MLL-AF9 Leukemia Cells Research Articles

H2-K1 on MLL-AF9 Leukemia Cells Facilitates the Escape of NK Cell-Mediated Immune Surveillance

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, and prognosis is poor; 5-year survival approximately 30%. Recently, there is an emerging recognition of the innate immune system, in particular Natural killer (NK) cells and macrophages, for immune surveillance against AML, but the mechanistic basis for this is mostly unknow. Identifying how AML cells escape immune surveillance by NK cells may translate into new treatment opportunities for AML patients. To identify new therapeutic opportunities, we recently performed an in vivo CRISPR/Cas9 screen targeting 961 cell surface genes using the MLL-AF9 AML mouse model. One of the top leukemia stem cell dependencies in the bone marrow niche was H2-K1, an ortholog of human HLA-A, a classical MHC class-I molecule. In validation experiments, we observed a seven-fold depletion (p<0.001) of H2-K1 sgRNA-expressing c-Kit +Cas9 + leukemia cells in the bone marrow. In contrast, genetic disruption of H2-K1 did not impact the growth and survival of MLL-AF9 leukemia cells in culture. Given the known suppressive role of MHC class-I molecules for immune cells, we speculated that H2-K1 mayprovide inhibitory signals that counteract immune-surveillance mechanisms in the bone marrow niche. To test this hypothesis, we depleted NK cells and macrophages prior to transplantation of the c-Kit + MLL-AF9 leukemia cells. Macrophage depletion by clodronate liposomes did not affect the in vivo expansion of H2-K1 sgRNA-expressing leukemia cells demonstrating that macrophages were not suppressed by H2-K1 on the leukemia cells. In contrast, NK1.1 antibody-mediated depletion of NK cells fully rescued the depletion of H2-K1 sgRNA-expressing leukemia cells in vivo. These findings suggest that H2-K1 expression on MLL-AF9 leukemia cells inhibits NK cells in this model. Consistent with these findings, H2-K1 knockdown in leukemia cells triggered a two-fold increase of INFγ production (p<0.01) in the NK cells, accompanied by augmented apoptosis of the leukemia cells (p<0.01). Given that NK cells have been shown to be dysfunctional in AML patients, we next explored whether leukemia development affects NK cell maturation. The expansion of leukemia cells in the mice skewed NK cells towards a M1 (CD27 +CD11b -) state and decreased the level of the more cytotoxic M2 (CD27 +CD11b +) and M3 (CD27 -CD11b +) populations. H2-K1 disruption in the leukemic cells restored the level of M2 and M3 NK cell population in the bone marrow. Notably, restoration of matured NK cell populations was accompanied by an increased expression of NKG2D, an activating receptor, indicating a more cytotoxic state of the NK cells. In line with these findings, ablation of H2-K1 in leukemia cells induced JAK/STAT and NF-κβ signaling in the NK cells. Taken together, our study identifies that H2-K1 on MLL-AF9 leukemia stem cells facilitates immune evasion by suppressing NK cells. H2-K1 alters the maturation and activation of NK cells in the bone marrow niche. These findings increase our understanding of how leukemia cells escape immune surveillance and suggest that the identification of corresponding mechanisms in human AML could pave the way for new therapies that boost the endogenous NK cells by restoring immune surveillance mechanisms.

SAMHD1 Inhibitor Combined with Immune Checkpoint Inhibitor Elicits an Anti-AML Immunity

Despite advances in targeted therapy and use of molecular/cytogenetic risk stratification to guide treatment, clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Harnessing the immune system with CAR-T or immune checkpoint inhibitors (ICIs) has been proven successful in other hematological malignancies and solid tumors. The use of these immunotherapies for the AML treatment remains challenging due to lack of selective target antigens or low expression of immune checkpoint proteins. Recently, one strategy proposed to transform AML from immune-cold to immune-hot is to trigger a type-I interferon (IFN-I) response via a dsDNA related cGAS/STING signaling or dsDNA related RIG-I/MDA5 signaling. We recently reported that the dNTP and Ara-CTP (active form of cytarabine [AraC]) hydrolase SAMHD1, whose expression levels are elevated in AML patients relative to normal donors, limits IFN-I and T cell responses in murine AML model ( Blood, 2022, 140 [Supplement 1]: 679-680). We also found that innate immune activation seen following SAMHD1 knockdown is associated with its catalytic activity inhibition in cancer cells, providing a rationale for the development of SAMHD1 inhibitor. To identify SAMHD1 inhibitor, we conducted a virtual screen on 8,000,000 compounds based on SAMHD1 crystal structure (PBD 6TXC). The top 500 hits were then assessed in SAMHD1-WT- or H233A (SAMHD1 loss-of-function mutant) -expressing Molm13 cells using a phenotypic screen for AraC sensitization. Specifically, a SAMHD1 inhibitor should sensitize SAMHD1-WT (S-WT) but not H233A (S-H233A) cells to AraC treatment. Among the top 20 hits from phenotypic screen, our HPLC-based cell-free and cell-based SAMHD1 enzymatic assay revealed Samin27 as the most potent hit in blocking SAMHD1 hydrolysis and promoting AraCTP accumulation. Therefore, we assessed the efficacy of Samin27 combined with AraC in vivo. Samin27 administration (25mg/kg i.p. qd, starting on day 8 for 7 days) enhanced AraC efficacy (50mg/kg i.p. qd, starting 8th day for 5 days) in NSG mice implanted with Molm13 cells, as evidenced by extended survival relative to AraC alone. Moreover, the physical interaction between Samin27 and SAMHD1 protein were confirmed in a Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR) titration assay. RNA-seq results revealed Samin27 ex-vivo treatment remarkably upregulated IFN-I response genes in Molm13 cells, consistent with transcriptome results of SAMHD1-knockdown (S-KD) Molm13 cells. SAMHD1 activity is known to restrict replication of LINE-1 (L1), which is the only autonomously active transposable elements (TE) in mammals; elevated retrotransposition induces DNA damage and promotes cytoplasmic DNA accumulation, resulting in an IFN-I response via cGAS signaling. Next, to explore the mechanism underlying SAMHD1-inhibition induced immunity, we asked if SAMHD1 loss promoted retrotransposition and upregulated TE transcripts in leukemia cells. Through re-analysis of RNA-seq data in Molm13 cells, we found SAMHD1 inhibition upregulated most L1 elements and LTR-containing endogenous retrovirus (ERV) subfamilies. To determine if Samin27 induced immunity via L1 upregulation, we employed 3TC treatment (3TC, the nucleoside reverse transcriptase inhibitor) to block L1 upregulation. 3TC treatment blocked Samin27-mediated IFN-I response genes upregulation. Given that IFN-I response can stimulate innate immune cells (macrophage and T cell) cross-priming, potentially rendering AML cells more susceptible to anti-CD47 monoclonal antibody (aCD47) treatment, we then assessed whether combining a SAMHD1 inhibitor with an aCD47 act synergistically. We first implanted murine MLL-AF9 (MA9) leukemia cells into WT B6 mice. Then we treated leukemic mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks), Samin27 (25mg/kg i.p. qd, 3 weeks) or combination. Notably, pharmacological inhibition of SAMHD1 remarkably inhibited leukemia progression, decreasing leukemia burden (Fig. A.B). In another cohort, combination treatment extended mouse survival (Fig. C). Collectively, our results may provide a rationale for combining Samin27 with aCD47 against AML population, where single anti-CD47 treatment shows little effects.

Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development.

Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing the transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNA sequencing data from mouse MLL-AF9 leukemic cells and bone marrow aspirates, representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle-promoting protein, CCNA1. Overexpression of TRIM13 initially prompted a proliferative burst in AML cells, which was followed by exhaustion, whereas loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development, in part, by repressing TRIM13 expression and that this relationship is necessary for leukemic progression.

Loss of Wdr5 attenuates MLL-rearranged leukemogenesis by suppressing Myc targets

WD repeat domain 5 (WDR5) is a prominent target for pharmacological inhibition in cancer through its scaffolding role with various oncogenic partners such as MLL and MYC. WDR5-related drug discovery efforts center on blocking these binding interfaces or degradation have been devoted to developing small-molecule inhibitors or degraders of WDR5 for cancer treatment. Nevertheless, the precise role of WDR5 in these cancer cells has not been well elucidated genetically. Here, by using an MLL-AF9 murine leukemia model, we found that genetically deletion of Wdr5 impairs cell growth and colony forming ability of MLL-AF9 leukemia cells in vitro or ex vivo and attenuates the leukemogenesis in vivo as well, which acts through direct regulation of ribosomal genes. Pharmacological inhibition of Wdr5 recapitulates genetic study results in the same model. In conclusion, our current study demonstrated the first genetic evidence for the indispensable role of Wdr5 in MLL-r leukemogenesis in vivo, which supports therapeutically targeting WDR5 in MLL-rearranged leukemia by strengthening its disease linkage genetically and deepening insights into its mechanism of action.

Acute Myeloid Leukemia Cells Rely on the Glycolytic Enzyme PGK1 to Support Energy Production and Amino Acid Metabolism

Acute myeloid leukemia (AML) cells frequently display an increased dependence on glucose compared to healthy hematopoietic stem and progenitors (HSPCs). Furthermore, increased glucose metabolism is associated with worse outcomes in AML. However, the upstream regulators that drive glucose metabolism as well as how glucose is utilized in AML remains largely unknown. Here, we have discovered that the oncogenic transcription factor (TF), JUN supports AML, in part, by regulating glycolytic enzymes that utilize glucose to support both cellular energetics and amino acid metabolism. Having previously reported that certain aggressive AML sub-types depend on JUN, we next set out to identify JUN-regulated transcriptional programs that support AML. To do so, we engineered the human AML cell line, THP-1 to express either control (shNT) or JUN-targeting shRNAs and then performed RNA-seq on cells from each condition. Next, we carried out a functional enrichment analyses and discovered that genes encoding numerous enzymatic regulators of glycolysis were significantly down-regulated by JUN-inhibition. Using qPCR validation, we confirmed that JUN inhibition results in reduced mRNA expression of Hexokinase 1 & 2 (HK1 & HK2), Glucose-Phosphate Isomerase (GPI), Phosphofructose Kinase (PFKP), Aldolase A (ALDOA), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Phosphoglycerate Kinase (PGK1), Enolase 1 (ENO1) and Pyruvate Kinase Muscle (PKM). ChIP assays confirmed that JUN directly localizes to the promoters of each of these genes except for ALDOA and PKM. To assess whether the JUN-dependent changes in glycolytic gene expression translated to defects in the ability of AML cells to catabolize glucose, we performed a glycolysis stress test on shNT- and shJUN-expressing human AML cell lines (e.g. THP-1, NOMO-1 and MOLM-14). From this assessment, we found that JUN inhibition resulted in a complete loss of glucose catabolism in all cell lines tested. We postulated that JUN activates glycolytic gene expression to support AML and thus, inhibition of JUN-regulated glycolytic genes will impede leukemia cell function. To test this hypothesis, we generated shRNAs targeting each of the JUN-regulated glycolytic enzymes. We then engineered mouse leukemia cells expressing the human leukemogenic allele, MLL-AF9 - found in both childhood and adult AML - to express these shRNAs and then assessed their colony forming capacity (CFC) in cytokine-enriched semisolid culture. In parallel, we also assessed how knockdown of each glycolytic enzyme impacted the CFC of healthy HSPCs. Notably, we observed that inhibition of each enzyme significantly impaired the CFC of MLL-AF9 leukemia cells but that inhibition of Pfkp, Aldoa or Pgk1 did not impact the CFC of healthy HSPC, suggesting that these enzymes were selectively important for AML. We next evaluated how knockdown of Pfkp, Aldoa or Pgk1 impacted leukemia propagation in vivo. Interestingly, inhibition of Pfkp or Pgk1, but not Aldoa, significantly delayed the onset of MLL-AF9 driven leukemia in vivo. Moreover, we found that a chemical inhibitor of PGK1, CBR-470-1, reduces the CFC of patient-derived AML cells in vitro. Since Pgk1 had not previously been studied in AML, we focused our attention on the its role in leukemia cell metabolism. We thus performed untargeted metabolomics of shNT- and shPgk1-expressing mouse MLL-AF9 cells using hydrophilic liquid chromatography mass spectrometry (HILIC-MS). PGK1 utilizes ADP to convert 1,3-bisphosphoglycerate into 3-phosphoglycerate (3-PG) and ATP. 3-PG can subsequently be used to generate phosphoenolpyruvate (PEP) or in the de novo synthesis of serine (Ser). As expected, we found that Pgk1 inhibition resulted in a significant decrease in 3-PG, PEP and Ser. We also observed that Pgk1 inhibition resulted in the decrease of several tricarboxylic acid cycle (TCA) intermediates that support oxidative phosphorylation energy production. Surprisingly, we also found that Pgk1 inhibition resulted in a marked decrease in several amino acids, including Alanine, Arginine and Histidine. We are currently investigating whether the decrease in these amino acids mediated by Pgk1 inhibition is due to an impediment of their biosynthesis or their consumption in gluconeogenic pathways. Collectively, our studies identify JUN as a previously unrecognized regulator of AML cell metabolism and shed new into how glucose is catabolized in AML cells.

Presence of the GFI1-36N single nucleotide polymorphism enhances the response of MLL-AF9 leukemic cells to CDK4/6 inhibition.

The zinc finger protein Growth Factor Independence 1 (GFI1) acts as a transcriptional repressor regulating differentiation of myeloid and lymphoid cells. A single nucleotide polymorphism of GFI1, GFI1-36N, has a prevalence of 7% in healthy Caucasians and 15% in acute myeloid leukemia (AML) patients, hence most probably predisposing to AML. One reason for this is that GFI1-36N differs from the wildtype form GFI1-36S regarding its ability to induce epigenetic changes resulting in a derepression of oncogenes. Using proteomics, immunofluorescence, and immunoblotting we have now gained evidence that murine GFI1-36N leukemic cells exhibit a higher protein level of the pro-proliferative protein arginine N-methyltransferase 5 (PRMT5) as well as increased levels of the cell cycle propagating cyclin-dependent kinases 4 (CDK4) and 6 (CDK6) leading to a faster proliferation of GFI1-36N leukemic cells in vitro. As a therapeutic approach, we subsequently treated leukemic GFI1-36S and GFI1-36N cells with the CDK4/6 inhibitor palbociclib and observed that GFI1-36N leukemic cells were more susceptible to this treatment. The findings suggest that presence of the GFI1-36N variant increases proliferation of leukemic cells and could possibly be a marker for a specific subset of AML patients sensitive to CDK4/6 inhibitors such as palbociclib.

Targeting PRMT9 Suppresses Acute Myeloid Leukemia Maintenance

AML is a heterogenous disease in which prognosis and treatment is determined by recurrent genetic mutations and chromosomal abnormalities. Some genetic alterations result in aberrant RNA translation, which is exploited by leukemia stem cells (LSCs) to produce short-lived oncoproteins (c-Myc, Mcl-1) to promote cell survival. Since LSCs are considered as the source of treatment failure and relapse, it is critical to understand how LSCs hijack the translational machinery in order to develop effective therapeutics in AML.Arginine methylation catalyzed by protein arginine methyltransferases 1-9 (PRMT1-9) regulates various activities, including RNA translation. We revealed that PRMT1 over activation contributes to leukemia maintenance (Blood, 2019; Blood, 2019). Unlike PRMT1, the recently described PRMT9, is not defined in leukemia. Follow up of AML patients from existing datasets (GSE12417, TARGET) confirms that patients with higher levels of PRMT9 correlates with decreased overall survival. In our AML cohort, we observed significantly increased PRMT9 mRNA levels in AML CD34 + cells relative to normal counterparts (Fig. 1A), spurring our interest in evaluating its function in AML. We thus developed a Prmt9 conditional KO mouse (Mx1-Cre/Prmt9 f/f) and crossed it with an MLL-AF9 (MA9) knock-in mouse to generate Prmt9-KO/MA9 mice for further transplant assay. As a result, Prmt9-KO significantly delayed leukemogenesis in recipient mice carrying MA9 transplants evidenced by prolonged survival (Fig. 1B). Prmt9-KO impaired leukemia-initiating activity evidenced by secondary transplantation that significant less residual CD45.2 + MA9 leukemia cells were found in BM of secondary recipients receiving Prmt9-KO cells relative to those of Prmt9-WT controls. We next validated PRMT9 function in human AML. PRMT9-KD potentially impaired survival of primary AML CD34 + cells, whereas large sparing normal counterparts (Fig. 1C). Moreover, we designed a WT PRMT9 construct and corresponding catalytically dead mutant, both resistant to shPRMT9 (WT-R and MUT-R). Unlike MUT-R, WT-R expression rescued survival effects seen following shPRMT9 treatment, indicating that PRMT9 catalytic activity is required for AML survival (Fig. 1D). To define PRMT9 downstream effectors, we searched for methylated substrates in Molm13 with inducible PRMT9-KD or corresponding controls by performing SILAC analysis coupled with quantitative Mass-Spec (MS) (Fig. 1E). Complete analyses of normalized methyl peptides SILAC ratios revealed more prominent down-regulation of R-methylation in PRMT9-KD cells, with 49 methyl sites downregulated. Notably, methylated PAPB1 C-terminus peptide containing R493 was most significantly depleted upon PRMT9-KD. PABP1 potentiates translation initiation by binding to the poly(A) mRNA tail and interacting with factors like eIF4G. We thus generated an anti-R493 methylation antibody. Indeed, PRMT9 was confirmed to catalyze R493 methylation through in-vitro and cellular methylation assays (Fig. 1F, G). To define the function of R493 methylation, we ectopically expressed either WT PABP1 or the R493K mutant in Molm13 cells and then engineered those cells to express shPABP1 to KD endogenous PABP1. Notably, unlike WT PABP1, expression of R493K suppressed protein synthesis and increased apoptosis after endogenous PABP1-KD, suggesting that PRMT9 mediated PABP1-R493 methylation promotes AML viability (Fig. 1H, I).To discover PRMT9 inhibitors, we conducted a structure-based virtual screen of 960,000 compounds from NCI and Zinc compound libraries (Fig. 1J). We requested the top 300 candidates to assess their anti-leukemia activity in Molm13 cells (Fig. 1K). The top 20 most effective compounds were further analyzed via PRMT9 methylation assay (R493 antibody detection). NSC641396 exhibited the most potent PRMT9 inhibitory effects, as evidenced by decreased PABP1 R493 methylation levels at a dose <1 µΜ (Fig. 1L). NSC641396 treatment on AML CD34 + cells altered polysome profiling, decreased protein biosynthesis and reduced levels of short-lived proteins, suggesting translation inhibition (Fig. 1M).Taken together, our results suggest that PRMT9 plays an oncogenic role in AML or LSC maintenance, by promoting PABP1 methylation mediated translation activity. Further studies are needed to explore if targeting PRMT9 with newly identified lead compound ablates LSCs activity. [Display omitted] DisclosuresMarcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

In Vivo crispr Screening Identifies GLUT1 As a Key Metabolic Regulator of MLL-AF9-Driven Acute Myeloid Leukemia

Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting.The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation.Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production.To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136).Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. DisclosuresNo relevant conflicts of interest to declare.

Histone Acetyl-Transferase Kat2a Regulates Transcriptional Heterogeneity and Impacts Self-Renewal of Acute Myeloid Leukemia Cells in a Disease-Specific Manner

Acute Myeloid Leukemia (AML) is a heterogeneous disease characterized by transcriptional and epigenetic dysregulation. Lysine acetylation is a highly dynamic post-translational modification of histones that correlates with active transcription. Promoter acetylation influences heterogeneity, or noise, of gene expression, which has been postulated to alter the balance of cell fate decisions.Here we show that Kat2a, the mammalian orthologue of founder yeast histone acetyl-transferase and candidate noise regulator Gcn5, controls the frequency of self-renewing leukemia-propagating cells (LSC) in a retroviral mouse model of MLL-AF9 leukemia. Conditional Kat2a gene knockout (KO) results in delayed leukemia initiation with erosion of self-renewing LSC in secondary transplants. Accordingly, serial re-plating of Kat2a KO MLL-AF9 -transduced mouse bone marrow cells associates with an increased probability of cell differentiation.Through single-cell RNA-sequencing transcriptional analysis, we identify a distinct cell clustering organization in Kat2a KO primary MLL-AF9 leukemias, with relative depletion of cell subsets characterized by higher expression of self-renewal-associated genes. Transcriptionally-defined cell compartments are dissociated from conventional cell surface phenotyping, compatible with a functional definition of LSC. Kat2a loss associates with global enhancement of transcriptional heterogeneity amongst MLL-AF9 leukemic cells, which may underlie destabilization of self-renewal programs and allow exploitation of differentiation networks.Intriguingly, we observe that loss of Kat2a in the more protracted AML1-ETO9a retroviral model of AML, instead promotes transformation in vitro, and accelerates disease initiation in vivo, once again with unmodified representation of conventional cell surface phenotypic markers when compared to Kat2a wild-type (WT) leukemias.Taken together, our data position histone acetyl-transferase Kat2a as a novel multifaceted regulator of cell fate decisions in AML, and suggest a differential contribution of transcriptional activation to the implementation of leukemic programs in a disease-specific manner. DisclosuresPrabakaran:Noncodomics: Other: Co-founder of Noncodomics, a data analysis company.

A CHAF1B-Dependent Molecular Switch in Hematopoiesis and Leukemic Pathogenesis

Children with Down syndrome (DS) are at substantial risk of developing acute leukemia, which suggests that there are leukemia promoting genes on chromosome 21. Chromatin Assembly Factor 1B (CHAF1B), which resides in the DS critical region, is a member of the heterotrimeric CAF1 chromatin assembly complex that is responsible for depositing H3/H4 heterodimers at the replication fork during S-phase. We previously demonstrated that CHAF1B is overexpressed in acute megakaryocytic leukemia cells with trisomy 21 and have now discovered that it is also elevated in AML cell lines and primary tumor samples. This elevated expression is correlated with poor prognosis but the mechanism by which CHAF1B may promote leukemogenesis is unknown. In this study, we demonstrate that CHAF1B acts as regulator of transcription, maintaining an undifferentiated state by competing with CEBPA, FLI1, and RUNX2 for occupancy at the promoters and enhancers of myeloid differentiation genes.To study CHAF1Bin the context of normal hematopoiesis, we generated Chaf1b floxed mice and found that homozygous deletion was associated with rapid pancytopenia and bone marrow failure due to a cell cycle arrest and cell death. These effects were associated with downregulation of cell adhesion and metabolic pathways, as well as upregulation of genes related to sensitivity to stress and inflammatory stimuli. On the other hand, consistent with a pro-leukemic function, overexpression of CHAF1B promoted serial replating in vitro and was associated with the upregulation of genes involved in metabolism and proliferation. However, CHAF1B overexpression alone was not sufficient to promote leukemia in vivo .Since CHAF1B expression is elevated in cell lines with MLL rearrangements and primary AML samples, we next studied its requirements in the murine MLL-AF9 AML model. First, we found that overexpression of CHAF1B enhanced the formation of MLL-AF9 leukemic cells (LCs) in vitro and significantly accelerated disease in vivo . In contrast to the cell death and cell cycle arrest observed in HSPCs following Chaf1b deletion, there were no changes in proliferation or survival of LCs at 48 hours following Chaf1b deletion. Instead, we found Chaf1b deletion led to terminal myeloid differentiation of LCs, suggesting that CHAF1B has a major role in maintaining leukemic stem cells. In fact, heterozygous deletion of Chaf1b or overexpression of a dominant negative allele was sufficient to substantially reduce colony formation, as well as completely prevent leukemia in vivo .Transcriptome analysis of Chaf1b- deficient LCs revealed an increase in expression of over 1500 genes, including many of those associated with differentiation of myeloid leukemia cells. In comparison, we only identified 255 significantly downregulated genes, suggesting that CHAF1B primarily functions as a transcriptional repressor. By ChIP-seq, we determined that CHAF1B, but not the DNA replication factor PCNA, co-occupied the promoter and enhancer regions of many of these upregulated genes. Surprisingly, by ATAC-seq we found a widespread decrease in accessibility at CHAF1B binding sites following its knockdown, suggesting that other mechanisms are likely the key contributors to the differentiation phenotype. Motif analysis of CHAF1B-bound peaks in MLL-AF9 cells revealed specific enrichment of differentiation-driving transcription factor binding sites, including CEBPA, FLI1, and RUNX2. Therefore, we hypothesized that CHAF1B competes with transcription factors for occupancy at promoters of differentiation genes, preventing their expression and maintaining a leukemia stem cell state. We tested our hypothesis by comparing the occupancy of CEBA, FLI1, and RUNX2 at CHAF1B bound sites by ChIP-seq in cells replete or deficient for CHAF1B. We found that occupancy of these factors was significantly enhanced at CHAF1B-bound regions in LCs following Chaf1b deletion, and that this was associated with concomitant increases in expression of the differentiation target genes. We conclude that CHAF1B maintains the leukemic stem cell state by preventing differentiation through direct competition for chromatin occupancy with transcription factors at regulatory regions of genes that promote terminal maturation. DisclosuresNo relevant conflicts of interest to declare.

LSD1 Inhibition Exerts Its Anti-Leukemic Effect By Recommissioning PU.1- and C/Ebpα-Dependent Enhancers in AML

Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. We aimed to investigate the chromatin changes induced by an irreversible inhibitor of LSD1 (GSK-LSD1) on rMLL+ leukemias. For this, we treated rMLL+ murine AML and patient-derived xenografts with the GSK-LSD1 inhibitor in vivo and assessed chromatin accessibility changes using the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq). Treated mice exhibited a lower proportion of MLL-AF9+ leukemia cells detected in the bone marrow (BM) and signs of myeloid differentiation. Treated mice also had markedly improved survival with a median survival of 78 days vs. control mice (39 days). After 6 weeks of treatment with GSK-LSD1, mice xenotransplanted with human rMLL+ AML samples showed a significant decrease in engraftment and myeloid differentiation. Serial transplantation of murine AML revealed eradication of LSC upon GSK-LSD1 treatment of donor mice. Most importantly, LSD1 inhibition resulted in gain in chromatin accessibility in both murine and PDX-AML cells in vitro as well as in vivo . We then wanted to compare the effect of another anti-leukemic epigenetic drug, also known to induce myeloid differentiation of MLL-AF9+ cells, such as the DOT1L inhibitor EPZ4777. In contrast to GSK-LSD1, EPZ4777 treatment caused a global decrease in chromatin accessibility. The intersection of sites with dynamic accessibility upon DOT1L and LSD1 inhibition showed minimal overlap between the 2 treatments. The distribution of genomic regions with dynamic accessibility upon these drugs indicated that DOT1L inhibition affects a greater number of sites in promoter regions, whereas LSD1 inhibition affected a higher proportion of loci occupied within the intergenic and intronic regions. Furthermore, we sought to identify TF motif signatures at GSK-LSD1-induced dynamic sites. The most highly enriched motifs captured were those for myeloid TFs PU.1 and C/EBPα-β. We then performed ChIP-seq to identify sites of C/EBPα and PU.1 occupancy in the setting of GSK-LSD1 treatment. Interestingly, we observed a redistribution of PU.1 and C/EBPα occupancy upon treatment, consistent with the role of LSD1 in regulating the binding and activity of TFs. While PU.1 ChIPseq signal increased at dynamic ATAC-seq peaks in response to LSD1 inhibition, no increase in C/EBPα occupancy was observed. These dynamic sites also demonstrates GSK-LSD1-dependent co-occupancy of PU.1 and C/EBPα. We next sought to delineate whether PU.1 and C/EBPα are critical for these dynamic changes in chromatin accessibility in response to LSD1 inhibition. We performed ATAC-seq on PU.1 URE KO MLL-AF9 leukemias and C/EBPα KO MLL-AF9 leukemias treated with vehicle or GSK-LSD1. The increase in chromatin accessibility in TF-WT MLL-AF9 leukemias upon GSK-LSD1 was abrogated in both PU.1 URE KO and C/EBPα KO MLL-AF9 leukemia cells. We next assess the impact of TF-depletion on sensitivity to LSD1 inhibition. We treated PU.1 URE KO leukemias cells with GSK-LSD1 for 48 hours in vitro . These cells were more resistant to LSD1 inhibition when compared to PU.1 URE heterozygous and WT AML cells. In vivo, we reported loss of survival benefit upon GSK-LSD1 treatment in PU.1 URE KO MLL-AF9+ mice vs. WT mice. Similarly, C/EBPα KO leukemia cells were more resistant to LSD1 inhibition when compared to WT leukemia cells. After 2 weeks of treatment in vivo, C/EBPα KO leukemic mice also showed no decrease in MLL-AF9 allele burden. Taken together, our comparative investigations into the chromatin-modifying effects of two distinct epigenetic therapies targeting either LSD1 or DOT1L is illustrative of the concept that multiple paths toward myeloid differentiation can potentially be engaged to achieve therapeutic efficacy in AML. Our findings reveal that pharmacologic inhibition of LSD1 depends on balanced expression of the TFs PU.1 and C/EBPα, which can be used as predictors of response in AML aberrantly expressing these factors. DisclosuresMohammad:GSK: Employment. Ye:Incyte: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl:Celgene: Consultancy; Bayer Healthcare: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding. Kruger:GSK: Employment. Armstrong:Imago: Consultancy; Cyteir: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Combinatorial Use of Bexarotene Plus ATRA for the Treatment of Acute Myeloid Leukemia

Aim: All-trans retinoic acid (ATRA) results in high response rates in acute promyelocytic leukemia (APL), but has not had a significant impact on non-APL forms of acute myeloid leukemia (AML), even though retinoic acid receptors have been implicated in hematopoietic maturation and self-renewal. RXRs bind as heterodimers with RARs, and are expressed at higher levels in AML cells than are RARs. We sought to determine whether natural RXR ligands exist in primary leukemia cells, whether RXR ligands might augment or inhibit leukemic cell growth, and whether RXRs might be rational targets for AML therapeutics.Background: Retinoid receptors are nuclear hormone receptors, induced during terminal myeloid maturation with highest expressed in M4 and M5 AML. Retinoids serve as intracellular messengers and are activating ligands for the retinoic acid receptors (RAR) and retinoid X receptors (RXR).Methods: We developed an in vivo reporter assay that measures intracellular, activating nuclear receptor ligands in mouse bone marrow cells. We generated transgenic mice with GFP regulated by a UAS promoter. The yeast Gal4 transcription factor recognizes UAS promoter sequences. When a fusion of the Gal4-DNA binding domain (DBD) and a retinoid receptor ligand-binding domain (LBD) is expressed in UAS-GFP bone marrow cells, and an activating ligand is present, the cells express GFP. Leukemia was generated in bone marrow cells from these mice and from Mx1-Cre x Rxrαfl/fl x Rxrβfl/fl mice using MSCV-MLL-AF9 retrovirus.Results: We found that intermediate levels of RXRA ligands are present in mouse MLL-AF9 leukemia cells in vivo. To determine if activation of RXRA is required for MLL-AF9 cell growth, we transduced MSCV-MLL-AF9 retrovirus into bone marrow cells from Mx1-Cre+ x Rxrαfl/fl x Rxrβfl/fl mice and transplanted these into recipient mice. Mx1-Cre is “leaky.” Without external activation of Mx1-Cre (i.e. without pIpC treatment) we found that all subsequent leukemias were associated with deleted (“floxed out”) Rxra and Rxrb alleles, suggesting that the deleted allele was positively selected for. In secondary transplants, we found that MLL-AF9 leukemias with deleted Rxrα and Rxrβ were associated with shorter survival when compared with MLL-AF9 leukemias with wild type Rxrα and Rxrβ, suggesting that naturally occurring RXR ligands suppressed leukemic growth. To determine if pharmacologic activation of retinoid receptors could inhibit MLL-AF9 cell growth, we treated MLL-AF9 cells ex vivo with ATRA and bexarotene. We found that ATRA modestly inhibited growth, and this effect reached a plateau at 62 nM (this may partially explain the limited clinical efficacy of ATRA in non-APL AML) (see Figure). In contrast, the EC50 of bexarotene was 125 nM and the EC50 decreased to 54 nM when bexarotene was combined with 62 nM ATRA. Higher doses of ATRA decreased the bexarotene EC50 further to 43 nM and resulted in near complete culture collapse. Finally, to determine whether combination bexarotene and ATRA inhibited cell growth by inducing differentiation or apoptosis, we examined cytomorphology, cell division by Celltrace Violet staining (similar to CFSE), and Annexin V. Unexpectedly, we observed no evidence of differentiation, but rather increased Annexin V associated with ATRA and bexarotene-induced growth arrest.Conclusion: RXRs act as tumor suppressors in MLL-AF9 murine leukemia cells in vivo, and the activation of RXR/RAR heterodimers with bexarotene and all-trans retinoic acid leads to synergistic apoptosis and growth arrest. Additional experiments are now being performed to test the efficacy of this dug combination in vivo and assess their potential use in elderly relapsed/refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Macrophage Involvement in the Response of Acute Myeloid Leukaemia to Chemotherapy

Acute myeloid leukemia (AML) is a clonally heterogeneous neoplasm with poor survival rates. Although chemotherapy often leads to clinical remission, the relapse rate of AML is very high and usually fatal. Chemotherapy is usually effective at debulking AML blasts however a small population capable of re-initiating the leukemia (LIC) survive ultimately causing relapse. Mouse models of AML suggest that LIC are protected from chemotherapy by a protective niche similar to hematopoietic stem cell (HSC) niches. As 1) bone marrow (BM) macrophages are key regulators of HSC niche function and 2) tumor-associated macrophages promote chemotherapy resistance in several models of solid tumors (e.g. breast, lung & ovarian) we investigated a potential role of BM non-malignant macrophages in promoting chemoresistance of AML blasts.Using a mouse model of AML driven by the MLL-AF9 oncogene in which mouse HSC are transduced with MSC-MLL/AF9-ires-GFP and transplanted into syngeneic recipients, we examined how AML progression alters the phenotype of non-malignant GFP-negative BM macrophages by flow cytometry. 18 days post-transplant, GFP+ leukemia burden in the BM was in the 40-60% range. In non-malignant GFPneg BM macrophages, cell surface expression of MHC class II, CD80, L-selectin and neuropilin-1 was significantly decreased suggesting that AML blasts alter the function of BM macrophages.To determine whether BM macrophages could have any role in AML response to chemotherapy, we first determine whether non-malignant BM macrophages were depleted in vivo by treatment with cytarabine (5 days, 100mg/kg) and doxorubicin (3 days, 1mg/kg). Two days after cytarabine+doxoR treatment, most CD11b+ F4/80- Ly6G+ granulocytes and CD11b+ F4/80+ CD169- monocytes were ablated from the BM with 0.1% granulocytes and 1.0 % monocytes surviving after treatment. In contrast CD11b- F4/80+ CD169+ VCAM1+ macrophages were the most resilient myeloid cells in the BM with 9.5% still present 2 days after chemotherapy.To further investigate a possible role of these chemotherapy-resilient CD169+ macrophages in promoting response of AML blasts to chemotherapy, we transplanted MLL-AF9 leukemia cells generated with wild-type HSC into CD169DTR/WT mouse recipients in which one CD169 allele is knocked-in with human diphtheria toxin receptor (DTR). This enables specific depletion of CD169+ macrophages in vivo by injecting diphtheria toxin (DT). In this model, DT treatment targets host CD169+ macrophages but not the AML blasts as they do not contain the DTR knock-in. In the absence of chemotherapy, depletion of recipient CD169+ macrophages did not alter leukemia burden in the BM, spleen or blood 48 hours post depletion and did not alter mouse survival (median survival 29-30 days in both CD169-DTR and WT recipients treated with DT or saline).However when cytarabine + doxoR were administered 48 hours after DT administration, median survival of CD169-DTR recipients was significantly increased to 39.5 days compared to wild-type recipients 35 days, p<0.001. Furthermore leukemia burden in both the blood and spleen was significantly reduced.To determine whether CD169 could by itself mediate lower response of AML to chemotherapy, we transplanted MLL-AF9 cells generated from WT HSC into CD169DTR/DTR mice which are knocked-out for both CD169 alleles. Deletion of both CD169 alleles in recipient mice did not change mouse survival in response to chemotherapy in the absence of DT. Therefore absence of CD169 is not sufficient to prolong mouse survival in response to chemotherapy.In conclusion, non-malignant CD169+ macrophages may protect MLL-AF9 leukemia blasts from the cytotoxic effects of cytarabine and doxorubicin. The mechanisms of this chemoprotection remain to be determined but do not involve a direct effect of CD169 on AML blasts. DisclosuresNo relevant conflicts of interest to declare.

AML Associated Mesenchymal Stroma Cells Support Growth of AML Cells As a Result of Activated Notch Signaling and This Can be Targeted By Dexamethasone

Introduction: Acute myeloid leukemia (AML) is a hematological malignancy characterized by poor prognosis. Interaction with bone marrow mesenchymal stromal cells (BM-MSC) supports leukemic cells to evade chemotherapy-induced apoptosis. Of particular interest, massive parallel transcriptome sequencing of MSCs from mice and patients with preleukemic syndromes identified inflammatory signaling in inducing genotoxic stress, leukemogenesis, and clinical outcome. Recently, Dexamethasone (Dex), an anti-inflammatory drug, demonstrated antileukemic activity and improved clinical outcome in AML patients. These observations intrigued us to further investigate leukemia-BM niche-associated signaling and to identify signaling cascades supporting leukemic cells and BM-MSC interaction. Methods: BM-MSC from AML patients at diagnosis, or in remission were cultured alone or together with human AML cell lines (Kasumi-1 and M-07e) in direct contact or separated by transwells. Following transplantation experiments, BM-MSC were analyzed by microarray analysis. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML MSC. The functional characterization of BM-MSC and AML cell interaction was investigated using in vitro (cell proliferation and apoptosis) and in vivo (leukemia development, drug response, and survival) analysis. Results: To functionally evaluate the role of BM-MSC, we co-cultured MSCs from AML (AML-MSC) or non-leukemic (non-AML-MSC) patients with Kasumi-1 or M-07e cells. AML-MSCs significantly supported proliferation of AML cells by altering cell cycle distribution from G0/G1 to S phase (30.5 vs 51.3%, p&amp;lt;0.01). Strikingly, the cell cycle profile was unchanged when the cells were separated by a transwell. Consistently, in vivo,MLL-AF9 leukemic cells (Lin-cells transduced with an MLL-AF9 oncogene) and AML-MSCs succumbed to leukemia only when cotransplanted (40 vs 143 days, p&amp;lt;0.05). To gain further insights into leukemic cell-induced changes in MSC, we performed gene expression profiling in MSCs sorted from secondary transplanted leukemic (AML-MSC, n=3) and healthy (HD-MSC, n=3) mice. GSEA analysis identified hallmark-Notch signaling to be significantly overrepresented in AML-MSC as compared to controls (p&amp;lt;0.005, NES: 1.7). Increased Notch-1 and -2 expression could be verified at both the mRNA and protein levels in AML-MSCs. To functionally determine the contribution of Notch activation in MSC, we ectopically expressed the Notch intracellular domain (Notch-ICN) in a murine stromal cell line, MS-5 (Notch-ICN). Underscoring a role for Notch signaling in AML-MSC interaction, Notch-ICN expression significantly supported the proliferation of both MLL-AF9 leukemic cells and MSCs. A role for Notch signaling in the modulation of the inflammatory response has been previously reported. Dex an anti-inflammatory corticosteroid enhanced clinical outcome in AML patients. We, therefore, investigated whether Notch-dependent AML-MSC interaction could be therapeutically targeted using Dex. Interestingly, Dex treatment affected Notch1 expression (52%; p&amp;lt;0.005) and proliferation of leukemia cells (63%; p&amp;lt;0.001). Finally, we evaluated the therapeutic efficacy of Dex by treating mice transplanted with MLL-AF9 leukemic cells. Interestingly, mice treated with Dex exhibited significantly prolonged survival (73 vs 21 days, p&amp;lt;0.01) and had a lower percentage of blasts (50%, p&amp;lt;0.01) and MSC compared to leukemic mice receiving vehicle treatment. Our data thus identified Dex mediated abrogation of AML-MSC interaction is in part mediated by affecting Notch signaling. Conclusion: Our study demonstrated distinct functional differences between AML and non-AML-MSC and direct AML-MSC interaction as an indispensable factor in influencing cell proliferation, leukemogenesis, and survival in mice. We further identified and confirmed the role of Notch signaling in AML-MSC interaction. Therapeutically, Dex abrogated AML-MSC interaction, decreasing AML cell viability, and leukemic mice survival. Further studies are warranted to better understand how aberrant Notch signaling in MSC contributes to AML development and whether the use of Dex alone or in combination with Notch inhibitors could circumvent chemoresistance and relapse in AML patients. Disclosures Lenz: Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy; Agios: Research Funding; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria, Research Funding; AQUINOX: Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy.

RNA Binding Protein CELF2 Mutation Cooperates with MLL-AF9 in Driving Acute Myeloid Leukemia Development

Acute myeloid leukemia (AML) is an aggressive form of hematologic malignancies, caused by the accumulation of immature leukemic blasts in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission rate and 5-year-survival rate. MLL-AF9 mutations occur in approximately 10% of all acute leukemia patients, and are associated with poor therapy response and prognosis. Deregulated expression of ELAV-like family protein 2 (CELF2) has been identified in AML patients with MLL-AF9. As an RNA binding protein, CELF2 has been shown to regulate RNA splicing and embryonic hematopoietic development. However, the role of CELF2 in AML progression is still largely unknown. In this study, we first analyzed genetic data of 160 AML cases in the Cancer Genome Atlas in which CELF2 deletion account for 10% of the total cases. The survival time of patients with CELF2 deletion was much shorter than that of the others (P &amp;lt; 0.001). Interestingly, the expression of CELF2 was significantly decreased in different sub-type of acute myeloid leukemia cells, and the lowest expression of CELF2 was seen in MLL-AF9 (MA9) AML cells, when compared with that of normal myeloid cells. These results suggested that CELF2 deletion/mutation may play an important regulatory role in MA9-induced AML progression. To evaluate the potential role of Celf2 in the hematopoietic malignant transformation, we generated hematopoietic specific Celf2-deficient mice (Vav1-Cre;Celf2fl/fl) and found that Celf2-deficient hematopoietic stem cells (HSCs) over-expanded upon transplantation indicating a role of Celf2 in HSC proliferation. Consequently, the percentage of Celf2-KO HSC-derived myeloid cells was significantly increased in peripheral blood and bone marrow of recipient mice when compared with that of control mice. Celf2-KO Lin- cells were then infected by MA9-containing retrovirus and transplanted into lethally irradiated mice to generate Celf2-KO+MA9 mice. All Celf2-KO+MA9 mice succumbed to AML in 35 days, and their survival time is shorter than that of Celf2-WT+MA9 mice. The Celf2-KO+MA9 mice showed more aggressive phenotypes of AML than Celf2-WT+MA9 mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher extramedullar infiltration in spleen, liver and lung. To quantify the functional LSCs in Celf2-KO+MA9 mice and control MA9 mice, extreme limiting dilution transplantation assay was performed. The penetrance rate of leukemia in mice that received 1x103Celf2-KO+MA9 cells was significantly higher than that of control mice (100% versus 50%). The frequency of functional LSCs was significantly increased in Celf2-KO+MA9 mice as compared to that of the control, supporting a role of loss of Celf2 in promoting leukemia blast self-renewal. To investigate the underlying molecular mechanisms of Celf2 deletion in accelerating MA9 AML progression, we performed RNAseq to analyze the transcriptome programing changes associated with Celf2 in isogenic Celf2-WT+MA9 or Celf2-KO+MA9 AML cells. Consistent with severe extramedullar infiltration of leukemia cells in Celf2-KO+MA9 mice, gene set enrichment analysis showed up-regulated leukocyte activation and migration in Celf2-KO+MA9 leukemia cells. We next analyzed the downstream signaling pathways targeted by Celf2, and found that the phosphorylation of S6 was increased in Celf2-KO+MA9 cells, suggesting that Celf2 deletion might promote leukemia progression by activating mTORC1 signaling pathway. To confirm this, we treated Celf2-KO+MA9 mice with MA9 inhibitor (EPZ5676) and mTORC1 inhibitor (Rapamycin), and found a collaborative response to the treatment with a significant attenuation in leukemia burden in vivo. In conclusion, we found that CELF2 low expression is associated with AML patients with MA9 mutation. Celf2 deletion in mice induced myeloid-biased differentiation of HSCs. Notably, Celf2 deficiency promoted MA9-induced AML progression by activating mTORC1 signaling pathway independent of MA9 signaling pathway. Combinational therapy with MA9 inhibitors and mTORC1 inhibitors could significantly decrease the Celf2-KO+MA9 leukemia progression in vivo, indicating that combinational usage the drugs may have synergistic benefit for MA9 AML patients with low CELF2 expression. Disclosures No relevant conflicts of interest to declare.

SUV39H1 regulates the progression of MLL-AF9-induced acute myeloid leukemia

Epigenetic regulations play crucial roles in leukemogenesis and leukemia progression. SUV39H1 is the dominant H3K9 methyltransferase in the hematopoietic system, and its expression declines with aging. However, the role of SUV39H1 via its-mediated repressive modification H3K9me3 in leukemogenesis/leukemia progression remains to be explored. We found that SUV39H1 was down-regulated in a variety of leukemias, including MLL-r AML, as compared with normal individuals. Decreased levels of Suv39h1 expression and genomic H3K9me3 occupancy were observed in LSCs from MLL-r-induced AML mouse models in comparison with that of hematopoietic stem/progenitor cells. Suv39h1 overexpression increased leukemia latency and decreased the frequency of LSCs in MLL-r AML mouse models, while Suv39h1 knockdown accelerated disease progression with increased number of LSCs. Increased Suv39h1 expression led to the inactivation of Hoxb13 and Six1, as well as reversion of Hoxa9/Meis1 downstream target genes, which in turn decelerated leukemia progression. Interestingly, Hoxb13 expression is up-regulated in MLL-AF9-induced AML cells, while knockdown of Hoxb13 in MLL-AF9 leukemic cells significantly prolonged the survival of leukemic mice with reduced LSC frequencies. Our data revealed that SUV39H1 functions as a tumor suppressor in MLL-AF9-induced AML progression. These findings provide the direct link of SUV39H1 to AML development and progression.

Zoledronic acid inhibits the growth of leukemic MLL-AF9 transformed hematopoietic cells

A leukemic in vitro model produced by transducing Cord Blood derived-hematopoietic CD34+ cells with the MLL-AF9 translocation resulting in the oncogenic fusion protein, is used to assess for sensitivity to Zoledronic acid. These cells are practically immortalized and are of myeloid origin. Proliferation, clonogenic and stromal co-culture assays showed that the MLL-AF9 cells were considerably more sensitive to Zoledronic acid than normal hematopoietic CD34+ cells or MS-5 stromal cells. The MLL-AF9 cells were notably more inhibited by Zoledronic acid when cultured as colonies in 3 dimensions, requiring cell-cell contacts compared to suspension expansion cultures. This is coherent with the mechanism of action of Zoledronic acid inhibiting farnesyl diphosphate synthase which results in a block in prenylation of GTPases such that their role in the membrane is compromised for cell-cell contacts. Zoledronic acid can be proposed to target the MLL-AF9 leukemic stem cells before they emerge from the hematopoietic niche, which being in proximity to bone osteoclasts where Zoledronic acid is sequestered can be predicted to result in sufficient levels to result in an anti-leukemic action.

Tudor Domain-Focused CRISPR Dropout Screen Identifies SGF29 As a Novel Essential Gene in MLL-Rearranged Leukemias

Leukemic patients with MLL gene rearrangements (MLL-r) suffer from unfavorable prognosis and lack of effective targeted therapy. Recent studies suggest that MLL-r leukemias are frequently associated with epigenetic dysregulations; therefore, targeting epigenetic machineries may provide therapeutic opportunities to improve outcomes for MLL-r leukemias. Tudor domains are chromatin reader modules that recognize the methylated-lysine/arginine on histones. Given the crucial roles of histone methylations in controlling gene expression, we hypothesized that certain Tudor domain-containing genes may play essential roles in MLL-r leukemias. To identify a Tudor domain-containing gene(s) that is crucial for MLL-r leukemic cells, we custom-built a CRISPR library of 1,000 sgRNAs that targets the exon regions of all known Tudor domains (total 56 Tudor domains in 33 genes) with an average density of 17.7 sgRNAs per Tudor domain (Fig. 1A &amp; B). We delivered this sgRNA library by lentiviral transduction into mouse MLL-AF9 leukemic cells constitutively expressing Streptococcus pyogenesCas9 (SpCas9). We then compared the frequencies of each integrated sgRNA sequence before versus after 12 days of culture using high-throughput sequencing (NextSeq, Illumina), and identified Sgf29 (also known as Ccdc101) as a novel essential gene for MLL-r leukemia. Using SpCas9-mediated gene depletion (individual sgRNA co-expressed with a TagRFP reporter) and high-throughput flow cytometric growth competition assay (Attune NxT, ThermoFisher), we validated that SGF29 is essential in both mouse (MLL-AF9) and human (MV4-11 and MOLM-13) MLL-r leukemic cells. We also observed a stronger dependency to SGF29 in MLL-r leukemic cells compared to other cell types including T-cell lymphoma (Hut78), colon cancer (SW620), breast cancer (MDA-MB-231), lung cancer (H661) and glioblastoma (U251) using the growth competition assay. This suggests that targeting SGF29 may represent a novel therapeutic opportunity for MLL-r leukemias. Mechanistically, we found that CRISPR depletion of Sgf29 in MLL-AF9 cells induced morphological differentiation by Wright-Giemsa staining, and resulted in up-regulation of Cd11b (a myeloid-differentiation marker) and down-regulation of c-Kit (a hematopoietic progenitor and leukemic stem cell marker) by flow cytometry. To identify functionally important regions of Sgf29, we performed a saturation CRISPR-Cas9 gene body scan (also known as CRISPR-tiling screen) that targets all available "NGG" protospacer adjacent motifs (PAM) along the Sgf29 exons (total 147 sgRNAs; average targeting density of 6.0 bp per sgRNA) in MLL-AF9SpCas9+cells (Fig. 1A &amp; C). After high-throughput sequencing of the integrated sgRNA sequences, we mapped the data onto a three-dimensional (3D) crystal structure of SGF29 (PDB: 3ME9) and found that the CRISPR-scan hotpots (i.e.the essential regions) are centered around the H3K4me3-recognition pocket of SGF29. Using RNA-seq and gene set enrichment analysis (GSEA), we observed that the proto-oncogene Myc and Myc-regulated gene sets were significantly down-regulated in the Sgf29 depleted condition. Importantly, ectopic expression of Myc cDNA significantly rescued the lethality induced by Sgf29 depletion. Our current analyses suggest that SGF29 may serve as an upstream regulator of MYC to support the continuous proliferation of the leukemic cells. Based on our 3D CRISPR-scan analysis, we reasoned that development of small molecules binding to the "CRISPR-hot" region may disrupt the normal function of SGF29 (Fig. 1C). Therefore, we performed a virtual screen by docking 1.7 million diverse compounds into this H3K4me3-recognizing pocket of SGF29 using AutoDock Vina (The Scripps Research Institute). By combining the CRISPR-tiling screen, 3D structural analysis, and in silico compound docking, we prioritized a list of candidate binders of SGF29 Tudor domain for in vitro binding and cell survival assays. These may pave the way to develop the first SGF29 inhibitor with the ability to suppress MLL-r leukemias. Taken all together, our data suggest the epigenetic reader protein, SGF29, is a novel therapeutic candidate for MLL-r leukemias. We anticipate that the Tudor domain-focused CRISPR dropout screening and CRISPR-tiling strategies can also be applied to identify therapeutically relevant Tudor domain-containing genes in other hematopoietic malignancies. Disclosures Armstrong: Janssen: Research Funding; OxStem Oncology: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership; Epizyme, Inc.: Consultancy, Equity Ownership; AstraZeneca: Research Funding; Novartis: Research Funding; Mana Therapeutics: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership.

Selective Dependency of MLL-Rearranged Leukemia on Immunoproteasome Function

Several cellular pathways control the fine balance between self-renewal and differentiation to maintain leukemia-initiating cell (LIC) function. To identify cellular dependencies with relevance for oncogenic fusion proteins, we performed global proteome profiling. Acute myeloid leukemia (AML) was induced by retroviral expression of either MLL-AF9 (MA9) or AML1-ETO9a (AE) in murine hematopoietic stem and progenitor cells (HSPCs) (Lineage-Sca1+Kit+, LSK) which were subsequently transplanted into irradiated syngeneic recipients. After onset of leukemia, LIC-enriched (GFP+ Kithigh) cells isolated from 4 different primary recipients (per oncogene) were analyzed by in-depth quantitative proteomic analysis using high-resolution mass spectrometry (MS). More than 3,000 proteins were quantified with 868 proteins being differentially expressed between MA9 and AE LIC-enriched populations. In MLL-rearranged (MLLr) cells, gene set enrichment analysis (GSEA) revealed significant enrichment of cellular functions related to protein degradation and proteasome function. As this enrichment is present in MLLr-leukemia but not AE-driven LICs, may indicate an oncogene specific vulnerability. Expression of proteasome subunits is highly heterogeneous between different cell types and therefore may also be influenced by the underlying differentiation stage or oncogenic fusion. In published AML gene-expression datasets, immunoproteasome (IP) subunits PSMB8/LMP7 (p=0.0003***), PSMB9/LMP2 (p=0.0007***) and PSMB10/MECL1 (p&amp;lt;0.0001****) showed significantly higher expression in MLLr compared to non-MLLr-AML. IP is a proteasomal variant constitutively expressed in cells of hematopoietic origin, induced under stimulation with pro-inflammatory cytokines and relevant for mediating stress-responses during inflammation and infection. To assess for functional dependency of MLLr cells on IP subunits we performed an in vitro CRISPR/Cas9 dropout screen in MLLr MOLM-13 cells. Genetic inactivation of PSMB8/LMP7 resulted in outcompetition with 3/5 sgRNAs, while there was less dependency detectable for the other subunits. Specificity of this finding was confirmed in 5 different cell lines (4 MLLr; 1 non-MLLr) by RNAi using 2 shRNAs against PSMB8/LMP7 versus non-targeting control. To confirm these findings in primary cells, we used a previously published conventional LMP7 knockout mouse model (Fehling et al., Science, 1994). LSK cells sorted from the bone marrow (BM) of LMP7 knockout and wildtype mice were retrovirally transformed with either MA9, MLL-ENL (ME) or NUP98-HOXA9 (as non-MLLr control) to assess for disease development by serial plating in methylcellulose. Only in MA9 or ME transformed cells LMP7-deficiency limited re-plating capacity to 2-4 rounds. When we injected 2,5x 104 MA9-infected LSK cells into sublethally irradiated recipient mice, recipients of MA9-LMP7-/- cells (n=12) and MA9-LMP7+/+ (n=12) showed development of AML. However, recipients of MA9-LMP7-/- cells had a significant delay in AML development (median survival 63.0 days for LMP7+/+ versus 92.5 days for LMP7 -/- animals, p=0.0387*). Besides the significant delay in AML development, disease penetrance was reduced by 50%, indicating that deficiency for LMP7 impairs development of MA9 driven AML. In contrast, immunophenotypic abundance of HSPCs in LMP7-/- versus LMP7+/+ animals revealed comparable numbers in all relevant subpopulations. Competitive transplantation of LMP7-/- BM into recipient hosts showed no competitive disadvantage or lack of self-renewal capacity compared to LMP7+/+ controls. Pharmacologic inhibition of IP function using the specific LMP7-inhibitor PR-957 (ONX-0914) resulted in significant delay of disease development in secondary recipient hosts. To assess its effect on LIC frequency we performed limiting dilution assays of MA9 leukemic cells in sublethally irradiated recipient mice. PR-957 treatment reduced LIC frequency compared to DMSO control (1/57410 vs. 1/4450). Pharmacologic inhibition of PSMB8/LMP7 in human MLLr leukemia cell lines induced cellular differentiation. Likewise, cell cycle and metabolism appeared affected, functions which could be confirmed by global transcriptome analysis. Taken together, our studies uncover a selective dependency of MLLr-leukemia on IP function and identify PSMB8/LMP7 as a tractable target. Disclosures Heidel: Celgene: Consultancy; Novartis: Consultancy, Research Funding; CTI: Consultancy.

SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation.

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML). We recently showed that the histone 3 lysine 9 methyltransferase SETDB1 negatively regulates the expression of the proleukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on AML. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in AML patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with AML, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML.