ALBERT

Description: The Sox4 transcription factor mediates early B-cell differentiation. Compared with normal pre-B cells, SOX4 promoter regions in Ph+ ALL cells are significantly hypomethylated. Loss and gain-of-function experiments identified Sox4 as a critical activator of PI3K/AKT and MAPK signaling in ALL cells. ChIP experiments confirmed that SOX4 binds to and transcriptionally activates promoters of multiple components within the PI3K/AKT and MAPK signaling pathways. Cre-mediated deletion of Sox4 had little effect on normal pre-B cells but compromised proliferation and viability of leukemia cells, which was rescued by BCL2L1 and constitutively active AKT and p110 PI3K. Consistent with these findings, high levels of SOX4 expression in ALL cells at the time of diagnosis predicted poor outcome in a pediatric clinical trial (COG P9906). Collectively, these studies identify SOX4 as a central mediator of oncogenic PI3K/AKT and MAPK signaling in ALL.

Description: Abstract 3231 B cell lineage acute lymphoblastic leukemia (ALL) is a common malignancy in childhood, and the pre-B cell receptor (pre-BCR) signalling pathway was previously demonstrated to function as a tumor suppressor. The transcription factor PAX5, a key regulator of B cell development, is frequently involved in chromosomal rearrangements of leukemic blasts. Using high resolution single nucleotide polymorphism (SNP) genomic microarray by us and other groups, several candidate partner genes fused to PAX5 have been detected in samples of pediatric ALL, such as TEL, FOXP1, AUTS2, and C20orf112. Recently, we studied the fusion gene PAX5/TEL and its role in leukemic evolution by Affymetrix HG-U133 plus 2.0 Array of the ALL cell line Nalm6 transfected with a PAX5/TEL construct. PAX5/TEL reduced the expression of PAX5 and its downstream target genes (e.g. CD79A, BACH2, CD19). Moreover, we demonstrated a dominant negative impact of the PAX5/TEL-fusion protein on the binding affinity of wild-type PAX5 to the promoter of CD79A (Iwanski et al., 2009 ASH Abstract No. 3455). To expand our findings, we analyzed the gene expression profile of pediatric ALL samples carrying PAX5/TEL (PAX5/TEL+, n=2) compared to samples with normal PAX5 (n=7) from a genomic ALL study. Samples with normal PAX5 were selected from among 95 B-ALL patients with normal PAX5 status, based on characteristics that most closely matched the two PAX5-TEL+ patients including cytogenetics and current risk stratification. Gene expression data were compiled using the Affymetrix HG-U133A Array, and a heatmap based on the Top 200 probes with the highest expression levels from both sample sets was generated (TIBCO Software Inc.). Notably, the downregulated genes included Bruton agammaglobulinemia tyrosine kinase (BTK; -2.8 fold, FDR 〈 0.2), an important regulator of pre-BCR signaling, Spleen tyrosine kinase (SYK, -2.3 fold, FDR 〈 0.2), and IGHM (-5,9 fold; FDR 〈 0.1), but also significantly up-regulated expression of genes involved in myeloid differentiation, namely Myeloperoxidase (MPO, +24.2 fold, FDR 〈 0.2), and CCAAT/enhancer binding protein alpha (CEBPA, +3.2 fold, FDR 〈 0.3), as well as the erythroid genes Aminolevulinate delta-dehydratase (ALAD, +12.2 fold, FDR 〈 0.2) and the Erythropoietin receptor (EPOR, +7.0, FDR 〈 0.3). Additionally, we performed a meta-analysis comparing deregulated genes detected in the Nalm6-microarray (PAX5/TEL-MIGR vs. empty vector) to the data from the human B-ALL samples (PAX5/TEL+ vs. normal PAX5). Overall, we identified a set of 35 overlapping genes (FDR 〈 0.3) that were deregulated in both data sets (21 downregulated, 14 up-regulated). Notably, 10 out of the 21 (47%) downregulated genes are known to be involved in B cell development and BCR signaling, some of them well-recognized as direct PAX5 target genes (e.g. CD79A, CD19, BACH2). Moreover, reporter gene assay with a luciferase reporter construct containing cDNA of the CD19 promoter with PAX5 binding sites (luc-CD19) was performed in Nalm6 cells. Since these cells already express a high level of endogenous PAX5, transcriptional activity of the luc-CD19 reporter plasmid was relatively high in the Nalm6 cells transfected with empty vector, as compared to 293T cells. In contrast, PAX5/TEL-transduced Nalm6 cells displayed a significantly reduced transcriptional activation of the reporter construct (P 〈 0.01). We also explored if mutation and/or deletion of PAX5 (mut/del PAX5) may have an impact on genes involved in B cell development and the pre-BCR/BCR pathway. Hence, expression files from human ALL samples with mut/del PAX5 (n=50) and B-ALL samples with normal PAX5 (n=95) were analyzed. Notably, only two genes that are known to be involved in B cell development and the pre-BCR pathway were significantly downregulated in samples with mut/del PAX5 compared to normal PAX5, namely CD72, a B cell specific repressor gene activated by PAX5 (-1.51 mean fold, FDR = 0.05), and immunoglobulin heavy constant delta (IGHD), a gene involved in pre-BCR signalling (-1.6 mean fold, FDR = 0.18). These findings suggest no strong influence of mut/del PAX5 on the expression of downstream genes involved in pre-BCR signaling. In conclusion, our results provide further insights into the dominant-negative role of PAX5/TEL and link this fusion gene with the pre-BCR pathway. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 863 Background: The SOX4 (SRY-related HMG-box) transcription factor is expressed in early B- and T- cell development. In the absence of SOX4, B cell development is arrested at the pro-B to pre-B cell transition. In Sox4−/− mice, pro-B cells fail to proliferate in response to IL7 and to expand and differentiate past the pre-B cell receptor checkpoint. Interestingly, SOX4 functions a transcription factor yet closely interacts with membrane-proximal cytokine receptor signaling. The PDZ domain-containing adaptor protein syntenin (SDCBP) recruits the Sox4 protein directly to the cytoplasmic tail of the cytokine receptors. With regard to its receptor-proximal activation, Sox4 resembles the SMAD and STAT transcription factors. A role for Sox4 in acute myeloid leukemia was proposed based on the identification of viral insertions, and Sox4-overexpression causes myeloid leukemia. In this study, we found SOX4 was strongly upregulated upon tyrosine kinase inhibitor (TKI) treatment in Ph+ ALL and high expression of Sox4 correlates with poor clinical outcome of patients with ALL. Results: To elucidate the potential contribution of Sox4 to BCR-ABL1-driven B cell lineage leukemia, we studied a conditional Sox4 knockout mouse model. To this end, we transformed bone marrow B cell precursors from Sox4fl/fl mice with BCR-ABL1 to test its function in a mouse-model for Ph+ ALL. Tamoxifen (4-OHT)-inducible activation of Cre and subsequent deletion of loxP-flanked Sox4 alleles in B cell precursor ALL resulted in reduced mRNA levels of multiple genes that play a known role in survival signaling in Ph+ ALL cells including BCLXL (BCL2l1), Mapk8, Map2k6, PI3K/AKT (Pik3r2, Pik3r3, Rps6ka2, Mtor). Overexpression of Sox4 had the opposite effect and resulted in increased mRNA levels of BCLXL (Bcl2l1), Mapk8, PI3K/AKT (Pik3r2, Pik3r3). Single-locus quantitative ChIP confirmed binding of SOX4 to BCL2l1, PIK3R3 and MAPK8 promoters in human Ph+ ALL cells via its putative DNA binding motif. Transcriptional regulation of components within the PI3K/AKT pathway is indeed functionally relevant since Cre-mediated deletion of Sox4 caused multiple de-phosphorylation events including AKTS473, SRCY416 and RPS6S235/6. In addition, inducible deletion of Sox4 resulted in strong upregulation of both Arf and p53 protein, suggesting that Sox4 mediates survival signaling both by activation of BCLXL (Bcl2l1) and repression of Arf/p53. Consistent with Sox4-mediated negative regulation of Arf/p53, deletion of Sox4 resulted in G0/G1 arrest of BCR-ABL1 ALL cells and loss of self-renewal capacity, failure to form colonies in methylcellulose replating assays and delayed the onset of disease and substantially prolonged overall survival of recipient mice. In agreement with reduced expression of BCLXL (Bcl2l1) and increased levels of Arf/p53, the overall outcome of Sox4 deletion is rapid loss of viability and apoptosis in BCR-ABL1-transformed ALL but not normal pre-B cells. Loss of viability upon Sox4 deletion in the leukemia cells was rescued by transduction with constitutively active (myristoylated; CA) mutants of AKTCA and the p110 catalytic subunit of PI3K (p110αCA) and as well with BCLXL. Clinical relevance: These findings are also relevant to human disease since promoter CpG methylation analysis (HELP assay) revealed lower levels SOX4 promoter methylation in human Ph+ALL cells compared to their normal pre-B cell counterparts. In addition, samples from patients with high risk ALL (n=31) show a trend towards higher SOX4 mRNA levels compared to patients with intermediate risk ALL (n=23, p=0.07; ALL REZ BFM 2002). Analyzing data from a larger study based on 207 patients with ALL (COG P9906) showed a statistically significant association between high mRNA levels of SOX4 and poor clinical outcome (shorter overall survival). Conclusion: Collectively, these findings identify SOX4 as a critical upstream regulator of survival signaling in Ph+ ALL. Pathways affected by SOX4 include PI3K/AKT signaling downstream of BCR-ABL1, activation of BCLXL (BCL2L1) and negative regulation of Arf and p53. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 528 Background: Tyrosine kinase-driven B-cell lineage acute lymphoblastic leukemia such as Ph+ ALL, is associated with particularly poor clinical outcome and defined by deletions of the IKZF1 gene encoding the Ikaros transcription factor in 〉80% of the cases. While the role of Ikaros in lymphoid differentiation has been extensively studied, it remains elusive how Ikaros functions as tumor suppressor in Ph+ALL. Here we demonstrate that Ikaros orchestrates a set of gene expression changes that affects leukemia cell proliferation and survival at multiple levels. Results: A comprehensive gene expression analysis revealed that reconstitution of Ikaros function in patient-derived Ph+ ALL cells mimics gene expression changes induced by Pax5 (a B cell specific transcription factor), μ-chain and BLNK (pre-B cell receptor components), and treatment with Imatinib (a BCR-ABL1 kinase inhibitor), while it reverses Myc- and Stat5a/b-dependent survival signals. In combining seven experimental perturbations (reconstitution of Ikzf1, Pax5, μ-chain, Blnk, treatment with Imatinib or deletion of Stat5a/bfl/fl and Mycfl/fl), we identified a commonly regulated gene set, including known tumor suppressors SPIB, BTG1, and BTG2 and highly enriched for Myc-related gene signatures. These findings suggest that reconstitution of PAX5 and IKZF1 transcription factors converges with pre-B cell receptor-mediated transcriptional changes. We next connected our analysis at the transcriptional level with Ikaros-dependent changes at the level of tyrosine phosphorylation events: The BCR-ABL1 kinase in Ph+ ALL drives proliferation and survival through phosphorylation of Stat5, AKT, ERK, SRC and multiple other signaling intermediates. A global mass spectrometry analysis of phosphotyrosine proteins in patient-derived Ph+ ALL cells showed that reconstitution of Ikaros leads to global loss of phospho-tyrosine, in a comparable manner as treatment with TKI. The vast majority of Ikaros-mediated changes resulted in loss of tyrosine phosphorylation such as the SRC tyrosine kinase LCK (Y504 and Y192), the proximal BCR-ABL1 adapter molecule NEDD9 (Y166, Y240, Y344 and Y578) and ABL1 itself (Y257). For a small number of molecules, Ikaros-reconstitution resulted in increased tyrosine phosphorylation including the phosphatases Shp2 (Ptpn11Y584) and Ship2 (Inppl1Y1161). A focus phospho-tyrosine analysis of individual signaling molecules analysis revealed that Stat5, SRC, AKT and ERK are all affected by Ikaros-induced de-phosphorylation. These results suggest that that Ikaros induces dephosphorylation of multiple signaling intermediates in BCR-ABL1 signaling. To identify Ikaros-mediated gene expression changes that were directly induced by Ikaros promoter binding, we matched Ikaros-dependent gene expression changes with Ikaros ChIP-seq data. This analysis confirmed enriched binding of Ikaros on Btg1, Myc and Smyd2 promoters. Among Ikaros target genes, we identified a set of genes that predicts clinical outcome for adult and pediatric patients with ALL. According to this predictor, IkarosUP (FBXO33, HBP1, HIVEP1, DTX1, KIF13B, RHOQ) and IkarosDOWN (PFKP, ASNS, ETV6, PSTPIP2, SMYD2, CDKL5), patients with B-ALL were segregated into two groups with significantly different outcomes, with a 5-year RFS of 76% vs 29% (COG P9906 trial, n=110; p=0.006) and a 5-year OS of 39% vs 0% (ECOG E2993 trial, n=25; p=0.007). Further biochemistry and mouse experiments showed that both the ability of Ikaros to dephosphorylate Stat5 and to function as tumor suppressor requires cooperation with the pre-B cell receptor linker molecule BLNK but not the proximal effector kinase Syk. Conclusions: Importantly, Ikaros binds to and transcriptionally activates CDKN2A (Arf) and TP53 loci and thereby opposes the function of BCL6, which acts as transcriptional repressor at these loci. Comparing the gene expression microarray and ChIP-seq or ChIP-on-Chip data of BCL6 and Ikaros, we identified a set of target genes that is regulated by both BCL6 and Ikaros and found that for the vast majority of Ikaros/BCL6 target genes, Ikaros-reconstitution recapitulated a BCL6−/− situation, i.e. genes that are induced by Ikaros are repressed by BCL6. These findings are of potential clinical relevance since pharmacological inhibition of BCL6 restores sensitivity of IKZF1-mutant Ph+ ALL cells to conventional tyrosine kinase inhibitors. Disclosures: Druker: ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other.

Description: Abstract 872 Background: The unfolded protein response (UPR) is a cellular machinery required to salvage of ER stress and to promote cell survival. The pathway consists of three different components, namely inositol-requiring enzyme 1a (IRE-1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6) and converges at the level of its effector molecule X-box binding protein 1 (XBP1). Previous work identified Xbp1 as a central requirement of plasma cell development and as critical mediator of cell survival in plasma cell-derived multiple myeloma. RESULTS: While the role of Xbp1 in plasma cells and plasma cell malignancies is well established, we report here the unexpected finding of a central role of Xbp1 in the survival of pre-B cell-derived Ph+ ALL cells. Surprisingly, patient-derived Ph+ ALL cells express Xbp1 (and related molecules in the IRE1 pathway) at significantly higher levels than normal bone marrow pre-B cells. In addition, we found that high expression levels of Xbp1 at diagnosis predict poor poor overall survival (OS), relapse-free survival (RFS) of leukemia patients in two clinical trials for patients with high risk acute lymphoblastic leukemia (n=207; COG P9906 trial; p=1.12e-4 and ECOG E2993; n=215; p=2.48e-5). In addition, high levels of XBP1 correlated with positive minimal residual disease (MRD) status at day 29 after onset of chemotherapy. To study the function of Xbp1 in Ph+ ALL in a genetic experiment, we developed a Ph+ ALL leukemia model based on bone marrow progenitor cells from mice carring loxP-flanked allele of Xbp1 (Xbp1fl/fl). On the basis of this model, bone marrow B cell precursors were transformed by BCR-ABL1 in the presence of IL7. Inducible Cre-mediated deletion of Xbp1 was achieved by transduction of leukemia cells with tamoxifen (4-OHT)-inducible Cre. Interestingly, 4-OHT-induced deletion of Xbp1 in Ph+ ALL-like leukemia cells caused rapid cell death within two days of induction. Xbp1-deletion resulted in extensive apoptosis, cellular senescence and cell cycle arrest owing to increased levels of p53, p21 and Arf. Interestingly, similar observations were made in an in vivo setting where Xbp1-deletion resulted in prolonged survival of NOD-SCID transplant recipient mice (n=7; p=0.007). Mechanistically, deletion of Xbp1 leads to increased expression of the pro-apoptotic molecule CHOP as in plasma cells/multiple myeloma and phosphorylation of the stress MAP kinases p38 and JNK. CLINICAL RELEVANCE: To test the potential clinical relevance of these findings, we used a recently identified small-molecule inhibitor STF-083010 (Papandreou et al., 2011), which blocks the endonuclease activity of upstream molecule IRE-1, essential for the splicing of the active form of Xbp1. STF-083010 indeed inhibited splicing of XBP1 and overall mimicked findings in genetic experiments. Importantly, targeting of Xbp1 by STF-083010 also induced cell death in three patient-derived cases of Ph+ ALL carrying the T315I mutations, which confers far-reaching TKI-resistance. CONCLUSIONS: These findings identify Xbp1 as a fundamentally novel target for the therapy of TKI-resistant Ph+ ALL. Like plasma cells and tumor cells in multiple myeloma, Ph+ ALL cells are selectively sensitive to ER stress and critically dependent on Xbp1 and likely other factors of the UPR pathway. Clinical validation of this concept could lead to improved treatment options for patients with TKI-resistant Ph+ ALL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 776 Background: BCL6 is known as a protooncogene and transcriptional repressor in Diffuse Large B cell lymphoma (DLBCL), where BCL6 is frequently involved in chromosomal rearrangements. We recently found that BCL6 mediates a novel form of drug resistance to tyrosine kinase inhibitors (TKI) in Ph+ ALL and CML (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). This was based on the finding that Ph+ ALL and CML cells respond to TKI-mediated inhibition of BCR-ABL1 kinase activity by upregulation of BCL6, which protects from p53-mediated apoptosis. Our current study was prompted by the finding that high expression levels of BCL6 represent a predictor of poor clinical outcome in children with Non-Ph ALL. We therefore performed detailed studies of expression and function of BCL6 in various subtypes of Non-Ph childhood ALL. Results: In a gene expression analysis of 207 children with high-risk B cell precursor ALL (COG P9906 trial), we found that high expression levels of BCL6 at the time of diagnosis represents a strong predictor of poor overall (OS; p=0.007) and relapse-free (RFS; p=0.02) survival. Also, high expression levels of BCL6 are predictive of positive minimal residual disease (MRD+) status on day 29 after the onset of chemotherapy. For 49 patients in this clinical trial, matched sample pairs at diagnosis and relapse were available. In these patients, BCL6 expression was significantly higher at relapse compared to diagnosis (p=0.003). We next studied BCL6 mRNA (n=122) and protein (n=21) levels in a larger cohort of primary childhood ALL patient samples and found that BCL6 expression levels are particularly high in MLL-AF4 infant ALL. This finding was unexpected and we tested if the MLL-AF4 oncogene drives aberrant BCL6 expression in these cells. First, ChIP-analysis revealed that the chimeric oncoprotein MLL-AF4 directly binds to the BCL6 promoter, suggesting that MLL-AF4 may indeed drive BCL6 expression. In support of this hypothesis, BCL6 Western blot analyses of inducible MLL-AF4-transgenic pro-B cells demonstrated that activation of the MLL-AF4 transgene is sufficient to induce ∼10-fold upregulation of BCL6 protein levels. We conclude that aberrant expression of BCL6 in childhood ALL can be the direct consequence of MLL-AF4 activity. To investigate the potential function of BCL6, we used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6−/− mice. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transformed B cell progenitor cells from BCL6−/− and wildtype mice with oncogenic NRASG12D, which represents the most frequent RAS lesion in B cell lineage leukemia. Surprisingly, BCL6-deficiency results in a failure of NRASG12D ALL cells to initiate leukemia, while NOD/SCID mice that were transplanted with BCL6 wildtype NRASG12DALL succumbed to the disease. Clinical relevance: To verify if these observations are relevant to human disease, we transduced primary human childhood ALL xenografts with MLL-AF4 gene rearrangement with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if BCL6 inhibition is of potential use for children with MLL-AF4 leukemia, we used a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI, Cerchietti et al., Blood 2009). RI-BPI is able to inhibit BCL6 function and is currently under clinical development for the treatment of BCL6-dependent DLBCL. Treatment of the primary human MLL-AF4 ALL xenograft cells with RI-BPI compromised colony formation in methylcellulose and leukemia-initiation in transplant recipient mice and had a strong synergistic effect when combined with Vincristine, which represents the backbone for most high risk regimen in pediatric ALL. Conclusions: These findings identify BCL6 as a therapeutic target in high-risk childhood leukemia and its pharmacological inhibition as a novel strategy as therapeutic adjuvant. Interestingly, MLL-AF4 ALL cells exhibit constitutively high expression levels of BCL6. Aberrant expression of BCL6 in MLL-AF4 ALL is the direct consequence of MLL-AF4 activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors (e.g. RI-BPI) with standard chemotherapy as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures: No relevant conflicts of interest to declare.

Description: Initial cell surface expression of the pre-B cell receptor induces proliferation. After 2 to 5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). The mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. The checkpoint at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling induces cell-cycle exit through up-regulation of the transcriptional repressor BCL6. Inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Hence, pre-B cell receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage.

Description: Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422. Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM. Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02). We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC. Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. Disclosures Weigert: Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

Description: Background: The E3 ubiquitin ligase Fbxw7 is responsible for the proteasomal degradation of several oncogenes involved in cellular proliferation, growth and survival. Notably, Fbxw7 has been characterized as a tumor suppressor in a variety of cancers, including T ALL and CLL. In the absence of Fbxw7, hematopoiesis is defective leading to abnormal development of B and T cells. In these mice, hematopoietic stem cells have impaired self-renewal due to stabilization of c-Myc. Fbxw7 targets several well-characterized molecules (c-Myc, Mcl1, p100, cyclin E, mTOR, SREBP1) with essential functions in B cells. We have recently shown that glycogen synthase kinase 3 (Gsk3), which is negatively regulated by PI3K signaling, controls the metabolic needs of B cells (Jellusova et al., 2017 Nat. Immunol.). A number of Gsk3 substrates are phosphorylated and degraded via the proteasome through recognition of Fbxw7. We sought to investigate the GSK3-FBXW7 axis in B cells and its implications for B cell malignancies. Results: To elucidate the role of Fbxw7 in B cells, we studied Fbxw7 in a conditional knockout mouse model. We crossed Fbxw7fl/flmice with Mb1Cre mice to ablate Fbxw7 during early B cell development. In the absence of Fbxw7, B cells passed through their developmental stages and showed no significant signs of maturation defects in the bone marrow. However, mature recirculating B cells were nearly absent in Fbxw7-deficient animals. Correspondingly, peripheral B cells in the spleen were reduced and showed specific decreases in follicular B cells. Histological analysis of the spleen architecture showed intact B cell follicles with presence of marginal zone B cells. In B cell malignancies, mutations of FBXW7 have been reported in CLL, for which the normal counterpart are B1 cells. We analyzed the B1 cell compartment in the peritoneal cavity in the absence of Fbxw7. Loss of Fbxw7 resulted in drastic reductions of B1 cells in the peritoneal cavity. Both B1a and B1b cells were mutually affected in these mice. To further characterize this B1 cell defect, we generated a novel acute deletion model for Fbxw7. We crossed Fbxw7fl/flmice with hCD20Cre-ERT2 and administered tamoxifen by oral gavage to induce genetic ablation. Acute deletion of Fbxw7 lead to reduction of B1 cells within a few weeks, indicating the importance of Fbxw7 for the maintenance of B1 cells. When challenged with T cell-independent immunizations the overall antibody responses where reduced. BCR-stimulation of cells in vitro showed diminished growth and signaling potential of knockout cells. These cells have reduced calcium signaling and undergo massive apoptosis. The survival defect could be partially rescued with co-stimulation of IL-4 or ectopic expression of BCL2. Conclusion: Our findings identify Fbxw7 as a critical regulator of B cell development. Specifically, Fbxw7 regulates B cell signaling, development and maintenance of B1 cells. These findings are directly relevant to the pathogenesis of CLL. Loss of function mutations of FBXW7 in CLL likely occur later during tumor evolution and potentially contribute to the specific microenvironmental niche. Disclosures Rickert: Pfizer: Employment.