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Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

In an effort to identify novel drugs targeting fusion-oncogene–induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

Common approaches to alleviate acute graft-versus-host disease (aGvHD) focus on inducing immune tolerance by directly targeting immune cells. However, protecting key target tissues such as the gastrointestinal tract from immune-mediated damage may also reduce aGvHD severity. Using murine aGvHD models, Henden and colleagues present compelling evidence that interferon λ (IFN- λ) limits intestinal stem cell loss and promotes regeneration of gut epithelia, preserving mucosal barrier function. They propose IFN- λ therapy as a novel strategy to boost tissue protection., Key Points IFN-λ receptor signaling protects against lethal acute GI GVHD.Recombinant IL-29 treatment in the peri-transplant period promotes gut epithelial regeneration and is a promising therapy to limit GVHD., Visual Abstract, Immunopathology and intestinal stem cell (ISC) loss in the gastrointestinal (GI) tract is the prima facie manifestation of graft-versus-host disease (GVHD) and is responsible for significant mortality after allogeneic bone marrow transplantation (BMT). Approaches to prevent GVHD to date focus on immune suppression. Here, we identify interferon-λ (IFN-λ; interleukin-28 [IL-28]/IL-29) as a key protector of GI GVHD immunopathology, notably within the ISC compartment. Ifnlr1−/− mice displayed exaggerated GI GVHD and mortality independent of Paneth cells and alterations to the microbiome. Ifnlr1−/− intestinal organoid growth was significantly impaired, and targeted Ifnlr1 deficiency exhibited effects intrinsic to recipient Lgr5+ ISCs and natural killer cells. PEGylated recombinant IL-29 (PEG-rIL-29) treatment of naive mice enhanced Lgr5+ ISC numbers and organoid growth independent of both IL-22 and type I IFN and modulated proliferative and apoptosis gene sets in Lgr5+ ISCs. PEG-rIL-29 treatment improved survival, reduced GVHD severity, and enhanced epithelial proliferation and ISC-derived organoid growth after BMT. The preservation of ISC numbers in response to PEG-rIL-29 after BMT occurred both in the presence and absence of IFN-λ–signaling in recipient natural killer cells. IFN-λ is therefore an attractive and rapidly testable approach to prevent ISC loss and immunopathology during GVHD.

The dynamics of CD4⁺ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4⁺ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (T_H1) and follicular helper T (T_(FH)) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T_H1 and T_(FH) trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T_(FH) and central memory were revealed, with antimalarials modulating these responses and boosting T_H1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4⁺ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene–gene correlations (http://haquelab.mdhs.unimelb.edu.au/cd4_memory/).

TO THE EDITOR: Acute myeloid leukemia (AML) is a heterogeneous disease for which genetic profiles dictate clinical outcomes. Cytogenetic and molecular profiling in AML is a mandatory diagnostic and prognostic requirement, yet interpretation of these results is becoming increasingly complex. Next

Chemotherapy-resistant acute myeloid leukemia (AML) manifesting as primary refractory or relapsed disease carries a dismal prognosis and is often driven by clonal evolution. We have performed a genome-wide CRISPR knockout screen investigating resistance to conventional AML therapy, cytarabine and doxorubicin (AraC/Dox), in 2 independent human AML cell lines. Chemoresistant populations were enriched with gRNAs disrupting cell cycle arrest genes, including cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53. Here, we validate the direct contribution of these genes to chemoresistance and demonstrate that rationally designed therapeutic regimens targeting cell cycle enhances chemotherapy response in AML. Using CRISPR-mediated gene editing, we individually inactivated CDKN2A and CHEK2 in Cas9-expressing OCI-AML3 and MV4-11 cells. CDKN2A- and CHEK2-deficient cells demonstrated proliferative advantage in the presence of AraC/Dox in co-culture competition assays, confirming direct contribution of these gene knockouts to chemoresistance. Nil to modest reductions in apoptosis were seen in CDKN2A- and CHEK2-deficient cells treated with AraC/Dox for 72 hours compared to unedited controls. However, failure of accumulation of cells in the non-cycling G 0/G 1 proportion was seen in these edited cells after chemotherapy, corresponding with maintained DNA synthesis, as measured by BrdU incorporation, and a failure to downregulate phospho-Rb protein expression, indicating ongoing active cell cycling in spite of chemotherapy. These results confirm failure of cell cycle arrest as the major mechanism of resistance with inactivation of CDKN2A or CHEK2. To assess relevance of these genes in chemotherapy response in human AML, we analysed the effect of CDKN2A expression on prognosis. Reduced expression of CDKN2A conferred inferior overall survival in 3 independent clinical cohorts. Additionally, downregulation of CDKN2A and an increase in downstream cell cycling effector, cyclin-dependent kinase 6 (CDK6) was seen at relapse in paired diagnosis-relapse AML samples (Li et al. 2016, Nature Medicine). Further, CHEK2 mutations in clonal hematopoiesis are enriched in patients with solid organ cancers following chemo- or radiotherapy, functionally demonstrating chemoresistance over non-mutated cells. These data suggest CDKN2A and CHEK2 loss-of-function is relevant in promoting chemoresistance in human hematopoiesis. We therefore investigated whether therapeutically targeting cell cycle control pathways that converge on the G 1/S restriction point could synergise with cytotoxic chemotherapy, potentially circumventing chemoresistance. The addition of MDM2 inhibitor, nutlin-3a with AraC/Dox achieved striking synergism in TP53-competent cell lines in reducing viability and promoting apoptosis. Inhibition of CDK4/6 with palbociclib alone induced cell cycle arrest without apoptosis, however combination therapy with AraC/Dox significantly potentiated apoptosis. To functionally validate the role of CDKN2A itself, we examined the inhibitor of histone acetyltransferase KAT6A, WM-1119 (Baell et al. 2018, Nature), which transcriptionally upregulates CDKN2A. WM-1119 had anti-leukemic activity across multiple cell lines, corresponding with down regulation of cell cycling and upregulation of senescence signatures. The anti-leukemic activity was prevented by CDKN2A inactivation, indicating that CDKN2A upregulation is essential for WM-1119's anti-leukemic effect. WM-1119 enhanced activity of AraC/Dox in multiple cell contexts, mediated by cooperative induction of CDKN2A. Its efficacy and synergy with AraC/Dox was further demonstrated in MLL-AF9 primary murine AML ex vivo, demonstrating broader applicability of the approach. Our findings reveal defects in cell cycle arrest pathways as clinically relevant contributors to chemoresistance in AML. Combining agents to target cell cycle components improved in vitro anti-leukemic activity of chemotherapy and warrants further exploration of their translational potential. Disclosures Bullinger: Amgen: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Menarini: Consultancy; Hexal: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Astellas: Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Seattle Genetics: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Consultancy, Honoraria. Lane: BMS: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Novartis: Consultancy; Geron: Consultancy.

Introduction Despite the advent of targeted therapy for FLT3-mutated AML, unmet need still exists for patients unfit for intensive chemotherapy, with no evidence that overall survival (OS) can be improved by combining either venetoclax (Konopleva et al., ASH 2020) or gilteritinib (Astellas press release, December 2020) with azacitidine. Although gilteritinib has been shown to improve median OS from 5.5 to 9.8 months, the majority will relapse (Perl et al., 2019). Adaptive on-target gilteritinib resistance may be due to the FLT3-F691L gatekeeper mutation, whereas off-target resistance may be due to loss-of-function variants in CBL, which encodes an E3 ubiquitin-protein ligase that negatively regulates FLT3 (McMahon et al, 2019). Ponatinib is a type-1 FLT3 inhibitor that is active in vitro against FLT3 F691L (Smith et al., 2013) and had an overall response rate (ORR) of 43% in a small pilot phase-I study (Talpaz et al., 2011). Combination of a FLT3 inhibitor with azacitidine may antagonize the synergistic hypermethylation reported for FLT3-ITD in association with epigenetic mutations (Shih et al., 2015). CBL loss-of-function mutations may also enhance responsiveness to FLT3 inhibitors (Taylor et al, 2015). We thus hypothesize that the combination of ponatinib and azacitidine could mitigate the rapid evolution of drug resistance typical of more selective FLT3 inhibitors used as single agents. Methods A phase-Ib study was conducted with the primary objective safety and key secondary objective preliminary efficacy of azacitidine in combination with ponatinib in patients with FLT3-ITD AML failing prior therapy or unfit for intensive chemotherapy. Exploratory objectives included mechanisms of ponatinib resistance and responsiveness of CBL-mutant AML to FLT3 inhibition. At dose level 1 (DL1), patients received azacitidine 60 mg/m 2 on days 1-5 and 8-9 and ponatinib 30 mg daily on days 5-25 of each cycle. In patients not achieving CR or CRi after cycle 1, the ponatinib dose was increased to 45 mg during cycle 2. For dose level 2 (DL2), the dose of azacitidine was increased to 75 mg/m 2. Results Thirty-one patients were evaluable for response. Median age was 67 years (range, 26-87). Frequency of prior lines of therapy was 0 (15%), 1 (46%), 2 (23%) or 3 (8%). Four patients had a history of prior allogeneic hematopoietic cell transplant and one had previously received a FLT3 inhibitor. FLT3-ITD was present in 28 patients (median VAF 0.33; range, 0.009-17.95) and 3 had inactivating CBL mutations. A total of 20 patients were treated at DL1 and 12 patients at DL2. There were two grade-4 DLTs (raised AST/ALT [DL1] and tubulointerstitial nephritis [DL2]). Three grade-2 thromboembolic events were observed (two cannula-related DVTs and a distal lower-limb DVT). There were two grade-5 AEs (infection and cardiac failure), which were not considered drug related. The most common grade-3-4 AEs were febrile neutropenia (57%), neutropenia (47%), infections (47%), thrombocytopenia (40%) and anaemia (27%). Cardiac arrhythmias (atrial fibrillation/flutter, bradycardia, sinus tachycardia and ventricular tachycardia [1 patient]) were observed in 30% of patients. Of these, 80% were grade 1 or 2 and only one was considered by the investigator to be related to study treatment. Response was evaluable in 23 of 31 patients. Nine patients (39%) achieved CR or CRi, 3 (13%) achieved a PR and 8 (35%) achieved SD (ORR 52%). ORR at DL1 and DL2 was 43% and 66%, respectively. Median time to best response was 1.4 months (range 1.0-11.9). Median duration of best response was 12.9 months at both dose levels. Median OS for DL1 was 6.5 months and not reached for DL2. Despite shorter follow-up, DL2 patients experienced better OS than DL1 patients (p = 0.015). Responses were seen in 2 of 4 patients with post-allograft relapse. Two of three patients with a CBL mutation responded (1 CR and 1 CRi). Eradication of the CBL mutation was seen in one patient, who remains on therapy after 15 cycles. Molecular studies to investigate dynamic changes in molecular architecture are ongoing. Conclusions The recommended phase-II dose of ponatinib is 30 mg on days 5-25 and that of azacitidine is 75 mg/m 2 for seven doses each cycle. The ORR was 52% and durable disease control was observed, especially in patients receiving DL2. Preliminary efficacy was observed in CBL-mutated patients. Further clinical investigation of this regimen is warranted in patients with FLT3- or CBL-mutant AML. Figure 1 Figure 1. Disclosures Kipp: Novartis: Honoraria. Perkins: Celgene: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau. Lane: Novartis: Consultancy; Geron: Consultancy; BMS: Consultancy, Research Funding; Abbvie: Honoraria; Astellas: Membership on an entity's Board of Directors or advisory committees. Enjeti: Sanofi: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria; Roche: Speakers Bureau; Astra Zeneca: Honoraria. Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Reynolds: Novartis AG: Current equity holder in publicly-traded company; Abbvie: Research Funding; Alcon: Current equity holder in publicly-traded company. Wei: Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Membership on an entity's Board of Directors or advisory committees; Novartis, Abbvie, Celgene/BMS: Speakers Bureau; Former employee of Walter and Eliza Hall Institute: Patents & Royalties: Prof. Andrew Wei is a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax; Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Honoraria; Servier: Consultancy; Abbvie, Amgen, AstraZeneca, Celgene/BMS, Novartis, Servier and F. Hoffmann-La Roche: Research Funding. OffLabel Disclosure: Ponatinib - used as an experimental therapy for AML in combination with azacitidine

Many patients with ET are resistant to or intolerant of current standards of care (SOC) - hydroxyurea (HU), interferon, anagrelide - underscoring the need for novel therapies with distinct modes of action that reduce the risk of thrombosis, improve the patient's experience and favorably alter the natural history. Lysine-specific demethylase-1 (LSD1) is an enzyme critical for the self-renewal potential of malignant cells and hematopoietic differentiation, e.g., LSD1 licenses progenitors to mature into megakaryocytes, a cell central to ET pathogenesis. Bomedemstat is an orally active LSD1 inhibitor that reduced peripheral cell counts, splenomegaly, inflammatory cytokines, mutant cell burdens and improved survival in mouse models of MPNs (Kleppe et al. 2015; Jutzi et al. 2018). IMG-7289-CTP-201 is a global, open-label, Phase 2b study of bomedemstat taken once daily for 24+ weeks in patients with ET who are resistant to or intolerant of at least one SOC treatment (NCT04254978). Key eligibility criteria include patients who require cytoreduction, a platelet count >450 x 10 9/L, hemoglobin ≥10 g/dL and absolute neutrophil count ≥0.5 x 10 9/L. Key objectives are safety and response, defined as platelets ≤400 x 10 9/L without new thromboembolism or disease progression. Exploratory endpoints include durability of response, reduction in WBCs, changes in mutant allele frequencies (MAF), and symptom improvement. All patients start at a dose of 0.6 mg/kg/d that is titrated to a target platelet count of 200-400 x 10 9/L. At data cut-off (15July'21), 30 patients have enrolled. Baseline median age was 68 (42-84) years with 33% males; 77% were resistant to or intolerant of HU, 10% to anagrelide, 7% to interferon, and 3% each to busulfan and ruxolitinib. The Day 1 (washout up to 28 days) mean platelet, WBC and hemoglobin values were 876 x 10 9/L (457-2220), 9.7 x 10 9/L (4.4-30.6), and 13.0 g/dL (9.4-16.5) respectively. Among all patients, median MPN10 total symptom score (TSS) at baseline was 16 (0-74); TSS >10 was observed in 63% (19/30); median 30 (11-74). Genotyping by sequencing at screening (N=32) revealed mutations in JAK2 (50%) and CALR (44%) with a wide distribution of MAFs (1-85%). All patients were wild-type at the MPL locus, two patients were "triple-negatives" and five patients had copy number neutral loss of heterozygosity. Non-driver mutations were present in 31% (10/32) including EZH2, ASXL1, SF3B1 and TP53. Median time on study is 16 weeks (0-41). Platelet count was reduced in 92% (24/26) of patients treated for more than 6 weeks with 81% (21/26) achieving a platelet count of ≤400 x 10 9/L. Of the 9/30 (30%) patients with a Day 1 WBC count ≥10 x 10 9/L, 89% (8/9) had a WBC count 10 (19/30), at Week 12, 77% (10/13) had decreased scores and 46% (6/13) demonstrated >10-point improvement. Of patients resequenced (261 genes) at Week 24 (N=6), mutant allele frequencies were stable and no new mutations were detected. Enrollment is on-going; additional clinical and genetic data will be presented. The most common (reported by >15% of patients) treatment-emergent AEs deemed related were dysgeusia (40%), fatigue, thrombocytopenia (without bleeding), constipation, and diarrhea (each 17%). Six AEs ≥Grade 3 were reported in 5 patients, with 2 (dysgeusia and constipation) deemed related to bomedemstat by the Investigator. Two unrelated SAEs were reported: a lung infection and a pulmonary embolus. Four patients discontinued treatment, three due to AEs (nausea, dysgeusia x 2) and one withdrew consent on Day 1. Similar to an ongoing MF study of bomedemstat (NCT03136185), there have been no safety signals, DLTs, or deaths related to drug. At the time of data cut-off, 87% (26/30) of patients remain on study. To date, in a majority of patients who were resistant or intolerant to at least one standard of care, bomedemstat has shown to be well-tolerated, reduce platelets, improve symptoms, and moderate WBC counts while maintaining hemoglobin. For those patients treated for at least 6 weeks, 81% achieved a complete peripheral blood count remission without evidence of disease progression. The mutation burden remained stable despite high molecular risk mutations. Based on this promising data, a Phase 3 study of bomedemstat for the treatment of ET is being planned. Figure 1 Figure 1. Disclosures Ross: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lane: BMS: Consultancy, Research Funding; Geron: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Novartis: Consultancy. Gerds: Constellation: Consultancy; AbbVie: Consultancy; Brystol Myers Squibb: Consultancy; Sierra Oncology: Consultancy; Novartis: Consultancy; PharmaEssentia: Consultancy; Incyte: Research Funding; Constellation: Research Funding; Krtos: Research Funding; CTI Biopharma: Research Funding; Imago: Research Funding; Accutate: Research Funding. Halpern: Novartis: Research Funding; Bayer: Research Funding; Tolero Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Abbvie: Consultancy; Gilead: Research Funding; Agios: Consultancy; Imago Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Nohla Therapeutics: Research Funding; Pfizer: Research Funding. Shortt: Amgen: Research Funding; Astex: Research Funding; BMS: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Jones: Imago BioSciences: Current Employment, Current equity holder in publicly-traded company. Peppe: Imago BioSciences: Current Employment, Current equity holder in publicly-traded company. Natsoulis: Imago BioSciences: Current Employment, Current equity holder in publicly-traded company. Navarro: Imago BioSciences: Current Employment, Current equity holder in publicly-traded company. Hong: Imago BioSciences: Current Employment, Current equity holder in publicly-traded company; Genentech, Inc.: Ended employment in the past 24 months. Harrison: Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Constellation Pharmaceuticals: Research Funding; Incyte Corporation: Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Talpaz: Imago: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Grant/research support ; Constellation: Membership on an entity's Board of Directors or advisory committees. Rienhoff: Imago BioSciences: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Allogeneic bone marrow transplantation (allo-BMT) is a curative therapy for hematological malignancies, but is associated with significant complications, principally graft-versus-host disease (GVHD) and opportunistic infections. Natural killer (NK) cells mediate important innate immunity that provides a temporal bridge until the reconstruction of adaptive immunity. Here, we show that the development of GVHD after allo-BMT prevented NK-cell reconstitution, particularly within the maturing M1 and M2 NK-cell subsets in association with exaggerated activation, apoptosis, and autophagy. Donor T cells were critical in this process by limiting the availability of interleukin 15 (IL-15), and administration of IL-15/IL-15Rα or immune suppression with rapamycin could restore NK-cell reconstitution. Importantly, the NK-cell defect induced by GVHD resulted in the failure of NK-cell-dependent in vivo cytotoxicity and graft-versus-leukemia effects. Control of cytomegalovirus infection after allo-BMT was also impaired during GVHD. Thus, during GVHD, donor T cells compete with NK cells for IL-15 thereby inducing profound defects in NK-cell reconstitution that compromise both leukemia and pathogen-specific immunity.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a−/− Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer–driven inflammatory signaling.

IL-17-producing cells are important mediators of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Here we demonstrate that a distinct CD8(+) Tc17 population develops rapidly after SCT but fails to maintain lineage fidelity such that they are unrecognizable in the absence of a fate reporter. Tc17 differentiation is dependent on alloantigen presentation by host dendritic cells (DCs) together with IL-6. Tc17 cells express high levels of multiple prototypic lineage-defining transcription factors (eg, RORγt, T-bet) and cytokines (eg, IL-17A, IL-22, interferon-γ, granulocyte macrophage colony-stimulating factor, IL-13). Targeted depletion of Tc17 early after transplant protects from lethal acute GVHD; however, Tc17 cells are noncytolytic and fail to mediate graft-versus-leukemia (GVL) effects. Thus, the Tc17 differentiation program during GVHD culminates in a highly plastic, hyperinflammatory, poorly cytolytic effector population, which we term "inflammatory iTc17" (iTc17). Because iTc17 cells mediate GVHD without contributing to GVL, therapeutic inhibition of iTc17 development in a clinical setting represents an attractive approach for separating GVHD and GVL.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.

SummaryAcute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc−/− LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs.

Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Binding of erythropoietin (Epo) to its receptor (EpoR) is required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Janus kinase 2 (Jak2) tyrosine kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR-Jak2 independently of Stat5. Plcγ1-deficient pro-erythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined HSCs (Lin(-)Sca1(+)KIT(+)CD48(-)CD150(+)) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation, we assessed changes occurring at the global transcriptional and DNA methylation level after inactivation of Plcγ1. The top common downstream effector was H2afy2, which encodes for the histone variant macroH2A2 (mH2A2). Inactivation of mH2A2 expression recapitulated the effects of Plcγ1 depletion on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target mH2A2, as a 'non-canonical' Epo signaling pathway essential for erythroid differentiation.

The field of regenerative medicine holds considerable promise for treating diseases that are currently intractable. Although many researchers are adopting the strategy of cell transplantation for tissue repair, an alternative approach to therapy is to manipulate the stem cell microenvironment, or niche, to facilitate repair by endogenous stem cells. The niche is highly dynamic, with multiple opportunities for intervention. These include administration of small molecules, biologics or biomaterials that target specific aspects of the niche, such as cell-cell and cell–extracellular matrix interactions, to stimulate expansion or differentiation of stem cells, or to cause reversion of differentiated cells to stem cells. Nevertheless, there are several challenges in targeting the niche therapeutically, not least that of achieving specificity of delivery and responses. We envisage that successful treatments in regenerative medicine will involve different combinations of factors to target stem cells and niche cells, applied at different times to effect recovery according to the dynamics of stem cell–niche interactions.

Chromosomal translocations found in acute myeloid leukemia (AML) can generate oncogenic fusions with aberrant epigenetic and transcriptional functions. However, direct therapeutic targeting of leukemia fusion proteins has not been accomplished so far. Although high remission rates can be induced in patients diagnosed with AML1-ETO/t(8;21)-positive AML only half of them achieve long-term disease-free survival (Papaemmanuiel et al., NEJM, 2016). In the other half of these patients, the disease maintaining leukemia stem cell (LSC) clone is not eliminated by chemotherapy. A functional characteristic of LSCs is unlimited self-renewal capacity and several signaling pathways have been identified that maintain stem cell self-renewal. Targeting the oncogene induced self-renewal capacity of LSCs has great potential to eliminate the malignant clone and prevent relapse. To identify oncogenic cellular functions with relevance for LSC self-renewal, we performed global proteome profiling in murine AML1-ETO9a (AE) compared to MLL-AF9 (MA9) driven LSCs. Gene set enrichment analyses revealed a significant enrichment of calcium-dependent cellular functions and Phospholipase C (PLC)-signaling in AE LSCs. These data could be confirmed in sorted CD34+ blasts from AE-positive AML when compared to non-AE-AML. All PLC family members are regulators of Ca2+ homeostasis. However, when analyzing published AML gene expression datasets we found exclusively PLCG1 to be highly expressed in t(8;21) AML. Conditional activation of AE in embryonic stem cells resulted in induction of PLCG1 expression and PLCG1 was identified as a direct target of the AE fusion by ChIP-sequencing in AE-positive Kasumi-1 cells.Here, PLCG1 depletion resulted in reduced Ca2+ release, impaired proliferation and reduced colony formation in vitro. In a xenograft model, inactivation of PLCG1 resulted not only in delayed disease development (median survival shNT vs. shPLCG1: 135 days vs. not reached, p=0.02) but also in reduction of disease penetrance by 87%. Consistent with these results, transcriptome analysis revealed strong induction of gene sets related to myeloid differentiation and down-regulation of gene sets linked to proliferation, stemness and c-Myc targets. To confirm the functional role of PLCG1-signaling in AE-driven LSCs, we generated a new conditional knockout mouse model for Plcg1 and induced leukemia using the oncogenes AE and KRAS-G12D (AE/K). Genetic inactivation of Plcg1in vivo after engraftment of leukemic cells resulted in significant reduction of LSC numbers (p=0.04) and a reduction of disease penetrance by 67% in primary recipients. Isolated LSCs revealed induction of differentiation, loss of cell cycle activity and failed to re-establish disease in secondary recipients (Plcg1+/+ vs. Plcg1-/-: median survival 12 days vs. not reached; p=0.0001). In contrast, genetic deletion of Plcg1 appeared to be dispensable for normal murine HSC function during primary and secondary transplantation. Primary human t(8;21) AML cells (derived from 4 different donors) showed impaired colony forming capacity following PLCG1 inactivation in vitro irrespective of co-occurring mutations while colony formation of human CD34+ BM cells was not affected to a major extent. As Ca2+ signaling appeared deregulated in t(8;21) AML, we aimed to investigate the effects of pharmacologic Ca2+ inhibition as a tractable target downstream of PLCG1. To assess specifically for LSC function, we treated primary recipient mice with established AE/K-driven leukemia with the clinically approved calcineurin inhibitor ciclosporin (CsA), a compound that blocks intracellular Ca2+ release. CsA-treated animals showed reduction in total leukemic burden (spleen weight diluent vs. CsA, p=0.01) and LSC numbers (p=0.02). This resulted in increased survival of secondary recipient hosts (diluent vs. CsA: median 15 vs. 29 days, p=0.0002). These effects could not be observed for other oncogenes (e.g. MA9), confirming its specificity for AE-induced disease. Consistently, CsA treated primary human t(8;21)-positive AML blasts failed to form colonies in methylcellulose. In summary, our findings identified PLCG1-dependent Ca2+ signaling as a critical pathway for t(8;21) LSC maintenance and self-renewal. Most importantly, as PLCG1 is dispensable for maintenance of normal HSPCs, PLCG1 could serve as a novel therapeutic target in t(8;21) AML. Disclosures Döhner: Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bullinger:Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Abbvie: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Bayer: Other: Financing of scientific research; Pfizer: Honoraria.

Genetic aberrations and clonal evolution underpin chemoresistant and relapsed acute myeloid leukaemia (AML). We used a genome-wide CRISPR knock-out screen to identify genes which mediate resistance to Cytarabine (AraC) and Doxorubicin (Dox) in AML, informing combination therapies to potentially circumvent resistance. Cas9-expressing human AML cell lines, OCI-AML3 and MV4-11, were transduced with the Brunello gRNA library and treated with continuous or intermittent Dox/AraC (D/A), at synergistic doses sufficient to eliminate non-transduced controls, but allow outgrowth of resistant library-transduced populations. In resistant populations, gRNAs targeting AraC metabolism components, including deoxycytidine kinase (DCK) were enriched. Additionally, cyclin-dependent kinase inhibitor 2a (CDKN2A) and checkpoint kinase 2 (CHEK2) were major hits. CDKN2A and CHEK2 mediate apoptosis and cell cycle arrest in response to DNA damage and are clinically relevant in AML. In competition assays, CDKN2A and CHK2 inactivation by CRISPR led to a survival advantage over empty-vector controls in the presence of D/A, confirming their contribution to chemoresistance. This resistance was mediated by evasion of cell cycle arrest in response to chemotherapy, with minimal reductions in apoptosis seen. Given convergence of CDKN2A and CHEK2 pathways on cell cycle G1S transition, we hypothesised that the CDK4/6 inhibitor, palbociclib could enhance efficacy of D/A therapy in AML with deregulation of CDKN2A or CHEK2. Palbociclib induced a rapid G1 arrest in empty vector, CDKN2A- and CHEK2-inactivated cells by 24 hours. Further, it synergised with D/A over longer treatments (4-11 days), demonstrating the therapeutic potential of cell cycle inhibitors in combination with conventional agents to improve chemotherapy response in AML.

Myeloproliferative neoplasms (MPN) arise from the acquisition of genetic mutations in hematopoietic stem cells. Currently, patients receive treatment with cytotoxic agents, the JAK1/2 inhibitor, ruxolitinib, or pegylated forms of interferon alpha (IFN-α). Ruxolitinib is highly effective in treating constitutional symptoms and splenomegaly, however, MPN patients rarely achieve molecular remissions or cure. In contrast, treatment with IFN-α can induce deep molecular remissions, as measured by allelic burden of the JAK2V617F driver-mutation, but treatment may be limited by side effects. There are now clinical trials underway investigating clinical synergy of ruxolitinib and IFN-α in MPN patients. However, the clinical benefits of this combination are currently unknown and deserve preclinical evaluation. We used a preclinical murine model of Jak2V617F-driven MPN to investigate the mechanisms of action of ruxolitinib and IFN-α, in particular, the effects on haematopoietic stem and progenitor cell populations. We demonstrate that ruxolitinib targets progenitor populations but does not deplete MPN disease initiating long-term hematopoietic stem cells (HSCs). Jak2V617F/+ long-term HSCs show hypersensitivity to IFN-α treatment through enhanced STAT1 signaling. Using a novel murine pegylated IFN-α, we show specific targeting of Jak2V617F/+ long-term HSCs in vivo. This was mediated through the preferential induction of reactive oxygen species and DNA damage in Jak2V617F/+ HSCs compared to wild-type HSCs after chronic treatment, offering a novel mechanistic explanation for IFN-α induced reduction of JAKV617F allelic burden in MPN patients. Interestingly, we demonstrate that the target cell specificity of ruxolitinib vs. IFN-α avoids potential antagonism in vivo, a finding with clinical relevance to future efforts to combine these two agents in patients with MPNs.

One of the greatest challenges in drug development for Acute Myeloid Leukemia (AML) is the low success rate of therapeutic candidates in clinical trials. This is mainly due to insufficient preclinical testing using cell lines or transgenic murine models that do not reflect the molecular abnormalities of most AMLs. Here we generated a patient-derived xenograft (PDX) resource within highly immunocompromised mice that recapitulates the risk groups and genetic diversity found in large clinical trials. Bone marrow or blood samples from 50 patients were tested for engraftment in NSGS, NRGS, or NSG, that was defined by blast morphology, established donor chimerism, splenomegaly, anemia or thrombocytopenia. Regression analysis identified donor age as predictor for engraftment success. The overall engraftment success rate in NSGS/NRGS was 70%, with accelerated AML progression when compared to NSG. Pairwise analysis revealed a higher percentage of patient-derived CD34+ CD38- cells in NSG when compared to NSGS/NRGS. From the engrafting AMLs, 30 were randomly selected and molecularly characterized by transcriptional and mutational sequencing. The identities and frequencies of the mutations were similar to those observed in large AML trials, with NPM1 and DNMT3A mutations most frequently found. We validated the clinical significance of the generated AML PDX resource by performing a randomized, Phase II-like preclinical trial on standard chemotherapy (5+3: cytarabine and doxorubicin; 19 individual AMLs; 6 NRGS per treatment group). Relapse was determined by the underlying AML kinetics and closely correlated with prognosis subgroup. Altogether, the generated AML PDX resource faithfully recapitulates the risk groups and molecular abnormalities found in comprehensive clinical trials, and is therefore a valuable tool for adequately powered Phase II-like preclinical trials in AML.

In recent years, technical developments in mouse genetics and imaging equipment have substantially advanced our understanding of hematopoietic stem cells (HSCs) and their niche. The availability of numerous Cre strains for targeting HSCs and microenvironmental cells provides extensive flexibility in experimental design, but it can also pose significant challenges due to strain-specific differences in cell specificity. Here we outline various genetic approaches for isolating, detecting, and ablating HSCs and niche components and provide a guide for advantages and caveats to consider. We also discuss opportunities and limitations presented by imaging technologies that allow investigation of HSC behavior in situ.

The p21-activated kinases (Paks) are serine/threonine kinases that are major effectors of the Rho guanosine 5'\x{2011}triphosphatase, Rac, and Cdc42. Rac and Cdc42 are known regulators of hematopoietic stem and progenitor cell (HSPC) function, however, a direct role for Paks in HSPCs has yet to be elucidated. Lin(-)Sca1(+)c-kit(+) (LSK) cells from wild-type mice were transduced with retrovirus expressing Pak inhibitory domain (PID), a well-characterized inhibitor of Pak activation. Defects in marrow homing and in vitro cell migration, assembly of the actin cytoskeleton, proliferation, and survival were associated with engraftment failure of PID-LSK. The PID-LSK demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK), whereas constitutive activation of ERK in these cells led to rescue of hematopoietic progenitor cell proliferation in vitro and partial rescue of Pak-deficient HSPC homing and engraftment in vivo. Using conditional knock-out mice, we demonstrate that among group A Paks, Pak2(-/-) HSPC show reduced homing to the bone marrow and altered cell shape similar to PID-LSK cells in vitro and are completely defective in HSPC engraftment. These data demonstrate that Pak proteins are key components of multiple engraftment-associated HSPC functions and play a direct role in activation of ERK in HSPCs, and that Pak2 is specifically essential for HSPC engraftment.

Bone marrow (BM) or hematopoietic stem cell (HSC) transplantation is used as curative therapy for hematologic malignancies. Incorporation of gene therapy to drive tolerogenic expression of antigens is a promising strategy to overcome the limited long-term efficacy of autologous HSC transplantation for autoimmune diseases. HSC engraftment and tolerance induction is readily achieved after myeloablative or immune-depleting conditioning regardless of the cellular compartment in which antigen is expressed. It is unclear whether the efficiency of engraftment and tolerance induction is influenced by targeting antigen to specific cellular compartments. This is particularly important when using clinically feasible low-intensity conditioning aimed at preserving infectious immunity in individuals where immunologic memory exists to the autoantigen to be expressed. Here we demonstrate that, under immune-preserving conditions, confining expression of a transgenically expressed antigen to dendritic cells permits stable, long-term engraftment of genetically modified BM even when recipients are immune to the expressed antigen. In contrast, broader expression within the hematopoietic compartment leads to graft rejection and therapeutic failure because of antigen expression in HSCs. These findings are relevant to the clinical application of genetically engineered HSCs and provide evidence that careful selection of promoters for HSC-mediated gene therapy is important, particularly where tolerance is sought under immune-preserving conditions.

In this issue of Blood , Stoddart et al think outside of the stem cell “box” to identify a therapeutically tractable pathway in del(5q) myelodysplastic syndrome (MDS), through blocking pathologically activated Wnt signaling arising within the stem cell niche. 1

Hematopoietic stem cells (HSCs) engage in complex bidirectional signals with the hematopoietic microenvironment (HM), and there is emerging evidence that leukemia stem cells (LSCs) may use similar interactions. Using a syngeneic retroviral model of MLL-AF9 induced acute myeloid leukemia (AML), we have identified 2 different stages of leukemia progression, propagated by “pre-LSCs” and established leukemia (LSCs) and compared the homing properties of these distinctive entities to that of normal HSCs. The homing and microlocalization of pre-LSCs was most similar to long-term HSCs and was dependent on cell-intrinsic Wnt signaling. In contrast, the homing of established LSCs was most similar to that of committed myeloid progenitors and distinct from HSCs. Although osteoblast-derived Dickkopf-1, a potent Wnt inhibitor known to impair HSC function, dramatically impaired normal HSC localization within the bone marrow, it did not affect pre-LSCs, LSC homing, or AML development. Mechanistically, cell-intrinsic Wnt activation was observed in human and murine AML samples, explaining the independence of MLL-AF9 LSCs from niche-derived Wnt signals. These data identify differential engagement of HM associated with leukemic progression and identify an LSC niche that is physically distinct and independent of the constraints of Wnt signaling that apply to normal HSCs.

In this issue of Blood , Wang et al report on the response of splenic-derived hematopoietic stem and progenitor cells from patients with myelofibrosis (MF) to the Janus kinase (JAK) inhibitor, AZD1480.[1][1] ![Figure][2] In MF, MPN disease-propagating stem cells aberrantly mobilize from the

Background: Myeloproliferative neoplasms (MPN) are a diverse group of hematopoietic stem cell disorders. JAK2V617F gain-of-function is the most prevalent mutation, accounting for more than 60% of MPNs. PRMT5 was initially identified as a JAK-binding protein. Its enzymatic function catalyses the symmetric di-methylation of arginine on a variety of substrates, including histones and proteins of the splicing apparatus. It has been proposed that mutant JAK2 can phosphorylate PRMT5, leading to loss of methylation activity and promotion of erythropoiesis (Liu F. et al. Cancer Cell 2011). Based upon this study, it was proposed that enhancing PRMT5 activity may be a useful therapeutic measure (Skoda RC et al. Cancer Cell 2011). Aim: To determine the role of PRMT5 in JAK2V671F mutant hematopoiesis. Hypothesis: Inhibition of PRMT5 will exacerbate JAK2V617F hematopoiesis R esults: Using a conditional null allele, we deleted Prmt5 in embryonic development with the hematopoietic-specific VavCre transgene. This led to embryonic lethality at E9.5 due to absence of erythropoiesis but not other lineages. Similar embryonic lethality was observed using the erythroid specific EpoRCre transgene. Following a 350,000-compound library screen, we developed a potent and selective SAM-dependent inhibitor (CTx034) of PRMT5 similar to that reported by Chan-Penebre E. at al. Nat. Chem. Biol. 2015. Consistent with the genetic evidence that PRMT5 is most important for erythropoiesis, CTx034 was a potent inhibitor of erythropoiesis in cultures derived from healthy human CD34+ cells. This suppression of erythropoiesis was associated with activation of p53. However, progenitor assays of bone marrow cells from patients with MPN showed that JAK2V617F erythropoiesis was more sensitive to CTx034 than normal erythropoiesis. We established JAK2V617F bone marrow chimeric mice to directly compare the in vivo effects of PRMT5 inhibition on mutant and wild-type erythropoiesis within the same animal. Remarkably, these studies showed normalization of spleen size and erythropoiesis, comparable to the current standard of care, Ruxolitinib (Figure 1A-B). Importantly, CTx034 was well tolerated in healthy animals with no suppression of hematopoiesis. One of the major therapeutic challenges for MPN is the eradication of the malignant clone, which is rarely achieved with Ruxolitinib. The addition of MDM2 inhibitors, which activate p53, are currently in trial. Importantly, CTx034 not only suppressed JAK2-mutant erythropoiesis but also activated p53 in JAK2-mutant progenitors, unlike Ruxolitinib (Figure 1C). This result strengthens the therapeutic rationale for PRMT5 inhibitors in MPN. To understand how CTx034 inhibits erythropoiesis, we initially considered direct methylation effects on JAK-STAT signalling and p53. Challenging previous reports, we could find no evidence that JAK alters PRMT5 activity, no evidence that PRMT5 inhibition perturbs JAK-STAT signalling and no evidence that PRMT5 methylates p53. To look more broadly, we performed RNA-seq analysis of CD34+ cells following 72 hours exposure to CTx034. Globally, this demonstrated a potent 'starvation' signal with suppression of protein synthesis despite activation of the upstream mTOR signalling pathway. This suppression of protein synthesis could be linked to three mechanisms. First, CTx034 inhibited methylation of the Sm core complex of the spliceosome, leading to alternate splicing (skipped exons and retained introns) affecting the elongation initiation factor 2 (EIF2) pathway. Second, PRMT5 directly interacts with the translation initiation complex (eIF4A, eIF4B, eIF4E and the poly(A)-binding protein 1, PABP1. Moreover, mass spectrometry identified PABP1 as a new target of PRMT5. Treatment with CTx034 did not alter protein abundance of any of these factors but decreased the RNA binding capacity of PABP1, thereby preventing the correct formation of the initiation of translation complex. Finally, CTx034 perturbed polysome formation with loss of methylation of RPS10. C onclusion: Challenging previous reports, we show that PRMT5 inhibitors are an attractive and novel therapeutic for JAK2V617F MPN by targeting initiation of translation, ribosome biogenesis and activation of p53. Disclosures Sonderegger: CRC Cancer Therapeutics: Research Funding. Cerruti:CRC Cancer Therapeutics: Research Funding. Toulmin:CRC Cancer Therapeutics: Research Funding. Lane:Novartis: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Stupple:CRC Cancer Therapeutics: Employment. Street:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Employment, Patents & Royalties. Jane:CRC Cancer Therapeutics: Patents & Royalties. Altura:MERCK: Employment. Nicholson:MERCK: Employment. Curtis:MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.

Introduction: Resistance to chemotherapy, manifesting as refractory or relapsed disease, remains the largest cause of mortality in acute myeloid leukemia (AML). Clonal evolution through the acquisition of new mutations, or selection of resistant clones, appears responsible in many cases of relapsed disease. We used a genome-wide CRISPR-Cas9 loss of function screen to identify deleted genes, which mediate resistance to Cytarabine (AraC) and anthracycline, Doxorubicin (Dox) in AML. Methods: Dose response was defined using MTS viability assays, with synergism determined using the Chou-Talalay method. For the screen, Cas9-expressing OCI-AML3 cells were transduced with the Brunello gRNA library, which contains 76,441 gRNAs targeting 19,114 genes (Doench et al. 2016, Nat Biotech). Cells were treated with continuous, intermediate-dose AraC/Dox (A/D) or with intermittent, high-dose A/D. Cell viability was monitored by trypan blue exclusion. gRNA representation was measured prior to, and at the end of treatment, using next-generation sequencing. Individual genomic targets were examined sequentially, using multiple gRNAs and correlated with effects on proliferation and cell cycle. Validation was performed in vivo, in independent cell lines (MV4;11) and in silico using bioinformatics on human clinical datasets. Differences in mean values were compared with t-tests. Results: AraC and Dox were synergistic at ratios 10:1, 20:1 and 40:1; the most synergistic ratio (40:1) was used for the screen. By 12 days, both continuous and intermittent dosing eliminated non-transduced controls and the bulk of library-transduced cells. Rapid resistance was seen in AraC monotherapy (10 days), however using combined AraC and Dox, a resistant population emerged by 20 days, mimicking dynamics of relapsed or refractory disease. Resistant populations also showed increased IC50 in vitro and resistance to in vivo A/D when transplanted into immunodeficient NRGS mice, compared to native cell lines (higher white cell counts (3.9 vs 1.9 x 109/L, p = 0.013), lower platelets (597 vs 978 x 109/L, p = 0.024) and higher bone marrow human CD45 chimerism (12.6 vs 1.7%, p = 0.007)). Deoxycytidine kinase (DCK) and cyclin-dependent kinase inhibitor 2a (CDKN2A) were the top hits, out of ~10 genes identified. DCK catalyses the rate-limiting step in metabolising AraC to its active form, and accordingly, AML cells with CRISPR-mediated DCK deletion were resistant to AraC, but also to combination A/D, demonstrating that resistance to a single agent may result in therapeutic failure of combination regimens. CDKN2A transcriptionally encodes p14ARF and p16INK4A, using common exons 2 and 3, but a distinct exon 1 and alternate reading frame. Enriched CDKN2A gRNAs identified in the screen were directed at common exon 2 and p16INK4A unique exon 1, with a p14ARF exon 1 gRNA not being represented in the Brunello library. p16INK4A regulates cell cycle and proliferation by inhibiting G1-S transition and p14ARF prevents p53 degradation, promoting apoptosis. CDKN2A is frequently deleted in multiple types of cancer. Functionally, CDKN2A exon 2 knockout, expected to disrupt both p14ARF and p16INK4A functions, led to chemotherapy resistance through enhanced proliferation and prevention of cell cycle arrest after low-dose and high-dose chemotherapy treatment. To delineate differential contributions of p14ARF and p16INK4A deletion to this resistance phenotype, we deleted p16INK4A unique exon 1. p16INK4A exon 1 deletion also conferred a proliferative advantage in low-dose chemotherapy, but not high-dose, suggesting that concurrent impaired function of p14ARF is required to confer resistance to high-dose chemotherapy. Finally, gene expression data from 3 independent AML patient cohorts (Verhaak, et al. 2009, Haematologica; Metzeler, et al. 2008, Blood; Bullinger, unpublished) demonstrated that low CDKN2A expression conferred inferior survival, confirming the clinical relevance of CDKN2A loss-of-function in AML. Conclusion: This study demonstrates the utility of genome-wide CRISPR screens to functionally capture genetic heterogeneity and evolution through chemotherapy treatment in AML. This approach can identify clinically relevant gene mutations and therapeutic vulnerabilities. Disclosures Bullinger: Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lane:Novartis: Consultancy; Celgene: Consultancy; Janssen: Consultancy, Research Funding.

Immunotherapy has revolutionised therapeutic approaches to fight cancer and, in certain diseases dramatically improves survival. Clinical responses to immune checkpoint blockade have in part been attributed to high mutational burden of tumours such as melanoma. High-risk acute myeloid leukaemia (AML) is defined by molecular and cytogenetic factors. AML has a low prevalence of somatic mutations and is predicted to have low immunogenicity. We aimed to determine how AMLs driven from different classes of oncogenes interact with endogenous anti-leukemic immune responses. Methods and Results We generated three oncogenically distinct models of AML: BCR-ABL+NUP98-HOXA9 (BA/NH9), MLL-AF9 (MA9), and AML1-ETO+NRASG12D (AE/NRAS), using retroviral transduced bone marrow transplanted into immune-competent, non-irradiated C57BL/6J (B6) mice or immune-deficient Rag2-/-γc-/- mice. Immunologic control of AML was dependent on the driver oncogene, as AE/NRAS AML was effectively controlled in B6, but not Rag2-/-γc-/-recipients, whereas survival of BA/NH9 AML recipients was similar between B6 and Rag2-/-γc-/-. MA9 AML had an intermediate phenotype (Figure 1A-C). To examine the mechanisms underlying immune escape in AE/NRAS, AML from immune-deficient or immune-competent hosts, was passaged through immune-competent hosts. Prior exposure to an intact immune system dramatically accelerated disease progression of AE/NRAS AML in subsequent B6 recipients, but this was not seen in passage through Rag2-/-γc-/- recipients. This demonstrates specific, functional immunoediting of AML resulting in evasion of immune control. Despite evidence of disease attenuation in immune competent hosts, functional immunoediting was not observed in MA9 AML. Antibody-mediated immune cell depletion experiments demonstrated that natural killer (NK) cells and T cells both contribute to the control AE/NRAS AML, whereas MA9 immune control was dependent on NK cells. As immunoediting was only seen in AE/NRAS model, this suggests that functional immunoediting in this model is primarily mediated by T cells. To characterise the mechanisms regulating immunoediting, we integrated proteomic and transcriptional analysis of immunoedited and non-immunoedited AE/NRAS AML. There was strong correlation between increased protein expression and transcriptional regulation. There was distinct regulation of inflammatory pathways between immunoedited and non-immunoedited AML. Immunoedited AE/NRAS cells showed increased IFN-γ-dependent response signatures, consistent with direct targeting of the leukemic cells by the immune system. Transcriptional analysis also showed modulation of expression of immune checkpoint molecules including upregulation of suppressive molecules Tim-3 and CD39 and downregulation of activating ligand CD137L. These findings were confirmed by cell-surface flow cytometry. Immunoedited AE/NRAS downregulated RAS signalling transcriptionally, with coordinate activation of MYC targets. In the murine AE/NRAS model, CD4+ and CD8+ T effector memory (TEM) cells (CD44+ CD62L-) demonstrated increased PD-1 expression compared to naïve mice. In addition, mice with high disease burden also had increased frequency of T cells co-expressing exhaustion markers PD-1, Tim-3 and LAG-3, consistent with suppression of the anti-leukemic effector immune response. To understand if these findings were relevant to AML in the clinic, we obtained single cell RNA-sequencing data from the CD45+ CD34- non-leukemic fraction of bone marrow in a patient with AML1-ETO AML at diagnosis compared to that in normal marrow. Single cell type classification and clustering using tSNE demonstrated remodelling of the immune microenvironment in AML with loss of NK cells, pre-B cells and skewing of T cell subsets. There was depletion of CD8+ TEM cells and greater proportions of CD4+ and CD8+ TEM cells expressing activation and exhaustion markers (IFN-γ, PD-1, LAG-3, TIM-3). Conclusions These data demonstrate that immune responses in AML are oncogene-specific and provide evidence that AE/NRAS AML cells undergo immunoediting over time in the presence of a competent immune microenvironment. Since AML is associated with alterations in T cell subsets, and changes in T cell activation and exhaustion states, these findings may inform translational strategies to use immunotherapies for patients with AML. Disclosures Smyth: Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding. Lane:Janssen: Consultancy, Research Funding; Celgene: Consultancy; Novartis: Consultancy.

Hematopoietic stem cells (HSC) require complex bidirectional signals from the hematopoietic microenvironment (HM) and there is emerging evidence that leukemia stem cells (LSC) reside in similar niches within the HM. Existing models of LSC-niche interactions have used xenograft transplantation experiments raising concerns about immunological or cross-species incompatibility. Moreover, the precise components that constrain LSC within the HM have not been fully characterized. We utilized a syngeneic retroviral model of MLL-AF9 induced acute myeloid leukemia (AML) to evaluate LSC-HM interactions and characterize the requirement of key self-renewal pathways in LSC-HM crosstalk. AML was generated using retroviral transduction of murine bone marrow or granulocyte macrophage progenitors (GMP) with the MLL-AF9 fusion oncogene. MLL-AF9 transformed TOPGal (Wnt reporter) bone marrow showed activated canonical Wnt signaling. LSC, (immunophenotype as lineagelowcKithighSca1-FcGRII/III+CD34+), were FACS purified and transplanted into lethally irradiated syngeneic mice expressing GFP in osteoblasts (2.3kbColl1alpha-GFP). The homing and microlocalization of LSC was examined using live, 3 dimensional two-photon confocal hybrid imaging of the calvarium. LSC localized near osteoblasts similar to GMP but farther than HSC (median distance 23.7µm LSC, 22.1µm GMP, 10.6µm HSC, p=ns LSC vs. GMP, p

SummaryWe report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor suppressor whose loss of function results in intestinal polyposis. APC is located in a commonly deleted region on human chromosome 5q, associated with myelodysplastic syndrome (MDS), suggesting that haploinsufficiency of APC contributes to the MDS phenotype. Analysis of the hematopoietic system of mice with the Apcmin allele that results in a premature stop codon and loss of function showed no abnormality in steady state hematopoiesis. Bone marrow derived from Apcmin mice showed enhanced repopulation potential, indicating a cell intrinsic gain of function in the long-term hematopoietic stem cell (HSC) population. However, Apcmin bone marrow was unable to repopulate secondary recipients because of loss of the quiescent HSC population. Apcmin mice developed a MDS/myeloproliferative phenotype. Our data indicate that Wnt activation through haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial transplantation strategies. These data provide a cautionary note for HSC-expansion strategies through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to the development of myeloid disease, and indicate that loss of function of APC may contribute to the phenotype observed in patients with MDS and del(5q).

The genetic events that contribute to the pathogenesis of acute myeloid leukemia are among the best characterized of all human malignancies. However, with notable exceptions such as acute promyelocytic leukemia, significant improvements in outcome based on these insights have not been forthcoming. Acute myeloid leukemia is a paradigm of cancer stem (or leukemia initiating) cells with hierarchy analogous to that seen in hematopoiesis. Normal hematopoiesis requires complex bidirectional interactions between the bone marrow microenvironment (or niche) and hematopoietic stem cells (HSCs). These interactions are critical for the maintenance of normal HSC quiescence and perturbations can influence HSC self-renewal. Leukemia stem cells (LSCs), which also possess limitless self-renewal, may hijack these homeostatic mechanisms, take refuge within the sanctuary of the niche during chemotherapy, and consequently contribute to eventual disease relapse. We will discuss the emerging evidence supporting the importance of the bone marrow microenvironment in LSC survival and consider the physiologic interactions of HSCs and the niche that inform our understanding of microenvironment support of LSCs. Finally, we will discuss approaches for the rational development of therapies that target the microenvironment.

Effective erythropoiesis requires an appropriate supply of iron and mechanisms regulating iron homeostasis and erythropoiesis are intrinsically linked. Iron dysregulation, typified by iron-deficiency anaemia and iron overload, is common in many clinical conditions and impacts the health of up to 30% of the world's population. The proteins transmembrane protease, serine 6 (TMPRSS6; also termed matriptase-2), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis, by regulating expression of the iron regulatory hormone hepcidin. We have performed a systematic analysis of mice deficient in these three proteins and show that TMPRSS6 predominates over HFE and TFR2 in hepcidin regulation. The phenotype of mice lacking TMPRSS6 and TFR2 is characterized by severe anaemia and extramedullary haematopoiesis in the spleen. Stress erythropoiesis in these mice results in increased expression of the newly identified erythroid iron regulator erythroferrone, which does not appear to overcome the hepcidin overproduction mediated by loss of TMPRSS6. Extended analysis reveals that TFR2 plays an important role in erythroid cells, where it is involved in terminal erythroblast differentiation and the regulation of erythropoietin. In conclusion, we have identified an essential role for TFR2 in erythropoiesis that may provide new targets for the treatment of anaemia.

Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF-induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically relevant cytokine-induced stress. These findings have direct relevance to HSC transplantation and the increasing clinical use of agents that modulate autophagy.

Interferon-α (IFNα) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNα on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNα treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNα treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNα on Jak2V617F mutant and normal hematopoiesis and suggest that IFNα achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNα and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy.

Replicative immortality depends on telomerase activation in the majority of cancers including acute myeloid leukemia (AML). Imetelstat is a covalently lipidated 13-mer oligonucleotide that competitively inhibits telomerase activity. Clinical efficacy of imetelstat has recently been reported in essential thrombocythemia and myelofibrosis. We investigated the efficacy of imetelstat in AML using a randomized trial in patient-derived xenografts (PDX). To establish an AML PDX cohort, primary bone marrow (BM) or peripheral blood (PB) samples from 31 AML patients were transplanted into NOD.Cg Prkdcscid Il2rgtm1Wjl Tg (CMV-IL3,CSF2,KITLG)1Eav/Mlo (NSGS). Engraftment was defined by reconstitution of BM and spleen with CD33+ donor cells, PB circulating blasts, anemia or thrombocytopenia. The success rate for engraftment was 70.4% and was independent of cytogenetics. Fifteen independent AML patient samples (n=12 recipients/sample) were tested for efficacy of imetelstat. After engraftment, mice were randomized and treated with imetelstat (15 mg/kg body weight) or control phosphate-buffered saline (PBS) intraperitoneally every 48-72h. Across the entire cohort, survival was improved for imetelstat- vs. PBS-treated PDX (Hazard ratio PBS: 5.299; 3.379 ± 8.312; p < 0.0001; Cox proportional hazards model). Imetelstat delayed the expansion of human AML cells in 14 PDX (93.3%). AML patient samples were divided into 2 groups based on survival outcomes, "Sustained responders" (imetelstat extended survival > 2-fold or > 4 weeks; 9 AML patient samples; 60%) or "Poor responders" (6 AML patient samples, 40%). European LeukemiaNet (ELN)-karyotypic subtypes differed trend-wise between groups: favourable (100% sustained, 0% poor response to imetelstat); intermediate-1, intermediate-2 and poor (45.5% sustained, 54.5% poor response to imetelstat; p = 0.1, Fisher's exact test). Next generation sequencing revealed baseline AML PDX genetic profiles. FLT3, NRAS, TET2, RAD21 as well as 15 genes annotated in DNA damage, cell cycle regulation and apoptosis were mutated exclusively in the sustained responders group. Poor responders revealed mutations in ETV6, FANCM, MKI67, WT1 and 16 additional genes regulating growth factor independence and evasion of apoptosis. Imetelstat response was associated with reduced quiescence and induction of γ-H2AX levels in AML populations (n=4-6 / group / 3 individual AML patient samples): γ-H2AX (MFI normalized to PBS; PBS: 1.0 ± 0.02; imetelstat: 1.24 ± 0.02; p < 0.0001, Student's t test), quiescence (%Ki67-diploid AML cells; PBS: 11.62% ± 0.87; imetelstat: 6.9% ± 0.68; p < 0.001, Student's t test). The presence of spliceosome mutations (SRSF2, U2AF1) was not correlated with response (p = 0.5; Fisher's exact test). To investigate the effects on normal human hematopoiesis, CD34-enriched cord blood was transplanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG, n=5-6/group/donor in 2 replicate experiments). Engraftment was confirmed at 4 weeks post-transplant by PB chimerism (>=1%). Mice were treated with imetelstat or vehicle control from 4 weeks. After 10 weeks treatment, BM cellularity and donor engraftment were significantly reduced whereas the frequency of the CD34+CD38low HSC-enriched population was unchanged (donor 1 PBS: 0.35% ± 0.03%, imetelstat: 0.23% ± 0.06%, p = 0.1; donor 2 PBS: 0.45% ± 0.05%, imetelstat: 0.92% ± 0.35%, p = 0.2, Student's t test). HSC cell cycle and γ-H2AX levels were unchanged. PB and spleen B-cell engraftment were reduced (donor 1 spleen PBS: 77.74% ± 4.66%, imetelstat: 45.48% ± 9.03%, p < 0.05; donor 2 spleen PBS: 93.95% ± 0.40%, imetelstat: 89.1% ± 2.52%, p = 0.09, Student's t test), whereas the myeloid lineage was elevated or relatively preserved (donor 1 spleen PBS: 3.29% ± 0.71%, imetelstat: 7.53% ± 1.6%, p < 0.05; donor 2 spleen PBS: 1.20% ± 0.12%, imetelstat: 4.49% ± 1.60%, p = 0.07, Student's t test). In summary, imetelstat demonstrates efficacy in a significant proportion of AML PDX (60%, 9 out of 15 individual AML patient samples). Robust responses to imetelstat were associated with favourable cytogenetic risk groups and mutations in pathways controlling DNA damage. The effects on normal human hematopoiesis were modest and predominantly seen in the B-lymphocyte lineage with relative preservation of myeloid and stem cell populations. Further study is warranted to understand the preclinical and clinical efficacy of imetelstat in AML. Disclosures Lane: Janssen: Other: i have done consulting (once) for janssen..

Myeloid malignancies arise following the sequential acquisition of somatic mutations within hematopoietic stem and progenitor cells (HSPC). JAK2V617F is commonly found in myeloproliferative neoplasms (MPN) such as polycythemia vera, essential thrombocythemia and myelofibrosis. While other mutations (e.g. TET2, DNMT3A) have been found to co-occur in MPN HSPC, it remains unclear how they impact disease biology or progression from early stage disease (i.e. polycythemia or essential thrombocythemia) to advanced stage disease such as myelofibrosis or acute myeloid leukemia. DNMT3A methylates cytosine rich DNA residues (known as CpG islands, and often found in promoters of genes) leading to transcriptional repression. DNMT3A is also recurrently mutated at relatively low frequency in polycythemia vera (5-7%) but mutations are more common in advanced MPN (approximately 15% of MF and 17% of AML, Stegelmann et al. Leukemia 2011; Abdel-Wahab et al. Leukaemia 2011). These mutations are found in the methyltransferase domain and cluster around arginine 882 (e.g. R882H), resulting in loss of DNA binding and reduced catalytic activity. We used CRISPR-Cas9 gene editing technology to disrupt Dnmt3a function in mouse HSPC and assessed for cooperativity together with a conditional, knockin Jak2V617F allele. Jak2V617F/∆Dnmt3a-Cas9 but not Jak2V617F/Cas9 controls demonstrated increased HSPC self-renewal and proliferation properties in vitro as evidenced by serial replating in methylcellulose (>5 weeks) and increased colony forming unit capacity. Flow-cytometry analysis of Jak2V617F/∆Dnmt3a-Cas9 revealed enrichment in LKS+ (Lin-Sca-1highKithigh) cells 5 weeks after CRISPR-Cas9disruption of Dnmt3a, and this was associated with increased expression of stemness markers Kit and Cd34 in Jak2V617F/∆Dnmt3a-Cas9 cells. RNAseq was performed on early (week 1, P1) and late culture HSPC (week 5, P5) from Jak2V617F-Cas9 (P1 only) and Jak2V617F/∆Dnmt3a-Cas9 (P1, P5). This confirmed deletion of Dnmt3a in Jak2V617F/∆Dnmt3a-Cas9 but not in Jak2V617F/Cas9 controls. Transcriptional upregulation of Kit and Cd34 were confirmed, as well as other key stem cell genes such as Erg and Angpt1 in Jak2V617F/∆Dnmt3a-Cas9 P5. We observed denovo expression of imprinted genes Igf2 and H19 in Jak2V617F/∆Dnmt3a P5, suggesting impaired DNA methylation in this group. Jak2V617F/∆Dnmt3a-Cas9 P5 were significantly enriched for transcriptional pathways controlling cell cycle progression, oncogenic signatures, and DNA damage. Conversely, Jak2V617F/Cas9 controls were enriched for myeloid differentiation and normal progenitor cell signatures. To assess the effect of Dnmt3a loss on Jak2V617F driven MPN, we transplanted Jak2V617F/∆Dnmt3a-Cas9 or Jak2V617F/Cas9 LKS+ into irradiated B6 recipients. Recipients of Jak2V617F/Cas9 LKS+ developed early stage MPN reminiscent of polycythemia vera with high hemoglobin, white cell count and platelets and was sustained >32 weeks. In contrast, Jak2V617F/∆Dnmt3-Cas9 recipients exhibited a biphasic disease, reminiscent of human myelofibrosis. At 8 weeks, Jak2V617F/∆Dnmt3-Cas9 showed panmyelosis with thrombocytosis (1.38x106/µl vs. 1.14x106/µl controls, p=0.057). However, by 32 weeks, this mice became severely pancytopenic with progressive bone marrow failure (Hemoglobin 121g/L vs. 210g/L controls, p =0.0011; platelets 0.338x106/µl vs. 1.343x106/µl controls, p These data demonstrate new evidence linking loss of Dnmt3a with acquisition of self-renewal in combination with constitutively active Jak2V617F. Importantly, in vivo loss of Dnmt3a accelerates or induces myelofibrotic transformation of the underlying MPN. This work provides new understanding to the factors that promote advanced disease in MPN. Ultimately, such knowledge has the potential to inform the development of novel targeted therapeutic approaches for the treatment of transformed MPN, a highly chemorefractory disease associated with extremely poor prognosis in patients. Disclosures Lane: Janssen: Other: i have done consulting (once) for janssen..

Chaetocin is a fungal metabolite that possesses a potent antiproliferative activity in solid tumors by inducing cell death. Although recent studies have extended the role of chaetocin in tumors, the underlying molecular mechanisms such as the downstream cascade that induces cell death has not clearly been elucidated. In this study, we show that chaetocin is able to induce both apoptosis and autophagy in several hepatoma cell lines including HepG2, Hep3B and Huh7 cell lines. Moreover, we found that the inhibition of caspase-3/7 activity by z-VAD-fmk treatment was able to block chaetocin-mediated cell death, whereas blocking autophagy by Bafilomycin A1 or the knockdown of autophagy protein 5 enhanced cell death mediated by chaetocin. These findings suggest that chaetocin has a potent anticancer effect against hepatoma. Inhibition of autophagy may potentiate anticancer effects of chaetocin thus providing evidence that combined treatment with chaetocin and autophagy inhibitors will be an effective strategy for treating cancer.

SummaryThe mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition.

In the current model of the pathogenesis of polycythemia vera (PV), the JAK2V617F mutation arises in hematopoietic stem cells (HSCs) that maintain the disease, while erythroid precursor populations expand, resulting in excessive red blood cell production. We examined the role of these specific cell populations using a conditional Jak2V617F knockin murine model. We demonstrate that the most immature long-term (LT) HSCs are solely responsible for initiating and maintaining the disease in vivo and that Jak2V617F mutant LT-HSCs dominate hematopoiesis over time. When we induced Jak2V617F expression in erythropoietin receptor expressing precursor cells, the mice developed elevated hematocrit, expanded erythroid precursors, and suppressed erythropoietin levels. However, the disease phenotype was significantly attenuated compared with mice expressing Jak2V617F in LT-HSCs. In addition to developing a PV phenotype, all mice transplanted with Jak2V617F LT-HSCs underwent myelofibrotic transformation over time. These findings recapitulate the development of post-PV myelofibrosis in human myeloproliferative neoplasms. In aggregate, these results demonstrate the distinct roles of LT-HSCs and erythroid precursors in the pathogenesis of PV.

Hematopoietic stem cells (HSCs) interact with osteoblastic, stromal, and vascular components of the BM hematopoietic microenvironment (HM) that are required for the maintenance of long-term self-renewal in vivo. Osteoblasts have been reported to be a critical cell type making up the HSC niche in vivo. Rac1 GTPase has been implicated in adhesion, spreading, and differentiation of osteoblast cell lines and is critical for HSC engraftment and retention. Recent data suggest a differential role of GTPases in endosteal/osteoblastic versus perivascular niche function. However, whether Rac signaling pathways are also necessary in the cell-extrinsic control of HSC function within the HM has not been examined. In the present study, genetic and inducible models of Rac deletion were used to demonstrate that Rac depletion causes impaired proliferation and induction of apoptosis in the OP9 cell line and in primary BM stromal cells. Deletion of Rac proteins caused reduced trabecular and cortical long bone growth in vivo. Surprisingly, HSC function and maintenance of hematopoiesis in vivo was preserved despite these substantial cell-extrinsic changes. These data have implications for therapeutic strategies to target Rac signaling in HSC mobilization and in the treatment of leukemia and provide clarification to our evolving concepts of HSC-HM interactions.