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Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Pre-leukemic clones (i.e., clonal hematopoiesis; CH) are detectable years before the development of these aggressive malignancies, though the genomic events leading to transformation and expansion are not well-defined. Here, leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of pre-chemotherapy samples, we reconstruct the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy is revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan are hypermutated and enriched for complex structural variants (i.e., chromothripsis) while neoplasms with non-mutagenic chemotherapy exposures are genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to a discrete clinical exposure in each patient's life, we estimate that several complex events and genomic drivers are acquired after chemotherapy is administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected following reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA-damage. Overall, we reveal a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select pre-existing CH, but also promote the acquisition of recurrent genomic drivers.

The germinal center (GC) reaction plays an important role in generating humoral immunity and is believed to give rise to most B-cell lymphomas. GC entry and exit are tightly regulated processes, controlled by the actions of transcription factors such as BCL6. Herein, we demonstrate that protein arginine methyltransferase 5 (PRMT5), a symmetric dimethyl arginine methyltransferase, is also necessary for GC formation and affinity maturation. PRMT5 contributes to GC formation and affinity maturation at least in part through its direct interaction with and methylation of BCL6 at arginine 305 (R305), a modification necessary for the full transcriptional repressive effects of BCL6. Inhibition of PRMT5 in B-cell lymphoma lines led to significant upregulation of BCL6 target genes, and the concomitant inhibition of both BCL6 and PRMT5 exhibited synergistic killing of BCL6-expressing lymphoma cells. Our studies identify PRMT5 as a novel regulator of the GC reaction and highlight the mechanistic rationale of cotargeting PRMT5 and BCL6 in lymphoma.

HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

Background: Patients treated with cytotoxic chemotherapies and/or autologous stem-cell transplantation (ASCT) are at risk for therapy-related myeloid neoplasms (tMN). As these agents yield increased mutation burden in relapsed malignancies and leave evidence of exposure via mutational signatures, we studied the genomic and temporal relationship between chemo exposure and progression of clonal hematopoiesis (CH) to tMN. Methods: We analyzed 32 tMN whole genomes (WG) from 31 patients [27 acute myeloid leukemias (AML), 4 myelodysplastic syndromes]. For 7 patients with tMN post-high-dose melphalan/ASCT, we investigated the presence of antecedent CH using targeted sequencing (MSK-IMPACT; Bolton et al. Nat Gen 2020) on pre-melphalan blood mononuclear cells, granulocytes, or CD34+ apheresis samples. Results: TMN was diagnosed a median of 4.2 years (IQR, 2.6-6.6) following primary treatment. When compared to data from 200 de novo AML from TCGA (NEJM, 2013), tMNs had fewer mutations in FLT3 (9.7% v 28.0%; p = 0.028) and NPM1 (3.2% v 27.0%; p = 0.003). TP53 loss was enriched in tMNs (25.8% v 10.5%; p = 0.035 ). Mutational signature analysis revealed 5 known single base substitution (SBS) signatures in tMN: the hematopoietic stem-cell (SBS-HSC), aging (SBS1), melphalan (SBS-MM1), and platinum signatures (E-SBS1, E-SBS20) (Rustad et al. Nat Comm 2020, Pich et al. Nat Gen 2019). Complex structural variants (SV), defined as ≥3 breakpoint pairs involved in simultaneous copy number changes (Rustad et al. Blood Can Disc 2020), were observed in 7 tMNs; including chromothripsis in 6 tumors (19.4%), chromoplexy in 2 (6.5%), templated insertion in 1 (3.2%), and unspecified complex SV in 2 (6.5%). Chromothripsis has not been previously reported in de novo AML and, in 4 cases, involved chromosome 19 with hyper-amplification of the SMARCA4 locus (≥5 copies). CH variants that became clonal in tumor were seen in 5/7 pre-melphalan/ASCT samples and included mutations in TP53, RUNX1, NCOR1, NF1, CREBBP, DNMT3A, and PPM1D. Chemotherapy introduces hundreds of mutations, leaving each exposed cell with a unique catalogue (i.e., barcode). In fact, TMNs with evidence of chemo signatures had a higher mutation burden (median 1574 single nucleotide variants) than those without (median 938; p = 0.004). Detection of chemo signatures in bulk genome sequencing relies on one cell, with its catalogue of mutations, to expand to clonal dominance (Fig 1a, Landau et al. Nat Comm 2020). Given the long latency between exposure and tMN diagnosis, this single-cell expansion model was expected for all samples exposed to melphalan or platinum-based regimens (i.e., agents with a measurable signature). Strikingly, all patients with pre-tMN platinum exposure (n=7) had evidence of platinum SBS signatures whereas only 2 of 7 patients with prior melphalan/ASCT had a melphalan signature (SBS-MM1). As all platinum-exposed tMN had mutational evidence of exposure, a CH clone must have existed prior to exposure, supporting a single-cell expansion model. Absence of a chemo signature for 5/7 post-melphalan/ASCT tumors despite exposure implies tumor progression driven either by multiple clones in parallel (Fig 1b) or by an unexposed clone. As latency largely excludes the former, this suggests pre-tMN CH clones were re-infused during SCT, thus avoiding chemo exposure (Fig 1c). This is supported by two lines of evidence: 1) tMNs from 2 patients exposed to sequential platinum and melphalan/ASCT had platinum but not melphalan signatures confirming single-cell expansion of the pre-tMN CH clone post-platinum but with escape from exposure to melphalan in the ASCT (Fig 1d); 2) targeted sequencing of pre-tMN samples from melphalan/ASCT patients identified tMN genomic mutations at the CH level in 5/7 cases, including in all 3 tested apheresis samples - one of which (TP53) expanded to dominance without a melphalan signature. Conclusion: WG sequencing identified novel features of tMN revealing the key driver role of complex SV. Mutational signature analyses and targeted sequencing of pre-tMN samples can increase our understanding of tMN pathogenesis and demonstrate that tMNs arising post-ASCT are often driven by CH clones that re-engraft after escaping melphalan exposure. This mode of expansion suggests that a permissive, immunosuppressed, post-transplant environment might play a more important role than chemotherapy-induced mutagenesis in tMN pathogenesis. Figure 1 Figure 1. Disclosures Diamond: Sanofi: Honoraria; Medscape: Honoraria. Watts: Rafael Pharmaceuticals: Consultancy; Genentech: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Aptevo Therapeutices: Research Funding. Kazandjian: Arcellx: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bradley: AbbVie: Consultancy, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bolli: Amgen: Honoraria; Takeda: Honoraria; Janssen: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy. Scordo: Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; Omeros Corporation: Consultancy; Angiocrine Bioscience: Consultancy, Research Funding; McKinsey & Company: Consultancy. Lahoud: MorphoSys: Membership on an entity's Board of Directors or advisory committees. Stein: Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy; Genentech: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Novartis: Consultancy; Astellas: Consultancy; Syndax Pharmaceuticals: Consultancy; PinotBio: Consultancy; Daiichi Sankyo: Consultancy; Syros Pharmaceuticals, Inc.: Consultancy. Sauter: Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Bristol-Myers Squibb: Research Funding; GSK: Consultancy; Gamida Cell: Consultancy; Celgene: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Hassoun: Celgene, Takeda, Janssen: Research Funding. Mailankody: Bristol Myers Squibb/Juno: Research Funding; Physician Education Resource: Honoraria; Plexus Communications: Honoraria; Takeda Oncology: Research Funding; Jansen Oncology: Research Funding; Fate Therapeutics: Research Funding; Allogene Therapeutics: Research Funding; Legend Biotech: Consultancy; Evicore: Consultancy. Korde: Medimmune: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Hultcrantz: Daiichi Sankyo: Research Funding; Intellisphere LLC: Consultancy; Curio Science LLC: Consultancy; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding. Shah: Bristol Myers Squibb: Research Funding; Janssen: Research Funding. Shah: Janssen Pharmaceutica: Research Funding; Amgen: Research Funding. Park: Servier: Consultancy; Affyimmune: Consultancy; Autolus: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy; BMS: Consultancy; Novartis: Consultancy; Kura Oncology: Consultancy; Curocel: Consultancy; Artiva: Consultancy; Innate Pharma: Consultancy; Intellia: Consultancy; Amgen: Consultancy; Kite Pharma: Consultancy. Landau: Genzyme: Honoraria; Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Sekeres: BMS: Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Ho: Blueprint Medicine: Membership on an entity's Board of Directors or advisory committees. Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Lesokhin: pfizer: Consultancy, Research Funding; Janssen: Honoraria, Research Funding; Iteos: Consultancy; Serametrix, Inc: Patents & Royalties; Genetech: Research Funding; Trillium Therapeutics: Consultancy; bristol myers squibb: Research Funding; Behringer Ingelheim: Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Landgren: Janssen: Other: IDMC; Janssen: Research Funding; Amgen: Honoraria; Celgene: Research Funding; Janssen: Honoraria; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria.

Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy. We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival. Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment. Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. Disclosures Neel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

Although the majority of patients with acute myeloid leukemia (AML) initially respond to chemotherapy, many of them subsequently relapse, and the mechanistic basis for AML persistence following chemotherapy has not been determined. Recurrent somatic mutations in DNA methyltransferase 3A (DNMT3A), most frequently at arginine 882 (DNMT3AR882), have been observed in AML and in individuals with clonal hematopoiesis in the absence of leukemic transformation. Patients with DNMT3AR882 AML have an inferior outcome when treated with standard-dose daunorubicin-based induction chemotherapy, suggesting that DNMT3AR882 cells persist and drive relapse. We found that Dnmt3a mutations induced hematopoietic stem cell expansion, cooperated with mutations in the FMS-like tyrosine kinase 3 gene (Flt3ITD) and the nucleophosmin gene (Npm1c) to induce AML in vivo, and promoted resistance to anthracycline chemotherapy. In patients with AML, the presence of DNMT3AR882 mutations predicts minimal residual disease, underscoring their role in AML chemoresistance. DNMT3AR882 cells showed impaired nucleosome eviction and chromatin remodeling in response to anthracycline treatment, which resulted from attenuated recruitment of histone chaperone SPT-16 following anthracycline exposure. This defect led to an inability to sense and repair DNA torsional stress, which resulted in increased mutagenesis. Our findings identify a crucial role for DNMT3AR882 mutations in driving AML chemoresistance and highlight the importance of chromatin remodeling in response to cytotoxic chemotherapy.

SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation–seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.

To mount an effective immune response against infectious pathogens or tissue injury, hematopoietic stem cells (HSCs) increase their proliferation and production of myeloid cells, including macrophages, which destroy the pathogens and repair the damaged tissue. Proper resolution of inflammation is essential to restore hematopoietic homeostasis, as unrestrained inflammation can result in life-threatening pathologies such as sepsis, autoimmune disorders and cancer. The molecular mechanisms that control the resolution of inflammation, and how these contribute to disease phenotypes, are poorly understood. BAF (SWI/SNF) complexes are ATPase dependent chromatin-remodeling complexes that play fundamental roles in transcription. BAF complexes use the energy of ATP to modulate the accessibility of transcription factors to DNA and thus, orchestrate the gene expression programs that control proliferation and cellular identity. Genes encoding for BAF subunits are frequently mutated in cancer and developmental disorders. In hematopoietic malignancies, loss-of-function mutations and low expression of specific BAF subunits have been reported in patients with anemia and bone marrow failure. Work from our lab previously demonstrated that the hematopoietic-specific BAF complex subunit Dpf2 cooperates with the transcription factor Runx1 to regulate myeloid differentiation. Based on these studies, we generated a hematopoietic-specific Dpf2 knock-out mouse model and found that mice lacking Dpf2 develop pancytopenia, anemia and an uncontrolled inflammatory response that results in early death. Dpf2-/- peripheral blood samples showed dysplastic features including increased number of polychromatophilic blood cells and Howell-Jolly bodies in erythrocytes. Histopathological analyses revealed the presence of fibrosis and prominent infiltration of histiocytes in multiple organs, including lungs, liver and spleen. Detailed chemical profiling of plasma showed increased levels of multiple pro-inflammatory cytokines, indicative of systemic inflammation. Flow cytometry analyses and Mass cytometry profiling further revealed an expansion of myeloid lineages, specifically monocytes and macrophages, concomitant with severe defects in lymphoid and erythroid differentiation. We also found that Dpf2-/-HSCs had increased serial re-plating capacity and a marked myeloid differentiation bias. To identify the transcription factor networks underlying these phenotypes, we performed RNAseq and ATACseq on control and Dpf2-/- HSCs. Gene Set Enrichment Analyses indicated that Dpf2-/- HSCs have extensive gene expression alterations in immune signaling and interferon response pathways, as well as leukocyte and erythroid differentiation. We also found that Dpf2 loss results in pronounced changes in expression and genomic accessibility of specific transcription factors that control macrophage differentiation and proliferation. Together, our mechanistic studies support a model whereby the absence of Dpf2 results in misregulation of the transcription factor networks that establish macrophage cell identity, leading to a marked increase in macrophage infiltrations and shortened survival of the mice. Treatment of the Dpf2-/-mice with clodronate-containing liposomes, which deplete macrophages from bone marrow and spleen, prolonged survival of the mice. Our work uncovers a novel role of Dpf2 in restraining inflammatory responses by controlling macrophage proliferation and function. Moreover, we propose that, in addition to their tumor suppressive roles in cancer, BAF complexes may have a central role in the prevention of immunopathologies. Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Scientific founder, fiduciary board of directors member, scientific advisory board member, shareholder, and consultant for Foghorn Therapeutics (Cambridge, MA). . Vega:NCI: Research Funding.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease., Several studies have recently demonstrated the role of the MSI2 RNA binding protein in normal and malignant haematopoietc stem cells. In this study, the authors show that MSI2 is required for maintaining myelodysplastic syndrome stem cells in mice and that MSI2 expression predicts poor prognosis in patients affected by this disease.

SUMMARY Acute myeloid leukemia (AML) is a heterogeneous and fatal disease with an urgent need for improved therapeutic regimens given that most patients die from relapsed disease. Irrespective of mutation status, the development of aggressive leukemias is enabled by increasing dependence on signaling networks. We demonstrate that a hyperactive signalosome drives addiction of AML cells to a tumor-specific Hsp90 species (teHsp90). Through genetic, environmental, and pharmacologic perturbations, we demonstrate a direct and quantitative link between hyperactivated signaling pathways and apoptotic sensitivity of AML to teHsp90 inhibition. Specifically, we find that hyperactive JAK-STAT and PI3K-AKT signaling networks are maintained by teHsp90 and, in fact, gradual activation of these networks drives tumors increasingly dependent on teHsp90. Thus, although clinically aggressive AML survives via signalosome activation, this addiction creates a vulnerability that can be exploited with Hsp90-directed therapy., Graphical Abstract

TET1/2/3 are methylcytosine dioxygenases that regulate cytosine hydroxymethylation. Tet1/2 are abundantly expressed in HSC/HPCs and are implicated in hematological malignancies. Tet2 deletion in mice causes myeloid malignancies, while Tet1-null mice develop B cell lymphoma after an extended period of latency. Interestingly, TET1/2 are often concomitantly downregulated in acute B-lymphocytic leukemia. Here, we investigated the overlapping and non-redundant functions of Tet1/2 using Tet1/2 double-knockout (DKO) mice. DKO and Tet2[superscript −/−] HSC/HPCs show overlapping and unique 5hmC and 5mC profiles. DKO mice exhibit strikingly decreased incidence and delayed onset of myeloid malignancies in comparison to Tet2[superscript −/−] mice and in contrast develop lethal B cell malignancies. Transcriptome analysis of DKO tumors reveals expression changes in many genes dysregulated in human B cell malignancies, including LMO2, BCL6, and MYC. These results highlight the critical roles of TET1/2 individually and together in the pathogenesis of hematological malignancies., National Institutes of Health (U.S.) (Grant HDO45022), National Institutes of Health (U.S.) (Grant CA084198), Simons Foundation

Background Epigenetic dysregulation is a hallmark feature of myelodysplastic syndrome (MDS) as epigenetic regulator genes, such as ASXL1, TET2, and SRSF2, are highly mutated in MDS patients. Tet2 (ten-eleven translocation-2, a methylcytosine deoxygenase) mutations are found in ~20% of MDS patients; they are also seen in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN). Loss of Tet2 in hematopoietic cells leads hematopoietic defects including enhanced hematopoietic stem cell (HSC) self-renewal, myeloid expansion and an increased propensity to develop MDS or AML. The lysine acetyltransferase (KAT) p300 (encoded by the EP300 gene) plays pivotal roles in essential cellular functions including proliferation, differentiation and signal transduction. However, the function of p300 in hematopoietic malignancies is clearly cell-context dependent, as we have shown that p300 functions as an oncogene in AML1-ETO-driven AML, and a tumor suppressor gene in NUP98-HOXD13-driven MDS. The EP300 and the related CREBBP genes are infrequently mutated in MDS patients. Methods To investigate the role of p300 in MDS driven by lack of Tet2, we generated a mouse model carrying a hematopoietic-specific EP300 conditional knockout on a Tet2-null background and characterized the effect of p300 loss on HSC function and the pathogenesis of MDS and AML. We also explored the therapeutic potential of manipulating p300 KAT activity to affect the course of MDS driven by Tet2 loss. Results Compared to the Tet2-/- mice, the EP300Δ/ΔTet2-/- (DKO) mice had shortened survival and an accelerated onset of AML, with increased blasts in the bone marrow and peripheral blood, anemia, splenomegaly, and universal presence of granulocytic sarcomas, demonstrating that depletion of p300 enhances the leukemogenicity of Tet2-/- HSC. The DKO mice showed enhanced hematopoietic stem/progenitor cell (HSPC, defined as Lin-Sca1+Kit+ cell) proliferation and an increased HSPC pool only 2 weeks after p300 deletion, far in advance of the development of any disease manifestations. In contrast, deletion of p300 in wild type (wt) mice affected neither HSPC proliferation, nor pool size. To further define the mechanisms by which these two genetic events cooperate to drive the development of MDS/AML, we assessed the DNA methylation status and gene expression signatures of HSPCs isolated from wt, Tet2-/- and DKO mice. As expected, loss of Tet2 leads to a genome-wide decrease in 5-hmC, whereas loss of p300 does not change 5-hmC distribution across the genome. Interestingly, DKO HSPCs showed local gains of 5-hmC at a variety of genes (compared to Tet2-null HSPCs), including at the c-Myb gene, which is a known oncogenic driver of AML. Gene Set Enrichment Analysis (GSEA) revealed that p300 loss in Tet2-/- HSPCs interferes with leukocyte differentiation, enhances proliferation and inhibits apoptosis. ChIP-X Enrichment Analysis (ChEA) further also indicated that among the differentially expressed genes, between DKO HSPCs and Tet2-null HSPCs, are potential Myb target genes. Given this data and the RNA-Seq and Q-PCR results that showed elevated levels of Myb mRNA in the DKO HSPCs compared to the Tet2-/- HSPCs, we examined the biological effects of knocking down (KD) Myb. Myb KD reduced the colony-forming capacity of the DKO HSPCs, but not the Tet2 -/- HSPCs, identifying its potential role in mediating the effect of p300 on MDS prone HSPCs. These results indicate that loss of p300 in Tet2-null HSPCs enhances their leukemogenicity through elevated Myb expression and activity. To determine whether increasing p300 KAT activity could have an opposite effect on MDS cells, we utilized I-CBP112, which binds p300 and increases its KAT activity and ability to stimulate H3K18 acetylation. Exposure to I-CBP112 did in fact eliminate the indefinite self-renewal capacity of Tet2-/- HSPCs, as measured by serial replating assays, further highlighting the importance of p300 KAT activity in regulating the behavior of Tet2-/- HSPCs. Additional in vivo studies of the effects of I-CBP112 on the course of MDS/AML driven by the absence of Tet2 are ongoing. Conclusions This work clearly indicates that the EP300 gene can play a tumor suppressor role in MDS/AML driven by loss of Tet2- by repressing Myb expression and activity. It also suggests that the enhancement of p300's KAT activity by use of small molecules, could be an effective therapeutic strategy for MDS/AML. Disclosures Cole: Abbvie: Consultancy; Acylin Therapeutics: Equity Ownership.

Background Myelodysplastic syndromes (MDS) are characterized by ineffective erythropoiesis with dysplastic bone marrow leading to peripheral cytopenia, risk of infection, and progression to acute myelogenous leukemia. Maitake mushroom beta-glucan, a dietary supplement, stimulates hematopoietic progenitor cell differentiation, granulocyte colony-stimulating factor production, and recovery of peripheral blood leukocytes after bone marrow injury. This phase II trial examined the effects of Maitake on innate immune function in MDS. Methods Myelodysplastic syndromes patients with International Prognostic Scoring System Low- and Intermediate-1-risk disease received oral Maitake extract at 3 mg/kg twice daily for 12 weeks. Primary endpoints included neutrophil count and function tested as endogenous or stimulated neutrophil production of reactive oxygen species (ROS) by flow cytometry compared with age-matched healthy controls (HC). ROS activators were Escherichiacoli, phorbol ester, and the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Complete blood counts, chemistry panels, iron studies, and monocyte function were evaluated. Results Of 21 patients enrolled, 18 completed the study and were evaluable. Maitake increased endogenous (basal) neutrophil (p = 0.005) and monocyte function (p = 0.021). Pre-treatment monocyte response to E. coli was reduced in MDS patients compared with HC (p = 0.002) and increased (p = 0.0004) after treatment. fMLP-stimulated ROS production response also increased (p = 0.03). Asymptomatic eosinophilia occurred in 4 patients (p = 0.014). Other changes in albumin, hemoglobin, and total protein were not clinically relevant. Conclusions Maitake was well tolerated. Enhanced in vitro neutrophil and monocyte function following treatment demonstrate that Maitake has beneficial immunomodulatory potential in MDS. Further study is warranted.

AML1-ETO (AE), a fusion oncoprotein generated by t(8;21), can trigger acute myeloid leukemia (AML) in collaboration with mutations including c-Kit, ASXL1/2, FLT3, N-RAS, and K-RAS. Caspase-3, a key executor among its family, plays multiple roles in cellular processes, including hematopoietic development and leukemia progression. Caspase-3 was revealed to directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3-compromised background and thereby accelerate leukemogenesis. Therefore, we developed a Caspase-3 knockout genetic mouse model of AML and found that loss of Caspase-3 actually delayed AML1-ETO9a (AE9a)-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation and/or progression of AML. We report here that loss of Caspase-3 triggers a conserved, adaptive mechanism, namely autophagy (or macroautophagy), which acts to limit AE9a-driven leukemia. Furthermore, we identify ULK1 as a novel substrate of Caspase-3 and show that upregulation of ULK1 drives autophagy initiation in leukemia cells and that inhibition of ULK1 can rescue the phenotype induced by Caspase-3 deletion in vitro and in vivo. Collectively, these data highlight Caspase-3 as an important regulator of autophagy in AML and demonstrate that the balance and selectivity between its substrates can dictate the pace of disease.

Small molecule protein arginine methyltransferase inhibitors (PRMTi) are being actively pursued for the treatment of a variety of cancers; however, the mechanisms of response to PRMTi remain poorly understood. CARM1, also known as PRMT4, is significantly overexpressed in AML, as well as many solid tumors, and regulates myeloid differentiation. We have shown the dependency of AML cells, but not normal blood cells, on CARM1 activity, based on CARM1 knockout, CARM1 knockdown, and chemical inhibition (Greenblatt et al. Cancer Cell 2018). These experiments showed that CARM1 regulates essential processes in leukemia cells, and is critical for leukemic transformation. Although several small molecule inhibitors of CARM1 have been reported recently, many display a lack of selectivity for CARM1 or fail to produce a biological response. The recent discovery of potent and selective CARM1 inhibitors (Drew et al., 2017), has made it possible to investigate the implications of pharmacological inhibition of CARM1 in vitro and in vivo. In vitro, a selective CARM1 inhibitor, EPZ025654, reduced the methylation of a CARM1 substrate, BAF155, in a time and concentration-dependent manner, while the specific histone targets of CARM1 remained unchanged. Translocation (8;21) AML samples in the Eastern Cooperative Oncology Group cohort, have significantly higher CARM1 expression compared to normal CD34+ controls. This led us to hypothesize that CARM1 is a direct target of the AML1-ETO fusion protein. Therefore, we assessed whether EPZ025654 could inhibit AML1-ETO driven gene expression. AML1-ETO specific target genes showed significant changes in expression following EPZ025654 treatment. AML1-ETO positive patient samples also displayed decreased colony formation in methylcellulose and increased myeloid differentiation in response to CARM1 inhibition. We next evaluated EZM2302, a compound structurally related to EPZ025654, that is highly orally bioavailable and is well tolerated in mice (Drew et al., 2017). We generated AE9a-GFP primary transplantation mice and treated them with 100 mg/kg of EZM2302 or vehicle twice-daily (BID). The inhibitor treated mice showed significantly improved survival as well as fewer GFP+ cells in the peripheral blood over time. GFP+ AE9a bone marrow cells also showed decreased colony formation in vitro and induced macrophage differentiation in methylcellulose. GFP+ cells were isolated by FACS and submitted for RNA-sequencing. Flow cytometry analysis post-treatment revealed a significant downregulation of c-Kit and increased differentiation of hematopoietic stem and progenitor cells. Resistance to epigenetic targeted therapeutics has been observed, often through the induction of kinase signaling pathways. Therefore, we explored synergistic combinations with CARM1 inhibition using RNA-sequencing and proteomics analysis in leukemia cell lines. We used L1000 profiling (Subramanian et al., 2017) to simultaneously profile the transcriptional response of 18 AML cell line and CD34+ cells after 6 days of treatment. The AML1-ETO positive cell lines exhibited an IC50 in the 0.4-3 μM range, while CD34+ cells and several AML cell lines appeared to be resistant to CARM1 inhibition. While gene expression changes resulting from alterations in RNA stability were observed, the most significant differences between sensitive and resistant cell lines were genes associated with the regulation of cell cycle progression. Gene expression changes were evaluated over time in an AML1-ETO positive cell line, SKNO-1. SKNO-1 cell lines showed an upregulation of a gene expression signature associated with PI3K/AKT/mTOR signaling, with the most significant gene expression changes occurring 7-14 days post treatment. We simultaneously profiled these cells using multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in response to CARM1 inhibition over time. A comparison of this response to chemical perturbation signatures in the L1000 database, identified several chemical inhibitors of the PI3K/AKT/mTOR axis that could reverse the gene expression changes induced by CARM1 inhibition. This finding elucidated a response mechanism for CARM inhibition and a synergistic therapeutic strategy that has the potential to improve CARM1 directed therapy. Disclosures No relevant conflicts of interest to declare.

The leukemogenic AML1-ETO fusion protein is produced by the t(8;21) translocation, which is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AETFC, that contains multiple transcription factors and cofactors. Among these AETFC components, E2A (also known as TCF3) and HEB (also known as TCF12), two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA (E-box) binding capacity to AETFC, and are functionally essential for leukemogenesis. However, we find that the third E protein, E2-2 (also known as TCF4), is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the basic helix-loop-helix (bHLH) DNA-binding domain of E2-2. Gene expression profiling and ChIP-seq analysis reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, consistent with the fact that E2-2 is a critical transcription factor in dendritic cell (DC) development, our studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with DC differentiation, and that restoration of E2-2 triggers a partial differentiation of the AML1-ETO-expressing leukemic cells into the DC lineage. Meanwhile, E2-2, but not E2A or HEB, represses MYC target genes, which may also contribute to leukemic cell differentiation and apoptosis. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates the development of leukemia. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a molecular heterogeneity of AETFC, which merits further study in different t(8;21) AML patients, as well as in its potential regulation of cellular heterogeneity of AML. These studies should improve our understanding of the precise mechanism of leukemogenesis and assist development of diagnostic and therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

Introduction: Additional sex combs-like 1 (ASXL1) is frequently mutated in a wide range of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasm, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). Notably, ASXL1 mutations are generally associated with the poor clinical outcomes. We and others have established Asxl1 mouse models and demonstrated that loss of Asxl1 leads to MDS-like disease, which can transform to myeloid leukemia in aged mice. These studies suggest that additional mutations may cooperate with Asxl1 loss to induce the leukemia transformation. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Methods: To identify cooperating events with ASXL1 mutations in myeloid malignancies, we recruited a cohort of 138 ASXL1 mutated patients and performed targeted exome sequencing. We then characterized the hematopoietic features using mouse models. A serial hematopoietic phenotypic analyses were used, including peripheral blood counts, flow cytometry, colony assay, morphology and transplantation assays. To decipher the molecular mechanisms by which loss of Asxl1 and Nf1 cooperate in promoting myeloid leukemia transformation, we performed RNA-seq and ChIP-seq to identify the differentially expressed genes and their associated histone modifications in four genotypes of mice, WT, Asxl1+/-, Nf1+/-, and Asxl1+/-;Nf1+/- mice. Finally, the leukemic mice were treated with pharmacologic inhibitors targeting both MAPK pathway and BET bromodomain in vivo as a proof-of-concept approach. Results: We analyzed the gene mutation profiles of 138 ASXL1 mutated patients based on targeted sequencing and found that 35 of these patients have gene mutations involving in RAS/MAPK signaling pathway, including NF1, NRAS, KRAS, PTPN11 or CBL. The incidence of AML was significantly higher in patients with RAS pathway mutations (48.6%) than in the cases without RAS pathway mutations (29.1%, p = 0.036, chi-square test). These data suggest that concomitant mutations of ASXL1 and RAS pathway genes associate with poor prognosis in myeloid malignancies. To validate the functional significance of cooperative mutations of ASXL1 and NF1, a negative regulator of the RAS signaling pathway, in the disease progression of myeloid leukemia, we generated Asxl1+/-;Nf1+/- and Mx1Cre;Asxl1fl/fl;Nf1fl/fl (Asxl1Δ/Δ;Nf1Δ/Δ) mice and performed hematopoietic phenotypic analyses. Asxl1 loss cooperates with haploinsufficiency of Nf1 to accelerate the development of myeloid leukemia in mice. Asxl1Δ/Δ;Nf1Δ/Δ mice displayed a rapid, progressive leukocytosis with severe anemia and thrombocytopenia 3-6 months after pIpC injection, indicative of aggressive myeloid leukemia with a 100% penetrance. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells lead to transcriptional activation of multiple pathways critical for leukemogenesis, such as MYC, NRAS, and BRD4. Convergent analysis of RNA-seq and ChIP-seq data reveal that the hyperactive MYC and BRD4 transcription program is correlated with elevated H3K4 tri-methylation at the promoter regions of genes involving these pathways. Importantly, pharmacological inhibition of both MAPK pathway and BET bromodomain prevents leukemia initiation and inhibits disease progression in Asxl1Δ/Δ;Nf1Δ/Δ mice, and significantly prolonged the survival of leukemic Asxl1+/-;Nf1+/- mice. Conclusion: This study sheds lights on the understanding of the cooperative effect between epigenetic alterations and signaling pathways in accelerating the progression of myeloid malignancies and provides a rationale therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations. Disclosures No relevant conflicts of interest to declare.

Aberrant expression of long non-coding RNAs (lncRNAs) might contribute to the development and progression of leukemia. However, functional studies on the actual role of lncRNAs during the development of leukemia remain scarce, and very few lncRNAs have been shown to be involved in leukemogenesis. HoxBlinc is an anterior HoxB gene-associated intergenic lncRNA. It is a cis-acting lncRNA and functions as an epigenetic regulator to coordinate anterior HoxB gene expression. Giving the dysregulation of HOXA/B genes is a dominant mechanism of leukemic transformation, HoxBlinc might be an oncogenic lncRNA of leukemia. To determine whether HOXBLINC lncRNA is aberrantly expressed in human AML samples, we performed RT-qPCR on bone marrow mononuclear cells (BMMNCs) from a cohort of 73 AML patients. A dramatic up-regulation of HOXBLINC was observed in over 60% of the patients. When TCGA-AML datasets of a cohort of 179 AML patients were analyzed for their HOXBLINC expression, a significant portion of these AML patients had high levels of HOXBLINC expression. Interestingly, AML patients with high HOXBLINC expression (the top thirty percentile of patients) had a significantly shortened survival as compared to patients with low HOXBLINC expression (the bottom thirty percentile). To investigate the impact of HoxBlinc overexpression on normal hematopoiesis and the pathogenesis of hematological malignancies in vivo, we generated a HoxBlinc transgenic(Tg) mouse model. Within 1 year of age, 67% of the HoxBlincTg mice (10 of 15) died or were sacrificed because of a moribund condition due to AML. We then assessed whether overexpression of HoxBlinc affects the pools of HSC/HPCs by flow cytometric analysis on the BM cells of young WT and HoxBlincTg mice (8-10 weeks of age). HoxBlincTg BM had a dramatically greater number of LT-HSC, ST-HSC, MPP cells, and a significantly higher percentage of GMP, but a lower percentage of MEP/CMP cell populations as compared to WT group. To determine the effect of HoxBlinc overexpression on the function of HSC/HPCs, we performed paired-daughter cell assay, replating assay and liquid culture on sorted LT-HSC, LSK or LK cells from young WT and HoxBlincTg mice, the results indicate that transgenic expression of HoxBlinc enhances HSC self-renewal and impairs HSC/HPC differentiation. To assess whether HoxBlinc overexpression-mediated changes in HSC/HPC function are cell-autonomous, we performed competitive transplantation assays to examine the repopulating capacity of HoxBlincTg BM cells. When the donor cell chimerism was analyzed kinetically in the PB of recipient mice, the CD45.2 cell population remained ~50% in mice receiving WT BM cells, whereas the CD45.2 chimerism in the recipients transplanted with HoxBlincTg BM cells steadily increased. Interestingly, mice receiving HoxBlincTg BM cells developed AML at 2-6 months after transplantation. Previous data reported that HoxBlinc can recruit the Setd1a/Mll1 histone H3K4 methyltransferase complex to mediate formation of the active topologically associated domain (TAD) in the anterior HoxB locus for transcription of the anterior HoxB genes. In this study, LSK or LK cells sorted from young WT and HoxBlincTg mice were analyzed by RNA-seq, ATAC-seq, H3K4me3 CHIP-seq and 4C analysis. Mechanistically, HoxBlinc overexpression alters HoxB locus chromatin three-dimensional organization to enhance enhancer/promoter chromatin accessibility and coordinate the expression of not only HoxB1-5 but also HoxA9, Runx1, Meis1 and so on, which are critical genes for HSC regulation and/or leukemogenesis. Our study provides novel insights into the HSC regulation by lncRNAs and identifies HOXBLINC, which coordinates to maintain an oncogenic transcription program for leukemic transformation, as a potent oncogenic lncRNA in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

ASXL2 is frequently mutated in acute myeloid leukaemia patients with t(8;21). However, the roles of ASXL2 in normal haematopoiesis and the pathogenesis of myeloid malignancies remain unknown. Here we show that deletion of Asxl2 in mice leads to the development of myelodysplastic syndrome (MDS)-like disease. Asxl2−/− mice have an increased bone marrow (BM) long-term haematopoietic stem cells (HSCs) and granulocyte–macrophage progenitors compared with wild-type controls. Recipients transplanted with Asxl2−/− and Asxl2+/− BM cells have shortened lifespan due to the development of MDS-like disease or myeloid leukaemia. Paired daughter cell assays demonstrate that Asxl2 loss enhances the self-renewal of HSCs. Deletion of Asxl2 alters the expression of genes critical for HSC self-renewal, differentiation and apoptosis in Lin−cKit+ cells. The altered gene expression is associated with dysregulated H3K27ac and H3K4me1/2. Our study demonstrates that ASXL2 functions as a tumour suppressor to maintain normal HSC function., ASXL2 mutations are mostly found in a subset of leukemia patients with certain genetic aberrations; however the role of this protein in normal hematopoiesis and related malignancies is still unclear. Here the authors use a knock-out mouse model to uncover the role of Asxl2 in hematopoiesis and leukemogenesis.

Multiple myeloma is a plasma cell neoplasm with an extremely variable clinical course. Animal models are needed to better understand its pathophysiology and for preclinical testing of potential therapeutic agents. Hematopoietic cells expressing the hypermorphic Rad50s allele show hematopoietic failure, which can be mitigated by the lack of a transcription factor, Mef/Elf4. However, we find that 70% of Mef−/−Rad50s/s mice die from multiple myeloma or other plasma cell neoplasms. These mice initially show an abnormal plasma cell proliferation and monoclonal protein production, and then develop anemia and a decreased bone mineral density. Tumor cells can be serially transplanted and according to array CGH and whole exome sequencing, the pathogenesis of plasma cell neoplasms in these mice is not linked to activation of a specific oncogene, or inactivation of a specific tumor suppressor. This model recapitulates the systemic manifestations of human plasma cell neoplasms, and implicates cooperativity between the Rad50s and Mef/Elf4 pathways in initiating myelomagenic mutations that promote plasma cell transformation.

We retrospectively assessed morphologic and cytogenetic responses to 5-azacytidine and decitabine in a cohort of 42 adult therapy-related myelodysplastic syndromes (tMDS) patients treated at Memorial Sloan-Kettering Cancer Center and in 2 industry-sponsored decitabine trials (D0007 and DACO-020). The overall response rate (complete remission+marrow CR+hematologic improvement) was 38%, including 6 patients with complete remission (14%), 6 with marrow CR with or without hematologic improvement (14%), and 4 with hematologic improvement alone (10%). We conclude that DNA methyltransferase inhibitors showed activity in tMDS that is roughly comparable to that seen in de novo MDS.

Abstract 4621 Fusion protein AML1-ETO resulting from t(8;21) translocation is highly related to leukemia development. It has been demonstrated that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. We have previously shown that the expression of AE9a, a splice isoform of AML1-ETO, can rapidly cause leukemia in mice. To understand how AML1-ETO is involved in leukemia development, we took advantage of our AE9a leukemia model and sought to identify its interacting proteins from primary leukemic cells. Here we report the discovery of a novel AE9a binding partner PRMT1 (protein arginine methyltransferase 1). PRMT1 not only interacts with but also weakly methylates Arginine 142 of AE9a. Knockdown of PRMT1 affects expression of a specific group of AE9a-activated genes. We also show that AE9a recruits PRMT1 to promoters of AE9a activated genes, resulting in enrichment of H4 Arg3 methylation, H3 Lys9/14 acetylation, and transcription activation. More importantly, knockdown of PRMT1 suppresses AE9a’s self-renewal capability, suggesting a potential role of PRMT1 in regulating leukemia development. Disclosures: No relevant conflicts of interest to declare.

Inhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1(-/-), p21(-/-), or Id1(-/-)p21(-/-) mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9-transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP(+) leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP(+) AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9-driven leukemia.

Despite advances in therapy, many patients with systemic light-chain amyloidosis (AL) die within 3 years from diagnosis. The humanized 2B6 monoclonal antibody (MoAb) is specific for the low-affinity IgG Fc receptor CD32B and effective in a human CD32B+ B-cell lymphoma murine xenograft model. Because MoAb therapy could improve outcomes in AL, we studied CD32B expression by clonal plasma cells obtained from 48 patients with AL. Transcript profiling showed that expression of CD32B was significantly higher than expression of all other Fc receptor family members. Reverse-transcriptase polymerase chain reaction (RT-PCR) using double-enriched CD138+ plasma cells showed uniform expression of the stable cell surface CD32B1 isoform at diagnosis and relapse, and flow cytometry showed intense CD32B cell surface staining on 99% of CD138+ plasma cells at diagnosis and relapse. These data provide a rationale for the novel therapeutic targeting of CD32B using the humanized 2B6 MoAb in patients with systemic AL-amyloidosis.

Transcriptional regulators are recurrently altered through translocations, deletions, or aberrant expression in acute myeloid leukemia (AML). Although critically important in leukemogenesis, the underlying pathogenetic mechanisms they trigger remain largely unknown. Here, we identified that Id1 (inhibitor of DNA binding 1) plays a pivotal role in acute myeloid leukemogenesis. Using genetically modified mice, we found that loss of Id1 inhibited t(8;21) leukemia initiation and progression in vivo by abrogating protein kinase B (AKT)1 activation, and that Id1 interacted with AKT1 through its C terminus. An Id1 inhibitor impaired the in vitro growth of AML cells and, when combined with an AKT inhibitor, triggered even greater apoptosis and growth inhibition, whereas normal hematopoietic stem/progenitor cells were largely spared. We then performed in vivo experiments and found that the Id1 inhibitor significantly prolonged the survival of t(8;21)(+) leukemic mice, whereas overexpression of activated AKT1 promoted leukemogenesis. Thus, our results establish Id1/Akt1 signaling as a potential therapeutic target in t(8;21) leukemia.

Hatlen et al. provide an integrative analysis of the mutational landscape of mouse and human AML and identify functionally relevant cooperation between AML1-ETO and PTPN11 D61Y. Based on these findings, they generate a novel mouse model of t(8;21)+ AML., t(8;21) is one of the most frequent chromosomal abnormalities observed in acute myeloid leukemia (AML). However, expression of AML1-ETO is not sufficient to induce transformation in vivo. Consistent with this observation, patients with this translocation harbor additional genetic abnormalities, suggesting a requirement for cooperating mutations. To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of AML1-ETO–driven mouse models of leukemia with the mutational profiles of human AML patients. We identified TET2 and PTPN11 mutations in both mouse and human AML and then demonstrated the ability of Tet2 loss and PTPN11 D61Y to initiate leukemogenesis in concert with expression of AML1-ETO in vivo. This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)+ AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML.

Natural killer T (NKT) cells are CD1d-restricted glycolipid reactive innate lymphocytes that play an important role in protection from pathogens and tumors. Pharmacologic approaches to enhance NKT cell function will facilitate specific NKT targeting in the clinic. Here we show that lenalidomide (LEN), a novel thalidomide (Thal) analog, enhances antigen-specific expansion of NKT cells in response to the NKT ligand α-galactosylceramide (α-GalCer) in both healthy donors and patients with myeloma. NKT cells activated in the presence of LEN have greater ability to secrete interferon-γ. Antigen-dependent activation of NKT cells was greater in the presence of dexamethasone (DEX) plus LEN than with DEX alone. Therapy with LEN/Thal also led to an increase in NKT cells in vivo in patients with myeloma and del5q myelodysplastic syndrome. Together these data demonstrate that LEN and its analogues enhance CD1d-mediated presentation of glycolipid antigens and support combining these agents with NKT targeted approaches for protection against tumors.

Deletions of the long (q) arm of chromosome 5 [del(5q)]occur in patients with myelodysplastic syndromes (MDS) including, but not limited to, those who meet the WHO definition of the 5q− syndrome. Del(5q) MDS patients frequently have symptomatic anemia, and its treatment has traditionally consisted of RBC transfusions and, for some, iron chelation therapy. Erythropoietin, darbepoetin, hypomethylating agents, and lenalidomide can enhance erythropoiesis in MDS patients with anemia, increasing hemoglobin levels and abrogating RBC transfusion requirements. Lenalidomide is particularly active in treating the anemia of del(5q) MDS, which is especially relevant given the low response rate to erythropoietin in this group of patients. In a recent study of 43 MDS patients, 10 of 12 patients (83%) with del(5q) MDS achieved sustained RBC transfusion independence (or a > 2 g/dL increase in hemoglobin), compared with 57% of those with a normal karyotype and 12% of those with other karyotypic abnormalities. Complete cytogenetic remissions were achieved in 75% (nine of 12) of the del(5q) MDS patients, suggesting that lenalidomide targets a fundamental pathogenetic feature of MDS that is more pronounced in the presence of chromosomal 5q deletions. This review highlights some issues about the classification and treatment of del(5q) MDS.

Background Fever during neutropenia and after neutrophil engraftment (postengraftment fever) occurs commonly during autologous transplantation (ASCT), but infections are infrequently identified. Tests that reliably exclude infection may reduce the cost and toxicity of unnecessary diagnostic testing and empiric treatment. We assessed whether serum levels of inflammatory cytokines could distinguish infectious from noninfectious causes of fever in patients undergoing ASCT. Methods Serum levels of IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12(p70), TNF-a and IFN-g were measured by sandwich ELISA at multiple predetermined times and at the onset of the first fever during neutropenia and after neutrophil engraftment in patients with hematologic malignancies undergoing ASCT. Standard clinical criteria were used to assess for the presence of infection. Results Seventy-two febrile episodes occurred in 54 of 65 enrolled patients; 29 (40%) of the episodes occurred after neutrophil engraftment. Infections were identified as the cause of 28% and 24% of the neutropenic and post-engraftment febrile episodes, respectively. The level of IL-12 decreased and that of IL-6 increased significantly during fever because of infection, such that the IL-12:IL-6 ratio accurately excluded infection. The area under the ROC curve for the IL-12:IL-6 ratio was 0.88 (95% CI 0.79 0.97). The sensitivity, specificity, positive predictive and negative predictive values associated with a cut-off ratio of 4.1 were 95%, 75%, 60%, and 97%, respectively. Discussion The IL-12:IL-6 ratio effectively discriminates infectious from non-infectious causes of fever during ASCT. It may be useful in assessing the probability of infection in patients with post-engraftment fever.

Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation., info:eu-repo/semantics/published

Previous studies provided evidence that MCA/129 fibrosarcomas and B16 melanomas grow 2- to 4-fold faster in acid sphingomyelinase (asmase)–deficient mice than in asmase+/+ littermates and are resistant to single-dose irradiation due to inability to mount an apoptotic response in tumor microvascular endothelium. However, others postulated the differences might be associated with a host antitumor immune response in asmase+/+ mice that is not expressed in asmase−/− mice due to phenotypic deficiency in antitumor immunity. The present studies demonstrate that none of the tumor–host combinations displayed the classic criteria of an immunogenic tumor because they lacked endotumoral or peritumoral infiltrates almost entirely. Furthermore, neither MCA/129 fibrosarcoma nor B16 melanoma tumors showed differences in growth or radioresponsiveness when implanted into mutant mouse models (Rag−/− and MEF−/−) lacking functional immune cell [natural killer (NK), NK-T, T, and B cells] populations. Additionally, development and function of B-, T-, and NK-cell populations in asmase−/− mice were normal, indistinguishable from their wild-type littermates. These data provide definitive evidence that MCA/129 fibrosarcomas and B16F1 melanomas do not elicit a host immune response in wild-type mice and that the asmase−/− phenotype is not deficient in antitumor immunity, supporting the notion that the patterns of tumors growth and radiation response are conditionally linked to the ability of the tumor endothelium to undergo ASMase-mediated apoptosis.

Recurrent cytogenetic abnormalities in leukemic blasts make these an attractive source for dendritic cells (DC) to induce a leukemia-specific immune response. In this study, three leukemic cell lines were investigated: Kasumi-1 and SKNO-1 (two acute myeloid leukemia (AML) cell lines carrying the (8;21)-chromosomal translocation, resulting in the expression of the leukemia-specific fusion protein AML1-eight-twenty-one) and REH, an acute lymphoblastic leukemia cell line with the (12;21)-chromosomal translocation and expression of translocation ETS-like leukemia-AML1. These fusion proteins are implicated in the pathogenesis of the leukemic state by recruiting corepressors and histone deacetylases (HDAC), which interfere with normal cell differentiation. In vitro generation of DC was achieved using a cytokine cocktail containing tumor necrosis factor α, granulocyte macrophage-colony stimulating factor, c-kit ligand, and soluble CD40 ligand; yet, addition of the HDAC inhibitor (Hdi) trichostatin A enhanced DC differentiation with retention of the fusion transcripts. These leukemic DC showed high-level CD83 and human leukocyte antigen (HLA)-DR expression and had a high allostimulatory potential. Only DC generated from these cell lines after Hdi induced blast-specific cytotoxic T cell responses in HLA-A-matched T cells with a cytotoxicity of 42% in parental Kasumi-1 and 83% in parental REH cells, respectively. This model system suggests that the Hdi supports the in vitro differentiation of DC from leukemic blasts with AML1-containing fusion proteins.

Insights into the pathogenesis of human leukemia have relied heavily on studies of the identified chromosomal translocations found in this group of malignant diseases. Acquired, balanced translocations in acute myelogenous leukemia (AML) generally involve transcriptional regulatory genes, whereas in the myeloproliferative disorders tyrosine kinases are frequently involved. These rearrangements alter the function of at least one and often both of the involved genes. In this review, we focus on the AML1-ETO (a.k.a. RUNX1-ETO) fusion protein, which is found in t(8;21)+ AML. Expression of AML1-ETO in human hematopoietic stem cells (HSCs) preferentially enhances their maintenance, as opposed to their differentiation. The direct effects of AML1-ETO on human and murine HSCs, and the potentially cooperating events that may contribute to its leukemogenic properties, are discussed.

Hematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

Here we show that patients with myelodysplastic syndromes (MDS) have a severe deficiency of glycolipid reactive Valpha24+/Vbeta11+ natural killer T (NKT) cells, but not NK cells or CD4+ or CD8+ T cells. Neither the blood nor marrow of MDS patients had detectable interferon-gamma-producing NKT cells in response to the NKT ligand, alpha-galactosyl ceramide, although influenza-virus-specific effector T-cell function was preserved. This severe and selective deficiency of an important immune regulatory cell may contribute to the pathogenesis of MDS.

ETS proteins (such as PU.1, Fli-1 and ETS-1) have been shown to play important roles in normal and abnormal hematopoiesis. We examined the expression of the ELF subfamily of ETS genes (ELF-1, MEF and NERF) in acute myeloid leukemia (AML) cells using Northern blot analysis. ELF-1 and MEF were expressed in all samples, whereas NERF was not. The relative expression (RE) of MEF, but not ELF-1, was significantly lower (P

The CCAAT/enhancer binding protein alpha (C/EBPalpha) belongs to a family of transcription factors that are involved in the differentiation process of numerous tissues, including the liver and hematopoietic cells. C/EBPalpha(-/-) mice show a block in hematopoietic differentiation, with an accumulation of myeloblasts and an absence of mature granulocytes, whereas expression of C/EBPalpha in leukemia cell lines leads to granulocytic differentiation. Recently, dominant-negative mutations in the C/EBPalpha gene and down-regulation of C/EBPalpha by AML1-ETO, an AML associated fusion protein, have been identified in acute myelogenous leukemia (AML). To better understand the role of C/EBPalpha in the lineage commitment and differentiation of hematopoietic progenitors, we transduced primary human CD34(+) cells with a retroviral construct that expresses the C/EBPalpha cDNA fused in-frame with the estrogen receptor ligand-binding domain. Induction of C/EBPalpha function in primary human CD34(+) cells, by the addition of beta-estradiol, leads to granulocytic differentiation and inhibits erythrocyte differentiation. Using Affymetrix (Santa Clara, CA) oligonucleotide arrays we have identified C/EBPalpha target genes in primary human hematopoietic cells, including granulocyte-specific genes that are involved in hematopoietic differentiation and inhibitor of differentiation 1 (Id1), a transcriptional repressor known to interfere with erythrocyte differentiation. Given the known differences in murine and human promoter regulatory sequences, this inducible system allows the identification of transcription factor target genes in a physiologic, human hematopoietic progenitor cell background.

We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF−/− mice have a profound reduction in the number of NK-T and NK cells. Purified MEF−/− NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNγ. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.

Background Particularly since the advent of lenalidomide, lower-risk myelodysplastic syndromes (MDS) patients with del(5q) have been the focus of many studies; however, the impact of age on disease characteristics and response to lenalidomide has not been analyzed. Methods We assessed the effect of age on clinical characteristics and outcomes in 286 lenalidomide-treated MDS patients with del(5q) from two multicenter trials. Results A total of 33.9, 34.3, and 31.8% patients were aged

RUNX1/CBFβ (core binding factor [CBF]) is a heterodimeric transcription factor complex that is frequently involved in chromosomal translocations, point mutations, or deletions in acute leukemia. The mixed lineage leukemia (MLL) gene is also frequently involved in chromosomal translocations or partial tandem duplication in acute leukemia. The MLL protein interacts with RUNX1 and prevents RUNX1 from ubiquitin-mediated degradation. RUNX1/CBFβ recruits MLL to regulate downstream target genes. However, the functional consequence of MLL fusions on RUNX1/CBFβ activity has not been fully understood. In this report, we show that MLL fusion proteins and the N-terminal MLL portion of MLL fusions downregulate RUNX1 and CBFβ protein expression via the MLL CXXC domain and flanking regions. We confirmed this finding in Mll-Af9 knock-in mice and human M4/M5 acute myeloid leukemia (AML) cell lines, with or without MLL translocations, showing that MLL translocations cause a hypomorph phenotype of RUNX1/CBFβ. Overexpression of RUNX1 inhibits the development of AML in Mll-Af9 knock-in mice; conversely, further reducing Runx1/Cbfβ levels accelerates MLL-AF9–mediated AML in bone marrow transplantation assays. These data reveal a newly defined negative regulation of RUNX1/CBFβ by MLL fusion proteins and suggest that targeting RUNX1/CBFβ levels may be a potential therapy for MLLs.

The t(15;17) translocation in acute promyelocytic leukemia (APL) yields a PML/RAR-α fusion messenger RNA species that can be detected by reverse transcription–polymerase chain reaction (RT-PCR) amplification. Breakpoints within intron 3 of PML produce a short PML/RAR-α isoform, whereas breakpoints within intron 6 result in a longer form. Using RT-PCR, serial evaluations were performed on the bone marrow of 82 patients with APL (median follow-up, > 63 months) who received retinoic acid (RA) induction followed by postremission treatment with chemotherapy, RA, and biologic agents. Sixty-four patients attained a clinical complete remission and had at least 2 RT-PCR assays performed after completing therapy. Forty of 47 patients (85%) with newly diagnosed APL who were induced using RA had residual disease detectable by RT-PCR before additional therapy. After 3 cycles of consolidation therapy, residual disease was found in only 4 of 40 evaluable patients (10%). Among newly diagnosed patients who had 2 or more negative RT-PCR assays, only 3 of 41 (7%) had a relapse, whereas all 4 patients (100%) who had 2 or more positive results had a relapse. Among 63 newly diagnosed patients, those who expressed the short isoform appeared to have shorter disease-free and overall survival durations than patients who expressed the long isoform. These data indicate that 2 or more negative RT-PCR assays on bone marrow, performed at least 1 month apart after completing therapy, are strongly associated with long-term remissions. Conversely, a confirmed positive test is highly predictive of relapse.

Background: Rearrangement between chromosome 8 and 21 is the most frequently observed chromosomal translocation. In AML patients, it leads to the formation of a novel fusion protein AML1-ETO. AML1-ETO functions as a transcriptional regulator to suppress myeloid differentiation and to promote self-renewal of hematopoiesis stem cells, which are of importance in leukemia development. However, AML1-ETO has no enzymatic function, thus targeting AML1-ETO directly is technically difficult. TAFII250, a largest subunit of the transcription factor IID complex (TFIID) serves to bring other components of basal transcription machinery to the preinitiation complex during the transcription initiation. And TAFII250 also plays a crucial role in the expression of genes involved in cell cycle and apoptosis. Yet despite the importance of these processes, there is no direct evidence implicating TAFII250 in cancer development. We have reported that acetylation of AML1-ETO on lysine 43 is critical for AML1-ETO mediated leukemogenesis and used a peptide pulldown strategy to identify proteins that preferring interact with acetylated AML1-ETO and identified TAFII250. Here, we are going to report the functions of TAFII250 in AML1-ETO induced leukemogenesis. Methods: We used Kasumi-1 and SKNO-1 cell lines derived from t(8; 21)AML patients, and a mouse AML1-ETO exon 9a (AE9a) expressing cell line developed from bone marrow cells of mice injected with bone marrow cells infected with AE9a to perform in vitro and in vivo studies. Results: We demonstrate that TAFII250 associates with AML1-ETO in AML cells through its bromodomain recognizing acetylated lysine 43 on AML1-ETO. The knockdown of TAFII250 completely abolished the proliferation of AML1-ETO expressing cells and has little influence on cell growth of non AML1-ETO expressing cells K562 and CD34+ cells. In addition, when we examined cleaved caspase 3 and PARP by western and measured annexin-V through flow cytometry, we found that the deficiency of TAFII250 induces apoptosis in AML1-ETO expressing cells. Further, the co-immunoprecipitation assay revealed that the deficiency of TAFII250 blocks the binding of AML1-ETO to CBFb (core binding factor beta subunit) and the recruitment of AML1-ETO at the promoter regions of a subset of its target genes. Consequently, loss of TAFII250 interferes with the expression of these genes. The depletion of TAFII250 also has negative effect on the self-renewal of leukemic cells, promoting their differentiation. Most importantly, the loss of TAFII250 severely impairs leukemia development in AE9a expressing cells. Conclusions: Together, these results reveal an essential role of TAFII250 in AML1-ETO mediated leukemogenesis and imply that TAFII250 might be a therapeutic target for AML1-ETO expressing AMLs. Disclosures No relevant conflicts of interest to declare.

Background: The FLT3-ITD (internal tandem duplication) is the most frequently observed molecular defect in acute myeloid leukemia (AML) and has been shown to be a poor prognostic factor in several studies. Many inhibitors of FLT3-ITD have been developed and tested in clinical trials; while a recent randomized study of PK412 (FLT3 inhibitor) showed significant clinical benefit in AML patients, many other studies have shown only modest effect. We and others have shown that the protein arginine methyltransferase 5 (PRMT5) is required to maintain self-renewal in many types of stem cells; we found that PRMT5 is expressed at much higher levels in hematopoietic stem cells than in differentiated cells and moreover, the expression of PRMT5 is increased in most acute myeloid leukemia cells and PRMT5 is also overexpressed in lymphoma and ovarian cancer cells. While we have been to define roles for PRMT5 in myeloproliferative disorders and normal hematopoietic stem and progenitor cells. The role of PRMT5 in AML has not been extensively studied. The mitogen activated protein kinases (MAPK) pathway is known to abnormally enhance the proliferation signal downstream of the FLT3-ITD. The dual-specificity mitogen activated protein kinase phosphatase-2 gene (DUSP4/MK-2) specifically dephosphorylates and inactivates MAPK. The role of DUSP4 in AML also is not clear. Here we reported that the role of PRMT5 and DUSP4 in AML with FLT3-ITD. Objective: The aim of the present study was to investigate whether the FLT3-ITD affects histone modification and/or the expression level of DUSP4 via effects on PRMT5 itself and also to explore whether the combination of a FLT3 inhibitor and a PRMT5 inhibitor could have synergistic anti-leukemic effects. Results: We showed that overexpression of FLT3-ITD increased the level of phospho-PRMT5 in 293T cells and that several FLT3 inhibitors reduced the phosphorylation of PRMT5 in MV4-11 cells. The FLT3-ITD binds PRMT5 when expressed in 293T cells and FLT3, FLT3-ITD, and FLT3-D835Y can directly phosphorylate PRMT5 in an in vitro kinase assay but this phosphorylation is inhibited by the AC220, in a dose-dependent fashion. Histone H2A arginine symmetric methylation, which is known to be a target of PRMT5, increases after treatment with CEP701 and AC220. These results suggest that the FLT3 inhibitor can enhance PRMT5 enzyme activity, and that FLT3-ITD inhibits PRMT5 enzymatic activity. We also showed that there was an enrichment of histone H2AR3/H4R symmetric di-methylation (sdm) at the promoter region of DUSP4. We also showed that absent PRMT5 increased the protein expression of DUSP4 and FLT3-ITD signaling suppression with AC220 decreased it through histone modification with Chip assay. We are able to show that PRMT5 inhibitor and sh-PRMT5 suppressed the cytokine signaling such as p-STAT5, p-Erk, and p-AKT that are known as signaling pathway that FLT3-ITD enhanced and DUSP4 suppressed. The PRMT5 was an essential gene for maintenance of MV4-11 cells. The sh-DUSP4 was able to partially rescue MV4-11 cells with treatment of sh-PRMT5. We showed that a FLT3 inhibitor and a PRMT5 inhibitor have synergistic anti-leukemic effects in MV4-11 cells. Conclusion: The FLT3-ITD affects the expression of DUSP4 through epigenetic modification such as the methylation status of histone H2AR3/H4R3 arginine symmetric methylation via PRMT5 in AML. The combination of an FLT3 inhibitor and PRMT5 inhibitor may have synergistic anti-leukemic effects in AML. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Background AML1-ETO (AE), a oncogenic protein generated by the t(8;21) translocation, causes acute myeloid leukemia (AML) in collaboration with other secondary events. The leukemogenicity of AE has been evaluated in multiple mouse models, such as expression of AE in Cdkn1a-null Hematopoietic stem cell (HSCs) and expression of AML1- ETO9a (AE9a), an alternatively spliced variant of AE, in WT HSCs. Both lead to the development of fully penetrant AML. Caspase-3 plays multiple roles in hematopoietic development and leukemia progression and treatment by affecting proliferation, self-renewal and differentiation. It has been shown that uncleaved caspase-3 levels are higher in the peripheral blood cells of AML patients compared to normal individuals, which suggests that the caspase pathway is dysregulated in AML. We and others have shown that Caspase-3 directly cleave AE in vitro, suggesting that AE may accumulate in a Caspase-3 compromised background and accelerate leukemogenesis. Methods We developed a Caspase-3 knockout genetic mouse model of AML based on fetal liver cell transplantation. In brief, fetal liver cells from WT or Caspase3-/- mice were transduced to express AE9a in vitroand 100,000 AE9a+ transduced cells were transplanted into lethally irradiated recipient mice by tail-vein injection. Results We found loss of Caspase-3 impaired leukemia stem cell (LSC) self-renewal and delayed AE9a-driven leukemogenesis, indicating that Caspase-3 may play distinct roles in the initiation or progression of AML. Moreover, we identified a new substrate of Caspase-3, ULK1, by in vitro cleavage assays and site-directed mutagenesis. ULK1 (serine/threonine UNC-51-like kinase) is the homology of Atg1 (the first autophagy related gene found in 1997) in mammalian cells, which is a direct target of mTOR and is responsible for initiation of the autophagic activity by forming a complex with mAtg13, FIP200 and Atg101. The induction of autophagy caused by upregulation of ULK1 in AE/AE9a-expressing Caspase-3-/- fetal liver cells acted to limit the leukemogenicity of AE9a in vivo. Inhibition of ULK1 by inhibitor or shRNAs could rescue the self-renewal capability induced by Caspase-3 deletion in serial replating assays. Unexpectedly, when we expressed AE/AE9a in fetal liver cells from WT and Caspase-3-/- mice, the protein levels were comparable suggesting the basal level of Caspase-3 didn't affect the expressing of AE/AE9a in fetal liver cells. Conclusion Autophagy may play a general role in the development and treatment of leukemia. In human AML, blasts display reduced expression of autophagy-related genes and decreased autophagic flux, indicating that low autophagy activity provides a general advantage for leukemia development. Beside this, a number of chemotherapy drugs have been reported to be able to induce leukemia cell death via activation of autophagy suggesting that autophagy plays critical roles in the leukemia treatment. Our study reveals that Caspase-3 regulates autophagy through its direct cleavage of ULK1 and this interaction dictates the pace of AE-driven leukemogenesis. Targeting this pathway may have therapeutic benefit for AML treatment. Disclosures No relevant conflicts of interest to declare.

Somatic mutations and chromosomal translocations of genes have emerged as major drivers in a range of hematopoietic malignancies. While ASXL1 is mutated in all forms of myeloid malignancies, ASXL2 is specifically mutated in t(8;21) AML patients. ASXL1 and ASXL2 mutations are mutually exclusive in t(8;21) AML. Despite the importance of ASXL2 mutations in clinical, it's role in leukemogenesis remain unknown. In the current study, we sought to dissect the role of ASXL2 in normal hematopoiesis and to identify the molecular mechanisms by which Asxl2 loss contributes to myeloid malignancies. In the current study, we utilized a mouse model of Asxl2 to characterize the hematopoietic features of in vivo. Asxl2-/- mice were characterized by pancytopenia and dysplastic features, including hyposegmented (bilobed) neutrophils with fine nuclear bridging (consistent with pseudo Pelger-Huët) and increased number of polychromatophilic red blood cells (RBCs), reminiscent of myelodysplastic syndrome (MDS). Flow cytometric analyses revealed that Asxl2-/- mice had an increased proportion of granulocytic/monocytic cells (Gr-1+/Mac1+) in the PB, BM and spleens compared to WT mice. The histologic analysis of the Asxl2+/- and Asxl2-/- spleen sections showed disrupted splenic architecture with an increased proportion of myeloid cells and massive accumulation of myeloperoxidase (MPO) positive cells in WT spleens. Asxl2-/- mice had an increased long-term (LH)-HSCs and granulocyte-macrophage progenitor (GMP) cells compared to WT mice.Consistently, the paired-daughter cell assays revealed that Asxl2-/- CD34-LSK BM cells had a higher proportion of cells with symmetric self-renewal capacity (SS, 62%) than WT cells (33%). In contrast, a significant reduction in the cells with symmetric differentiation potential was observed in Asxl2-/- HSCs (18%) compared to WT HSCs (40%), indicating a critical role of ASXL2 in the balance between the symmetric and asymmetric division of HSCs. Transplantation assays revealed that recipients transplanted with Asxl2-/- and Asxl2+/- bone marrow cells had shortened lifespan due to the development of MDS or AML, suggesting a cell-autonomous effect of Asxl2-loss in HSC/HPC functions. Furthermore, Asxl2-loss further increase the colony-forming potential and colony replating capacity of AML1-ETO expressing HSCs in vitro, suggesting a cooperative effect between AML1/ETO9a and Asxl2+/-to promote HSC self-renewal. RNA-seq analysis showed a unique signature of Asxl2-/- LK cells compared to WT LK cells. Gene set enrichment analysis revealed that altered expressed genes in Asxl2-/-LK cells were enriched in myeloid cell differentiation, hematopoiesis, apoptosis, and chromatin/nucleosome assembly signature. ChIP-seq analysis showed that differentially expressed genes were associated with dysregulated histone enhancer markers, including H3K27ac, H3K4me1, and H3K4me2. Further analysis demonstrated that the alteration of H3K27ac enrichment had a greater impact on gene expression, in comparison to H3K4me1/2. KEGG pathway analysis showed that genes with differential H3K27ac signals were enriched for hematopoietic cell lineage, cancer signaling pathway and myeloid leukemia development. IPA analysis further confirmed that genes with altered enrichment levels of were enriched in myeloid cell differentiation and apoptosis pathways. Altogether, these data suggest that ASXL2 regulates gene expression mainly through enhancer markers. Our results demonstrate that ASXL2 plays an important role in normal hematopoiesis, and Asxl2-loss in mice is sufficient to cause MDS-like disease and leukemia transformation. These results indicate that ASXL2 functions as a tumor suppressor in myelopoiesis. The Asxl2 knock-out mice present an ideal model for unveiling the mechanisms underlying the Asxl2-loss mediated multiple-step pathogenesis of myeloid malignancies and for testing novel therapeutic agents for myeloid malignant patients with ASXL2 alterations. Further studies to dissect the possible roles of ASXL2alterations in leukemogenesis and to identify therapeutic vulnerabilities they may create are ongoing. Disclosures No relevant conflicts of interest to declare.

Innate immune signals and p53 contribute to the 5q– myelodysplastic syndrome, an acquired bone marrow failure disorder (pages 49–58 and 59–66).

High-dose chemoradiotherapy (HDT) with autologous stem cell transplantation (ASCT) is the treatment of choice for patients with relapsed aggressive non-Hodgkin lymphoma (NHL). However, its role in the treatment of patients with primary refractory disease is not well defined. The outcomes of 85 patients with primary refractory aggressive NHL who underwent second-line chemotherapy with ICE with the intent of administering HDT/ASCT to those patients with chemosensitive disease were reviewed. Patients were retrospectively classified as induction partial responders (IPR) if they attained a partial response to doxorubicin-based front-line therapy or as induction failures (IF) if they had less than partial response. Forty-three patients (50.6%) had ICE-chemosensitive disease; there was no difference in the response rate between the IPR and the IF groups. Intention-to-treat analysis revealed that 25% of the patients were alive and 21.9% were event-free at a median follow-up of 35 months. Among 42 patients who underwent transplantation, the 3-year overall and event-free survival rates were 52.5% and 44.2%, respectively, similar to the outcomes for patients with chemosensitive relapsed disease. No differences were observed between the IPR and IF groups, and there were no transplantation-related deaths. More than one extranodal site of disease and a second-line age-adjusted International Prognostic Index of 3 or 4 before ICE chemotherapy were predictive of poor survival. These results suggest that patients with primary refractory aggressive NHL should receive second-line chemotherapy, with the intent of administering HDT/ASCT to those with chemosensitive disease. Newer therapies are needed to improve the outcomes of patients with poor-risk primary refractory disease.