ALBERT

Description: Key Points Our study demonstrates aberrant genome-wide deposition of histone 3 lysine 79 dimethylation on MLL-target genes in MLL-AF6–driven leukemia cells. We provide evidence that leukemia cells bearing the MLL-AF6 fusion are sensitive to genetic and pharmacologic DOT1L inhibition.

Description: Abstract 2384 The t(10;11)(p12;q23) and the t(10;11)(p12;q14), which encode the MLL-AF10 and CALM-AF10 fusion oncoproteins respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Patients with AML harboring either MLL-AF10 or CALM-AF10 rearrangements have a particularly poor outcome compared to patients whose leukemia cells do not harbor these translocations. Thus new therapeutic approaches are clearly needed for patients with leukemias bearing rearrangements of the AF10 gene. Previous studies have suggested that the histone H3 lysine 79 (H3K79) methyltransferase DOT1L is recruited by a part of the AF10 protein retained in leukemic MLL-AF10 and CALM-AF10 fusions, suggesting a role of this methyltransferase in transformation mediated by these fusion oncoproteins. The general amenability of enzymes to pharmacological inhibition makes DOT1L an attractive therapeutic target in leukemias bearing AF10-fusions. We decided to test whether transformation mediated by the MLL-AF10 or CALM-AF10 fusions can be impaired by Dot1l inhibition. We transformed bone marrow cells with either the MLL-AF10 or the CALM (400–648)-AF10 (677–758) fusion proteins that have been previously shown to immortalize hematopoietic progenitors in vitro and in vivo. In contrast to the large, highly clonogenic compact colonies observed in methylcellulose culture of wildtype transformed cells, Dot1l excision from the MLL-AF10 or CALM-AF10 immortalized progenitors led to the formation of small, diffuse colonies that had lost their replating capability. In MLL-AF10 or CALM-AF10 transformed cells, Dot1l- inactivation significantly reduced global H3K79 dimethylation as assessed by Western blotting, and Wright Giemsa staining confirmed morphological features consistent with myeloid differentiation. Moreover, the expression of MLL-AF10 and CALM-AF10 targets, such as Hoxa cluster genes and Meis1, decreased significantly after Dot1l inactivation as assessed by quantitative PCR. We then assessed the impact of Dot1l deletion on the in vivo leukemogenic activity of MLL-AF10 and CALM-AF10 transformed bone marrow cells. Ongoing experiments clearly demonstrate that Dot1l excision strongly impairs the initiation and maintenance of both MLL-AF10 as well as CALM-AF10 mediated murine leukemias. Having established that genetic inactivation of Dot1l inhibits H3K79 methylation and consequently transforming potential of the AF10-fusions, we investigated the efficacy of Dot1l inhibitors against MLL-AF10 or CALM-AF10 transformed murine bone marrow cells. Treatment of MLL-AF10 or CALM-AF10 transformed cells with EPZ004777, the first specific small-molecule inhibitor of Dot1l, suppressed expression of downstream oncogenic targets such as Hoxa cluster genes and Meis1, and selectively impaired the proliferation of MLL-AF10 and CALM-AF10 transformed, but not Hoxa9/Meis1 transformed cells. MLL-AF10 and CALM-AF10 transformed cells underwent differentiation and cell cycle arrest after EPZ004777 treatment. Results from in vivo colony-forming units-spleen (CFU-S) assays showed that pre-treatment of MLL-AF10 or CALM-AF10 transformed cells with the EPZ004777 inhibitor profoundly impaired their spleen-colony forming ability, suggesting that EPZ004777 may show in vivo efficacy against AF10-fusion transformed cells. Taken together, our results demonstrate that Dot1l inhibition impairs the in vitro and in vivo oncogenic activity of the MLL-AF10 and CALM-AF10 fusion oncogenes. These results indicate that patients with leukemias bearing chromosomal rearrangements of the AF10 gene may benefit from small molecule inhibition of H3K79 methylation. Disclosures: Olhava: Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

Description: A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are not very effective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10 positive leukemias which may lay the foundation for novel targeted therapies. The polycomb repressive complex 1 gene BMI1 is consistently overexpressed in CALM-AF10 leukemias. Previous studies have shown that CALM-AF10 leukemias express high levels of BMI1, regardless of whether the leukemias are myeloid or lymphoid. Our analysis of TCGA acute myeloid leukemia (AML) data confirmed that AML cells with AF10-rearrangements displayed significantly higher expression of BMI1 transcripts compared to cells from non AF10-rearranged AML patients. These observations indicate that BMI1 may be directly activated by AF10-fusion oncogenes as suggested by our previous studies. We sought to investigate the role of BMI1 in CALM-AF10 mediated leukemogenesis using murine and human models of CALM-AF10-mediated AML. First, we tested whether BMI1 deficiency can affect CALM-AF10 mediated oncogenic transformation of hematopoietic stem and progenitor cells (HSPCs). Towards this end, we retrovirally transduced fetal liver cells from Bmi1 wild-type, heterozygous or homozygous null mice with the CALM-AF10 fusion oncogene. Upon plating these cells in colony forming unit (CFU) assays, we observed a significant decrease in the colony formation capacity of the CALM-AF10 fusion transduced cells on a Bmi1 deficient background. Next, we performed Cre-recombinase mediated excision of Bmi1 of already transformed CALM-AF10 myeloid leukemia cells (Bmi1 floxed background). Bmi1 deletion led to a significant reduction in the number of total CFUs compared to Bmi1 wild-type cells, with a particularly striking reduction in the number of blast-like colonies. These experiments, using Bmi1 constitutive or conditional knockout-mice, revealed that CALM-AF10 transformed AML cells are dependent on Bmi1. Recently, selective pharmacological BMI1 inhibitors have been developed. We tested the impact of pharmacologic BMI1 inhibition on a panel of CALM-AF10-driven mouse leukemias with the small molecule inhibitor PTC-209. PTC-209 treatment increased gene expression of the known BMI1-repressed targets Cdkn2a (p16) and Cdkn1a (p21) and led to a dose-dependent decrease in cell proliferation. We also observed a marked increase in Annexin V+ cells upon PTC-209 treatment. In addition, cell-cycle analysis using BrdU incorporation assays revealed a significant decrease in cells in the S-phase, demonstrating that PTC-209 treatment leads to growth arrest and apoptosis in CALM-AF10 AML cells. In order to confirm these findings in human AML with CALM-AF10 rearrangements, we treated human CALM-AF10 positive AML cell lines P31, U937 and KPMOTS with PTC-209. Consistent with our results in the murine AML model, we observed a time and dose-dependent decrease in proliferation of these human cell lines upon PTC-209 treatment. Drug treated human cells also showed concomitant cell-cycle arrest and apoptosis induction, coupled with an increase in expression of BMI1 repressed tumor suppressor genes such as CDKN2A and CDKN1A. In summary, our results demonstrate that BMI1 is a bonafide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements and possibly other CALM-AF10 positive leukemias. We are now assessing clinical-grade BMI1 inhibitors for in vivo efficacy in mouse models of CALM-AF10-mediated AML. Disclosures 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: The MLLT10 gene, a known fusion partner for KMT2A, encodes AF10 protein, a transcription factor that binds unmodified histone H3 and regulates DOT1L expression. KMT2A-MLLT10 fusion portends adverse outcome, but MLLT10 function and prognostic implications in partnership with other genes has not been defined. In comprehensive transcriptome and karyotype evaluation of 2226 children and young adults (0-30 years), we defined the full spectrum of MLLT10 fusions, identified new fusion partners, and correlated MLLT10 structural variants with clinical outcome. We also evaluated transcription and methylation profiles to identify genes dysregulated in MLLT10 fusions with and without KMT2A. 2226 patients treated on Children's Oncology Group (COG) trials AAML0531 and AAML1031 were evaluated by transcriptome profiling and/or karyotyping to identify leukemia associated fusions and copy number changes associated with prognosis. Collectively, 127 patients (5.7%) had primary fusions involving MLLT10: 104 (82%) involving KMT2A (KMT2A-MLLT10), and 23 patients (18%) revealed other fusion partners (MLLT10-X). Alternate, recurrent fusion partners included PICALM (n=13), DDX3X (n=2), and TEC (n=2), while fusions with 6 other partner genes (DDX3Y, CEP164, NAP1L1, SCN2B, TREH, and XPO1) were each identified in single patients. Given the known association of KMT2A-MLLT10 fusions with adverse outcome, we sought to determine whether MLLT10-X had distinct characteristics and comparable outcomes. Initial comparison of disease characteristics in patients with and without KMT2A as fusion partner showed significant differences in age at diagnosis. Those with KMT2A-MLLT10 had a median age of 1.7 years (range 0-21.3), compared to 12.7 years (range 1.4-18.9) in those with MLLT10-X (p ≤ 0.001). There was no significant difference in gender, race, mutational status, or white blood cell count between these two cohorts. MLLT10 rearranged patients (n=127) demonstrated adverse outcomes, with 5-year event-free survival (EFS) of 18.6% vs. 49% in non-MLLT10 rearranged patients (N=1953, p6 logFC, or over 400x higher on average in MLLT10 rearranged patients. To determine if patients with MLLT10 fusions had distinct epigenetic profiles, we performed differential methylation analyses on samples from normal bone marrow and patients with 4 high-risk molecular features: MLLT10 rearranged, KMT2A rearranged, NUP98-NSD1 fused, and FLT3-ITD, across nearly 1 million CpG sites on the Infinium EPIC array (Illumina, CA). After fitting a multivariate model with all of the interacting molecular features, the 250 most discriminative regions were extracted and plotted (ComplexHeatmap) (Fig 1D). Strikingly, patients with MLLT10-X fusions cluster discretely with ultra-high-risk NUP98-NSD1 fusion patients, showing a broadly hypermethylated profile, while KMT2A-MLLT10 patients cluster within the larger KMT2A category and show far fewer hypermethylated regions. We identified patients with MLLT10 fusion partners not previously described, and compared them to other AML patients, as well as patients with known MLLT10 partners KMT2A and PICALM. All MLLT10-aberrant cases had poor EFS and OS, high RR, overexpressed HOXA genes, and distinct DNA methylation profiles, while patients with MLLT10-X fusions tend to be older children. Regardless of fusion partner, patients with MLLT10 fusions exhibit very high risk, and should be prioritized for alternative therapeutic intervention. Disclosures Farrar: Novartis: Research Funding. Deshpande:A2A Pharmaceuticals: Consultancy; Salgomed Therapeutics: Consultancy.

Description: Chromosomal translocations generating fusion proteins are frequently found in human leukemias. The fusion proteins play an important role in leukemogenesis by subverting the function of one or both partner proteins. The leukemogenic CALM-AF10 fusion protein is capable of interacting with the histone H3 lysine 79 (H3K79)–specific methyltransferase hDOT1L through the fused AF10 moiety. This interaction leads to local H3K79 hypermethylation on Hoxa5 loci, which up-regulates the expression of Hoxa5 and contributes to leukemogenesis. However, the long latency of leukemogenesis of CALM-AF10 transgenic mice suggests that the direct effects of fusion oncogene are not sufficient for the induction of leukemia. In this study, we show that the CALM-AF10 fusion protein can also greatly reduce global H3K79 methylation in both human and murine leukemic cells by disrupting the AF10-mediated association of hDOT1L with chromatin. Cells with reduced H3K79 methylation are more sensitive to γ-irradiation and display increased chromosomal instability. Consistently, leukemia patients harboring CALM-AF10 fusion have more secondary chromosomal aberrations. These findings suggest that chromosomal instability associated with global epigenetic alteration contributes to malignant transformation in certain leukemias, and that leukemias with this type of epigenetic alteration might benefit from treatment regimens containing DNA-damaging agents. This study is registered with www.clinicaltrials.gov as NCT00266136.

Description: The AF10/MLLT10 gene is recurrently involved in chromosomal rearrangements in human leukemia. AF10 rearrangements are linked to a poor prognosis in AML and T-ALL, underscoring the need to identify targeted therapies for AF10-fusion positive leukemia. Defining the molecular mechanisms of oncogenesis mediated by AF10-fusion proteins (AF10-FPs) may unravel novel actionable targets in leukemias with AF10-gene rearrangements. Towards this end, we established tetracycline (Tet)-inducible models of MLL-AF10 and CALM-AF10 AML and performed RNA-seq in AML cells treated with doxycycline (Dox) compared to vehicle treated counterparts. Since Dox treatment completely abrogates AF10-fusion gene expression from the Tet-regulated promoter, these models can be used to characterize the transcriptional landscape of potential AF10-FP target genes. We observed that among transcripts significantly downregulated upon Dox treatment, 168 genes were common in both the MLL-AF10 Tet-Off or CALM-AF10 Tet-Off conditions, indicating a high overlap between potential transcriptional targets of these distinct AF10-FPs. Expectedly, this list included genes previously implicated in leukemogenesis including Hoxa cluster genes, Meis1, Flt3, Mecom, Cd34, Gfi1b, Eya1 and Nkx2-3. Importantly, in addition to these well-characterized genes, we identified a number of novel pathways that were downregulated in the AF10-FP Tet-Off state. The most striking molecular signature of potential AF10-FP-regulated genes emerging from these analyses were factors involved in innate immunity and pro-inflammatory cytokine signaling. Prominent drivers of these molecular signatures included genes of the Jak/Stat and NFkB signaling pathways as well as Interferon response genes. We confirmed that AF10-FPs strongly activated Jak-Stat and NFkB signaling by performing Western blotting for key factors involved in these pathways. Since pro-inflammatory cytokines have been shown to play a role in AML cell survival, we tested the impact of cytokine depletion on murine AF10-FPs-driven AML cells. Proliferation assays demonstrated that AF10-FP-transformed cells could survive significantly better in cytokine-free medium compared to those transformed with other oncogenes such as MLL-AF9, which were completely dependent on cytokines for survival and proliferation in vitro. These results suggest that activation of cytokine signaling may contribute to increased survival of AF10-FP-driven AML cells. Next, we performed proteomic studies in which affinity-purified epitope-tagged AF10-FPs were evaluated for interacting proteins using Mass Spectrometry (MS). While studies on MLL-AF10 fusion are ongoing, our studies revealed that the strongest interactor of the CALM-AF10 fusion protein was the Janus kinase protein Jak1. We confirmed this finding by immunoprecipitation experiments in CALM-AF10 AML cells using a Jak1-specific antibody. Given the role of JAK1 in cytokine-mediated pro-inflammatory signaling, our findings indicate that CALM-AF10 may activate this pathway through direct recruitment of the Jak1 kinase. We sought to directly test the role of JAK1 in AF10-FP-mediated leukemogenesis. For this, we transformed bone marrow stem and progenitor cells from Jak1 floxed mice with the CALM-AF10 fusion. Deletion of Jak1 using Cre-recombinase in CALM-AF10 AML significantly reduced their proliferation in vitro. Furthermore, Jak1 deletion led to a highly significant reduction in the number of colony forming units (CFUs) from CALM-AF10 AML cells, with a particularly striking decrease in the number of blast-like colonies. We also observed a significant increase in differentiation of CALM-AF10 AML cells following Jak1 deletion, demonstrating that Jak1 activity is important for maintaining the CALM-AF10 leukemia cells in an undifferentiated state. Importantly, these results were recapitulated with two different small-molecule JAK1 inhibitors itacitinib and filgotinib that are being tested in clinical trials for a variety of human diseases. Treatment of CALM-AF10 AML cells with these selective JAK1 inhibitors led to a significant, dose-dependent decrease in proliferation accompanied by growth arrest and apoptosis. Taken together, our studies demonstrate that AF10 fusions activate pro-inflammatory signaling by co-opting the Jak-Stat pathway, presenting a potential therapeutic target in AF10-fusion-driven AML. Disclosures Levine: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Prelude: Research Funding; Loxo: Consultancy, Equity Ownership; Imago: Equity Ownership; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Gilead: Honoraria; Isoplexis: Equity Ownership; Epizyme: Patents & Royalties; Roche: Consultancy, Research Funding. Deshpande:A2A Pharma: Membership on an entity's Board of Directors or advisory committees; Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Description: We have demonstrated that the expression of the CALM/AF10 (C/A) fusion gene in murine BM cells results in an aggressive acute myeloid leukemia (AML). We sought to identify the domains involved in leukemogenesis mediated by the CALM/AF10 fusion gene. For this purpose we employed the CFU-S assay in which it was observed that C/A transduced BM cells generated an average of 75±23 day 12 CFU-S colonies per input 10^5 cells as compared to an average of 2 ±3 colonies with cells expressing the empty GFP vector (~ 37 fold increase; P 〈 0.0005) in the number of day-12 CFU-S content. Expression of the CALM gene truncated to the breakpoint of CALM/AF10 (CALMdelta3’) or the CALM/ AF10 fusion gene with a deleted octapeptide motif - leucine zipper domain (CALM/AF10 delta OM-LZ) gave 10 (±11) and 12 (±11) day12 CFU-S respectively per input 10^5 bone marrow cells showing that the AF10 portion of C/A, especially the OM-LZ region is necessary for the observed enhancement of d-12 CFU-S. BM cells transduced with a construct harboring the CALM gene fused only to the OM-LZ domain of AF10 (CALM+ OM-LZ) showed 68(±5) d-12 CFUS per input 10^5 cells (~34 fold Vs MIG; P=0.0006) comparable to the C/A fusion gene. We observed that the C/A fusion gene fails to show leukemic transformation of BM progenitors in CFC or proliferation assays in vitro in contrast to its striking leukemogenic potential in vivo. We tested different mutants of the C/A fusion gene using these assays and observed that the expression of a mutant of the CALM/AF10 fusion gene with a C-terminal portion of CALM (amino acids 400 – 648) fused to the OM-LZ motif of AF10, showed a significant increase in the number of secondary CFCs (32 fold Vs MIG; 15.38 fold Vs C/A), with the appearance of predominantly blast-like colonies. Interestingly, this mutant could also initiate leukemias in mice (n=5) with a latency and phenotype similar to mice injected with C/A transduced BM cells. Taken together, we demonstrate that the OM-LZ domain of AF10 is necessary for the expansion of early hematopoietic progenitors by CALM/AF10 and also sufficient for in vivo leukemic transformation. We also demonstrate that the deletion of amino acids 1 to 410 of CALM confers in vitro transformation potential to the C/A fusion gene. Our data identify the domains of C/A that are crucial for leukemogenesis and provide insights into the mechanism of transformation in t(10;11) (p13;q14) positive leukemia.