagens. The functional IL-3 and GM-CSF mRNAs were generated by mechanisms in which the splice donor apparatus of the IAP sequence has been used in IAP gag-to-IL-3 or -GM-CSF splicing events.
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Leslie, K. B., F. Lee, and J. W. Schrader. "Intracisternal A-type particle-mediated activations of cytokine genes in a murine myelomonocytic leukemia: generation of functional cytokine mRNAs by retroviral splicing events." Molecular and Cellular Biology 11, no. 11 (November 1991): 5562–70. http://dx.doi.org/10.1128/mcb.11.11.5562.
Full textAbstract:
Previously we have described the derivation of three distinct classes of leukemic cell clones from a single in vivo-passaged myelomonocytic leukemia, WEHI-274, that arose in a mouse infected with the Abelson leukemia virus/Moloney leukemia virus complex (K. B. Leslie and J. W. Schrader, Mol. Cell. Biol. 9:2414-2423, 1989). The three classes of cell clones were characterized by distinct patterns of growth in vitro, the production of cytokines, and the presence of cytokine gene rearrangements. However, all three classes of WEHI-274 clones bore a common rearrangement of the c-myb gene, suggesting that all were derived from the one ancestral cell and that at least three distinct and independent autostimulatory events were involved in the progression of a single myeloid leukemic disease. In this article, we demonstrate that the autocrine growth factor production by the WEHI-274 leukemic clones resulted from cytokine gene activations mediated by the insertion of an intracisternal A-type particle (IAP) sequence 5' to the interleukin-3 (IL-3) gene, in the case of the class I clone, or 5' to the gene for granulocyte-macrophage colony-stimulating factor (GM-CSF), in the case of the class II clones. IAPs are defective murine retroviruses encoded by endogenous genetic elements which may undergo transpositions and act as endogenous mutagens. The functional IL-3 and GM-CSF mRNAs were generated by mechanisms in which the splice donor apparatus of the IAP sequence has been used in IAP gag-to-IL-3 or -GM-CSF splicing events.
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Huang, Gang, Xiaomei Yan, Xinghui Zhao, Yalan Rao, Goro Sashida, and Rajeana Bowie. "Differential MLL Interaction and H3K4me3 Mark Maintenance at PU.1 Regulatory Region by AML Translocations-Associated Oncoproteins AML1-ETO and CBFβ-SMMHC." Blood 114, no. 22 (November 20, 2009): 674. http://dx.doi.org/10.1182/blood.v114.22.674.674.
Full textAbstract:
Abstract Abstract 674 The interplay between genetic and epigenetic regulators is a fundamental mechanism to ensure gene regulation and cell fate decision. Hematopoiesis and leukemia are excellent systems in which to study this process. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcription factor RUNX1 (AML1)/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. PU.1 is a critical downstream target gene for AML1 in adult hematopoiesis. AML1 regulated PU.1 through 3 AML1 binding sites in the PU.1 URE region (1). AML1 is responsible for the H3K4me3 mark at PU.1 URE and promoter region. AML1-ETO represses PU.1 expression through PU.1 URE region (2). Dysregulation of PU.1 levels cause leukemia in both mouse and human. PU.1 is absolutely required for the normal development of monocytic and B cell lineages, in which the core binding factor (CBF) fusions (CBFβ-SMMHC or TEL-AML1) or MLL fusions (MLL-AF9 or MLL-AF4) have been high frequently identified in human acute leukemias. These suggested that AML1/CBFβ and MLL might regulate PU.1 expression and AML1/CBFβ or MLL fusions might dysregulate PU.1 expression at genetic and epigenetic levels and eventually develop similar leukemias. We found that the AML1-ETO/CBFβ complex interacts with MLL in a similar manner as AML1/CBFβ (3), while the AML1/CBFβ-SMMHC complex interacts with MLL more strongly. Surprisingly, the CBFβ-SMMHC by itself interacts with MLL through the junction region of the fusion protein. The interactions with MLL by these fusion proteins complexes, AML1-ETO/CBFβ and AML1/CBFβ-SMMHC, correlate with their ability to maintain H3K4me3 levels at PU.1 URE and promoter regions in 416B cell lines stable expressing AML1-ETO or CBFβ-SMMHC. Taken together, our data indicate that AML1-ETO/CBFβ complex preserves the interaction with MLL, while the AML1/CBFβ-SMMHC complex enhances it. This suggests that these two leukemogenic fusions downregulate PU.1 expression through different epigenetic mechanisms in the presence of H3K4me3 mark at PU.1 regulatory region. The effects of these differential interactions on the H3K4me3 mark maintenance and on target gene expression, particularly PU.1, may be critical for the aberrant regulation that underlies the etiology of M2 and M4Eo acute myelogenous leukemia (AML). 1. Huang G, et al. Nat. Genet. 2008; 40: 51-60 2. Zhang P, et al. Blood 2008; 112: 594. 3. Huang G, et al. Blood 2008; 112: 282.
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Furugaki, Kouichi, Katerina Pokorna, Carole Le Pogam, Masayuki Aoki, Murielle Reboul, Véronique Bajzik, Patricia Krief, et al. "DNA vaccination with all-trans retinoic acid treatment induces long-term survival and elicits specific immune responses requiring CD4+ and CD8+ T-cell activation in an acute promyelocytic leukemia mouse model." Blood 115, no. 3 (January 21, 2010): 653–56. http://dx.doi.org/10.1182/blood-2007-08-109009.
Full textAbstract:
Abstract DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4+ or CD8+ cells abolished this effect. CD4+ depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4+ and CD8+ subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4+ cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester–based assays showed major histocompatibility complex–restricted APL-specific T cell–mediated immune responses. Sorted APL-specific CD8+CD107a+ T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA–treated mice were shown to secrete interferon-γ when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARα vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-γ–producing and cytotoxic T cells in the leukemic mice.
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Ziegler, S. F., S. D. Levin, L. Johnson, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, E. Baker, G. R. Sutherland, A. L. Feldhaus, and F. Ramsdell. "The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex." Journal of Immunology 152, no. 3 (February 1, 1994): 1228–36. http://dx.doi.org/10.4049/jimmunol.152.3.1228.
Full textAbstract:
Abstract CD69 is a rapidly induced T cell activation Ag that is also expressed in an inducible fashion on cells of most, if not all, hematopoietic lineages. Molecular cloning has shown that CD69 is a type II membrane glycoprotein that is a member of the C-type lectin family. In this report we have shown that induction of CD69 mRNA in activated murine thymocytes and T cells is very rapid, peaking between 30 and 60 min poststimulation, and transient, dropping to nearly resting levels by 8 h. An analysis of the mouse CD69 gene structure showed the gene to consist of 5 exons and have a phorbol ester-inducible promoter element within the first 700 bp upstream of the start of transcription. Chromosomal mapping placed the mouse CD69 gene on the long arm of chromosome 6 near the NK gene complex that contains the related NKR-P1 and Ly-49 gene families. The human CD69 gene mapped to chromosome 12p13 near the related NKG2 gene cluster and in a region associated with rearrangements in approximately 10% of cases of childhood acute lymphocytic leukemia.