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Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.

Thomas, Egan et al. report that hexokinase 2 localizes to the nucleus of leukaemic and normal haematopoietic cells to maintain stemness by interacting with nuclear proteins and modulating chromatin accessibility independently of its kinase activity.Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.

Continuous supply of immune cells throughout life relies on the delicate balance in the hematopoietic stem cell (HSC) pool between long-term maintenance and meeting the demands of both normal blood production and unexpected stress conditions. Here we identified distinct subsets of human long-term (LT)-HSCs that responded differently to regeneration-mediated stress: an immune checkpoint ligand CD112lo subset that exhibited a transient engraftment restraint (termed latency) before contributing to hematopoietic reconstitution and a primed CD112hi subset that responded rapidly. This functional heterogeneity and CD112 expression are regulated by INKA1 through direct interaction with PAK4 and SIRT1, inducing epigenetic changes and defining an alternative state of LT-HSC quiescence that serves to preserve self-renewal and regenerative capacity upon regeneration-mediated stress. Collectively, our data uncovered the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that balances blood cell demands with preserving a stem cell reservoir. Dick and colleagues identify human LT-HSC subsets with distinct quiescent states. They link these differences to INKA1-mediated downregulation of the transmembrane protein CD112 and its interaction with the protein deacetylase SIRT1. INKA1 is inversely correlated with the histone H4K16Ac mark, which then distinguishes ‘latent’ CD112lo LT-HSCs from CD112hi LT-HSCs that are more readily activated in response to hematopoietic stress.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Summary Age-related clonal hematopoiesis is a major risk factor for myeloid malignancy and myeloid skewing is a hallmark of aging. However, while it is known that non-cell-autonomous components of the microenvironment can also influence this risk, there have been few studies of how the spatial architecture of human bone marrow (BM) changes with aging. Here, we show that BM adiposity increases with age, which correlates with increased density of maturing myeloid cells and CD34+ hematopoietic stem/progenitor cells (HSPCs) and an increased proportion of HSPCs adjacent to adipocytes. However, NGFR+ bone marrow stromal cell (NGFR+ BMSC) density and distance to HSPCs and vessels remained stable. Interestingly, we found that, upon aging, maturing myeloid cell density increases in hematopoietic areas surrounding adipocytes. We propose that increased adjacency to adipocytes in the BM microenvironment may influence myeloid skewing of aging HSPCs, contributing to age-related risk of myeloid malignancies., Highlights • Aging increases adipose, myeloid, and CD34+ HSPC density in the human bone marrow • Human CD34+ HSPC niche is reticular, perivascular, and periadipocytic in aging • Aging increases maturing myeloid cell density surrounding adipocytes, In this article, Dick, Flores-Figueroa, and colleagues show that aging alters the spatial organization between CD34+ hematopoietic stem/progenitor cells and adipocytes within human bone marrow. An increased mature myeloid output correlates with these findings, raising the idea that these age-related changes may influence myeloid skewing and the accompanying risk of myeloid malignancy.

SUMMARYGene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSC) point to shared core stemness properties. However, discordance between mRNA and protein signatures underscores an important role for post-transcriptional regulation by miRNAs in governing this critical nexus. Here, we identified miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impaired B lymphoid differentiation and expanded long-term HSC. Integration of protein mass spectrometry and chimeric AGO2 eCLIP-seq identified TBL1XR1 as a primary miR-130a target, whose loss of function phenocopied miR-130a overexpression. Moreover, we found that miR-130a is highly expressed in t(8;21) AML where it is critical for maintaining the oncogenic molecular program mediated by AML1-ETO. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of novel genes and molecular networks underpinning stemness properties of normal and leukemic cells.HIGHLIGHTSmiR-130a is a regulator of HSC self-renewal and lineage commitmentTBL1XR1 is a principal target of miR-130aTBL1XR1 loss of function in HSPC phenocopies enforced expression of miR-130aElevated miR-130a levels maintain the AML1-ETO repressive program in t(8;21) AML

Bone marrow stromal cells (BMSCs) are a key part of the hematopoietic niche. Mouse and human BMSCs are recognized by different markers (LepR and NGFR/CD271, respectively). However, there has not been a detailed in situ comparison of both populations within the hematopoietic microenvironment. Moreover, dog BMSCs have not been characterized in situ by any of those markers. We conducted a systematic histopathological comparison of mouse, human, and dog BMSCs within their bone marrow architecture and microenvironment. Human and dog CD271+ BMSCs had a morphology, frequency, and distribution within trabecular bone marrow similar to those of mouse LepR+ BMSCs. However, mouse bone marrow had higher cellularity and megakaryocyte content. In conclusion, highly comparable bone marrow mesenchymal stromal cell distribution among the three species establishes the validity of using mouse and dog as a surrogate experimental model of hematopoietic stem cell-BMSC interactions. However, the distinct differences in adipocyte and megakaryocyte microenvironment content of mouse bone marrow and how they might influence hematopoietic stem cell interactions as compared with humans require further study.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.

SummaryIt is critical to understand how quiescent long-term hematopoietic stem cells (LT-HSC) sense demand from daily and stress-mediated cues and transition into bioenergetically active progeny to differentiate and meet these cellular needs. Here, we show that lysosomes, which are sophisticated nutrient sensing and signaling centers, are dichotomously regulated by the Transcription Factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSC and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, which promotes quiescence, self-renewal and governs erythroid-myeloid commitment. By contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism to drive LT-HSC activation. Collectively, our study identifies lysosomes as a central regulatory hub for proper and coordinated stem cell fate determination.

It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination., Cell Stem Cell, 28 (10), ISSN:1934-5909, ISSN:1875-9777

Inherited bone marrow failure syndromes (IBMFSs) such as Fanconi Anemia (FA) and Shwachman-Diamond syndrome (SDS) feature progressive cytopenia and a risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Using deep immunophenotypic analysis of hematopoietic stem cells (HSCs) and early progenitors in bone marrow samples from 13 FA/SDS patients without MDS/AML and 6 healthy controls, we revealed relative survival of progenitors that phenotypically resembled granulocyte-monocyte progenitors (GMP) in the patients. In contrast, HSCs, multipotent progenitors, common myeloid progenitors and megakaryocyte-erythroid progenitors were remarkably reduced in FA/SDS patients compared to controls. Whole exome and targeted sequencing of GMP-like cells in leukemia-free patients revealed a prominently higher mutation load in FA/SDS patient samples than in healthy controls. Patient samples also manifested molecular changes that are characteristic of AML: increased G>A/C>T variants, decreased A>G/T>C variants, increased trinucleotide mutations at Xp(C>T)pT, decreased lower mutation rates at Xp(C>T)pG sites compared to other Xp(C>T)pX sites and enrichment for cancer signature 1. AML cells have previously been reported as commonly harboring this cancer signature. Potential pre-leukemic targets in the GMP-like cells from FA/SDS patients included SYNE1, DST, HUWE1, LRP2, NOTCH2 and TP53. Serial analysis of GMPs from a SDS patient, who progressed to AML revealed a gradual increase in mutational burden, enrichment of G>A/C>T signature and emergence of new clones. Interestingly, the molecular signature of marrow cells from two FA/SDS patients with AML was similar to that of FA/SDS without transformation. The predicted founding clones in SDS-AML harbored mutations in several genes including TP53, while in FA-AML the mutated genes included ARID1B and SFPQ. In conclusion, we described an architectural change in the hematopoietic hierarchy of FA/SDS with remarkable preservation of GMP-like populations harboring unique mutation signatures. GMP-like cells might represent a cellular reservoir for clonal evolution. Disclosures No relevant conflicts of interest to declare.

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Pleiotropic cytokines like interleukin (IL)-3 dimerize their receptors to signal multiple functional activities of AML cells, however, the mechanistic link between cytokine receptor dimerization and LSC fate regulation is unknown. Here, we show that the Class I heterodimeric IL3Rα/βc receptors (IL-3R) assemble as hexamers and dodecamers that segregate proliferation versus differentiation signals, respectively. Receptor stoichiometry varies across the individual cells that make up the AML hierarchy, with LSCs presenting with the highest IL3Rα/βc ratio thereby driving resultant hexamer-mediated stemness programs and poor patient survival. Our study establishes a new signaling mechanism of cytokine pleiotropy and the biased instruction of cell fate that directs the AML hierarchy; a signaling mechanism that may be generalizable to other normal and transformed cellular hierarchies.

TO THE EDITOR: BCR-ABL -negative myeloproliferative neoplasms (MPNs) are a group of clonal stem cell disorders characterized by the overproduction of mature myeloid cells. However, MPN patients also have a 2.8- to 3.4-fold increased risk of developing a lymphoproliferative disorder (LPD) compared

Background The identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a "common retained region" within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies. Results In healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy. To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes. Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models. Conclusion We have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. Disclosures Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

ASH Abstract. Intro Acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) are poor prognosis hematological malignancies characterized by abnormal hematopoiesis and dysfunctions of the hematopoietic stem cell system. Chemotherapy remains the standard of care but is associated with side effects and often high rates of relapse. Today, less than a third of patients diagnosed with AML are cured. Bispecific T-cell engagers (BiTEs) are promising immunotherapeutic agents intended for cancer treatment. BiTEs are small molecules constructed of two single chain variable fragments (scFv) connected in tandem by a flexible linker that acts by retargeting T-cells against tumor cells. One scFv binds to CD3, while the second scFv binds to a tumor-associated antigen. This structure and specificity allow a BiTE construct to physically link a T-cell to a tumor cell, stimulating effector cell activation ultimately leading to cytokine production and tumor killing. Material BiTEs against CD34/CD3 and relevant controls were constructed by recombinant DNA technology and purified from the supernatants of transfected CHO cells following standard procedures. The scFv domain binding to CD34 is positioned N-terminally, and the scFv binding to CD3e C-terminally followed by a hexa-histidine sequence. Results By co-culturing T-cells and target AML cells for 48 h in the presence of increasing concentrations of BiTE or controls, we observed that CD34-BiTE efficiently triggered T-cell-mediated depletion of the CD34 hi and CD34 low cell lines, while negative controls killed none of the target cell lines. Next, we examined the T-cell activation and proliferation. We observed that both CD4+ and CD8+ T-cells presented high levels of CD25/CD69 expressions when the CD34+ cell lines were co-cultured with T-cells in the presence of the CD34/CD3 BiTE. No unspecific activation was found when CD34- cell line was used as target cell. Since CD34 is constitutively expressed by HSCs, the CD34-specific BiTE may deplete not only CD34 +AML blasts but also healthy HSCs. To test this, T-cells and HSCs were purified from PBSC grafts and co-cultured in the presence of either CD34/CD3 BiTE or controls. After co-culture, a significant depletion of CD34 + HSCs was observed for the CD34/CD3 BiTE. To address the potential of the anti-CD34 BiTE in vivo, we next established a human CD34 + cell line in NSG mice per intravenous injection and randomized into three different groups and started treatment the day after. Two groups of mice received two consecutive cycles of one intraperitoneal injection of freshly isolated human T-cells followed by daily intravenous injections of either BiTE or control. The mice were euthanatized at day 21 by which the AML burden was measured, and T-cells quantified. No side effects of the treatment, including after BiTE administration, was observed. There were statistically significant reductions of leukemia burden in both bone marrow and spleen in mice receiving T-cells and BiTE compared to T-cells only and control. Conclusions We show that the CD34/CD3 BiTE is able to promote T-cell activation and killing of CD34-expressing target cells with high efficacy in vitro and in vivo, supporting the translation of this drug into clinical trials. In this scenario, the treatment with CD34-targeting BiTE prior to HSCT would trigger the patient's T-cells to deplete CD34 + leukemic blasts and HSCs. As consequence, this adjuvant treatment would decrease the use of cytotoxic and cytostatic conditioning drugs before HSCT, reducing life-threatening complications such as GvHD and infections. Disclosures Arruda: Anocca: Current Employment, Research Funding. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mattsson: MattssonAB medical: Current Employment, Current holder of individual stocks in a privately-held company. Onfelt: Desumo: Current Employment, Current holder of individual stocks in a privately-held company. Uhlin: XNK therapeutics: Current Employment, Current holder of stock options in a privately-held company.

Introduction : Peripheral blood samples of healthy individuals may harbour detectable mutations in genes recurrently mutated in myeloid malignancies, a situation referred to as clonal hematopoiesis (CH). Risk factors for CH include increasing age as well as previous exposure to cytotoxic therapy. CH has been associated with an increased risk of overall mortality, including in the setting of autologous stem cell transplantation (ASCT) for non-Hodgkin lymphoma (Gibson et al, JCO, 2017). The excess mortality is largely driven by cardio-vascular disease, but may also be additionally attributable to an increased risk of myeloid malignancies that arise through the selection of CH subclones. Herein, we aimed to investigate the prognostic implications of CH after ASCT in an independent and diversified, large cohort of lymphoma patients using ultra-deep, highly sensitive error-correction sequencing. Methods : DNA was obtained from 420 residual apheresis products obtained from patients who had undergone autologous stem cell transplantation for lymphoma at the Princess Margaret Cancer Center between 2002 and 2018. Target DNA sequences corresponding to regions recurrently mutated in myeloid neoplasms (affecting n = 36 genes) were captured using single molecule molecular inversion probes (smMIPs) that incorporate molecular tagging. Single nucleotide variants and short insertions and deletions were identified using SmMIP-tools (Medeiros et al, bioRxiv, 2021), which implements a series of steps including probabilistic modeling of allele-specific error rates and generation of consensus sequences to suppress next-generation sequencing-associated errors. Given the high sensitivity and precision of our method, we did not prespecify a variant allele fraction cut-off. Results : All patients had relapsed/refractory lymphoma, except for 98 (23.3%) mantle cell lymphoma patients and one patient with extranodal NK/T-cell lymphoma where ASCT was part of frontline management. The most common conditioning regimens were high-dose melphalan and etoposide (77.5%) and high-dose melphalan and Ara-C (16.4%). We identified 275 high-confidence mutations in 181 out of 420 patients (43.1%), with 64 of these 181 patient samples (35.4%) having more than one mutation. The median age was higher in patients with CH than in patients without (55 years vs. 51, P = 0.002). The most frequently mutated gene were PPM1D (11.9%), followed by TET2 (11.4%), DNMT3A (8.8%), ASXL1 (5.2%) and TP53 (4.5%). The lymphoma subtype with the highest prevalence of CH was T-cell lymphoma (CH found in 72.2% of cases), followed by transformed indolent lymphoma (51.4%), mantle cell lymphoma (47.5%), diffuse large B-cell lymphoma (40.4%) and Hodgkin lymphoma (33.3%). While there was no difference in the number of CD34+ cells infused for patients with and without CH, the median time to neutrophil engraftment and the median time to platelet engraftment were significantly longer in patients with CH (11 days vs. 10 days, P = 0.025; and 14 days vs. 13 days, P < 0.001, respectively). The median follow-up of living patients was 4.2 years. Patients with CH had inferior 5-year OS from the time of first relapse (38.9% vs. 45.5%, P = 0.037) and from the time of ASCT (51.2% vs. 59.1%, P = 0.017, see figure). Five-year OS from ASCT was 47.5% vs. 53.7% in patients with 1 mutation and > 1 mutation, respectively, compared to 59.1% in patients without CH (P = 0.005). The presence of CH did not have an impact on the risk of post-ASCT relapse. In multivariate Cox regression analysis in which CH and age (as a continuous variable) were included, CH remained significantly associated with adverse OS post-ASCT (HR 1.39, 95% 1.02-1.91, P = 0.038). Only seven patients out of 420 (1.7%) developed a therapy-related myeloid neoplasm (TMN). The cumulative incidence of TMN was not significantly increased in patients with CH (10-year cumulative incidence 3.3% vs. 3.0% in those without CH, P = 0.433). Conclusions : Our results show that CH was associated with delayed neutrophil and platelet engraftment. Moreover, CH conferred an increased risk of death after ASCT that was not explained by lymphoma relapse. The risk of TMN was low in our cohort and CH was not a risk factor for TMN, an observation that is distinct from prior observations (e.g. Gibson et al, JCO, 2017 and Husby et al, Leukemia, 2020). Our results raise the possibility that the risk of TMN may be modulated by factors other than CH. Figure 1 Figure 1. Disclosures Minden: Astellas: Consultancy. Kuruvilla: Janssen: Honoraria, Research Funding; Antengene: Honoraria; AstraZeneca: Honoraria, Research Funding; Amgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Karyopharm: Honoraria, Other: Data and Safety Monitoring Board; Pfizer: Honoraria; AbbVie: Honoraria; TG Therapeutics: Honoraria; Medison Ventures: Honoraria; Merck: Honoraria; Gilead: Honoraria; BMS: Honoraria; Roche: Honoraria, Research Funding; Seattle Genetics: Honoraria. Crump: Roche: Research Funding; Epizyme: Research Funding; Kyte/Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Prica: Kite Gilead: Honoraria; Astra-Zeneca: Honoraria. Chen: Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Kridel: Gilead Sciences: Research Funding.

Acute myeloid leukemia (AML) is a heterogeneous, aggressive hematological malignancy with dismal prognosis where limited targeted therapies are currently available. Poly-(ADP-ribose)-polymerase (PARP) inhibition has emerged as an important therapeutic arsenal to target homologous recombination-deficient tumors. However, molecular understanding of PARP blockade in the context of epigenetic derangements and transcriptional plasticity in human elderly AML pathogenesis remains unexplored. KDM6 proteins are H3K27 demethylases that critically regulate chromatin architecture in multi-cellularity and tumorigenesis (Tran, Mol Cell Biol 2020). KDM6A escapes X-chr inactivation, and Utx-/- female mice spontaneously develop aging associated myeloid leukemia (Gozdecka, Nat Genet 2018; Sera, Blood 2021). In addition, KDM6A loss of function mutation is implicated in AML relapse (Stief, Leukemia 2020). In contrast, KDM6B primarily exerts an oncogenic function in heme-malignancies. Together, KDM6A and KDM6B play cell type-specific function in leukemia, and KDM6 proteins and associated signaling emerge as important focal point for developing molecular targeted therapy. We identify that KDM6 demethylase activity critically regulates DNA damage repair (DDR) gene expression program in AML. Transcriptome analysis indicated a significant downregulation of expression of DDR genesets in both KDM6A deficient human AML and Utx -/- pre-leukemic cells. Lentiviral shRNA screening performed in response to low-dose γ-irradiation in AML stem cells, revealed a radioprotective function of KDM6A. Expression of KDM6s is regulated by genotoxic stress in a time-dependent manner, and deficiency of JmjC catalytic function impaired DDR transcriptional activation and compromised repair potential. Mechanistically, quantitative ChIP experiments also revealed co-operation between KDM6A and SWI/SNF facilitating dynamic chromatin remodeling at TSS/promoter to induce DDR gene expression. To interrogate changes in chromatin accessibility we performed ATAC-seq analysis in KDM6 deficient AML. Motif enrichment highlighted that while KDM6A depletion led to reduced chromatin access to 140 transcription factors (TFs), only 56 TF binding sites showed increased accessibility. Overall, changes in chromatin accessibility, associated with a reduced binding of DDR regulatory TFs in KDM6 deficient AML, account for a compromised DDR function. In agreement with these findings an array of KDM6 deficient AML cells were more sensitive to PARP inhibition, and pre-clinical mice models xenotransplanted with KDM6A loss of function AML line showed an increased susceptibility to PARP blockade in vivo. FLT3-ITD positive AML with a lower KDM6A expression was more sensitive to olaparib. In addition, olaparib administration significantly reduced bone marrow engraftment of patient-derived xenografts of KDM6A-mutant primary AML. Interestingly, KDM6A expression is upregulated in venetoclax-resistant monocytic-AML compared to venet-sensitive primitive-AML. Using venet responsive isogenic lines we demonstrated that attenuation of KDM6 function increased mitochondrial activity, intracellular ROS levels, de-repressed BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6 loss resulted in transcriptional repression of BCL2A1, commonly associated with venet resistance (Zhang, Nat Cancer 2020). Corroborating these results, dual targeting of PARP with BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing primary AML apoptosis, and KDM6A loss further enhanced this synergism. In sum, our study illustrates a molecular mechanistic rationale in support for a novel combination targeted therapy for AML, and posit KDM6A as a molecular determinant for therapeutic efficacy. Intriguingly, KDM6A functions as a gatekeeper of BCL2 and BCL2A1 expression. Similar to TET2 although bi-allelic Utx loss causes evolution to myeloid neoplasms, minimal KDM6 activity is important for survival of human AML cells. KDM6s have been implicated in solid tumors, and both PARP and BCL2 inhibitors are being tested in cancer patients, underscoring a wider scope of application. To conclude, KDM6A unfolds to be a central regulator for susceptibility of AML to both PARP and BCL2 inhibition, expanding the possibility to characterize effective combination targeted therapy for AML in clinical settings. Disclosures Minden: Astellas: Consultancy. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Summary Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.

Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a primary AML culture model arranged in a hierarchy with defined stem cells. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. In contrast, other metabolic enzymes including PFK1, FH, PKM2, GPI1, ENO1, CS, ACO2, and SDHA1, 2 were not detected in the nucleus of these cells. We also detected HK2 in the nucleus of AML cell lines as well as 7 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals in 8227 and NB4 leukemia cells. This increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Nuclear HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with 3'UTR shRNA. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation with ATRA, and decreased CD34+CD38- 8227 stem cells without changing basal proliferation. Nuclear HK2 was also higher in normal human hematopoietic stem cells and multipotent progenitor fractions and declined as the cells matured. Over-expression of nuclear HK2 in normal cord blood increased the primary and secondary engraftment into mice. Transgenic mice over-expressing nuclear HK2 driven by a Vav promoter had increased hematopoietic stem cells in the marrow and decreased monocytes and lymphocytes in the peripheral blood. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2 (D209A D657A) and observed increased clonogenic growth and inhibited differentiation on ATRA treatment, nuclear HK2 function is independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry identified proteins related to chromatin organization and regulation to interact with nuclear HK2. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Citation Format: Geethu Emily Thomas, Grace Egan, Laura Gracia Prat, Aaron Botham, Veronique Voisin, Elias Orouji, Jordan Chin, Boaz Nachmias, Kerstin B. Kaufmann, Neil Maclean, Rose Hurren, Marcela Gronda, Xiaoming Wang, Dilshad H. Khan, Rashim P. Singh, Andrea Arruda, Mark Minden, Gary D. Bader, John E. Dick, Aaron D. Schimmer. The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal hematopoietic stem/progenitor cells to maintain stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3099.

AML is a stem cell disease wherein the properties of the disease-driving leukemia stem cells (LSCs) are reflected in the cellular hierarchies that they generate. We sought to understand how these hierarchies vary across patients and whether their characteristics are clinically relevant. To evaluate cellular hierarchies in AML, we re-analyzed the scRNA-seq data of 13,653 cells from 12 AML patients at diagnosis (van Galen, Cell 2019) and in particular the stem and progenitor blast populations. We identified three novel leukemia stem populations differing in their depth of quiescence, inflammatory signaling, and myeloid priming. Using the signatures of 7 malignant populations as well as 7 immune cell populations from this scRNA-seq data, we applied CIBERSORTx deconvolution (Newman, Nat Biotechnol 2019) on bulk RNA-seq data from multiple patient cohorts. This enabled determination of the relative abundance of each cell type in each patient, thereby capturing the "shape" of the leukemic hierarchies of hundreds of AML patients. AML patients within these cohorts clustered into 4 groups defined by different proportions of stem, progenitor, and mature blasts - each of which differed in their underlying genomic alterations and overall survival. Differences in chemotherapy response were mediated by specific cell types, notably a GMP-like blast population and a quiescent stem-like population (qLSPC). Dominance of the GMP-like population was associated with longer survival (HR -3.1, p=0.002), and these blasts were enriched among younger patients ( 65 years) and patients with adverse cytogenetic alterations. Critically, this qLSPC population was enriched at relapse as well as within a subset of pediatric AML patients that failed to respond to induction chemotherapy. We confirmed that qLSPCs were also enriched among functionally validated leukemia engrafting (LSC+) sorted AML fractions. Accordingly, a high LSC17 score was strongly correlated with qLSPC abundance and anti-correlated with GMP-like abundance, suggesting that the score may reflect the underlying cellular hierarchy of each AML patient. Next, we generated drug sensitivity profiles for each cell type by correlating ex vivo drug sensitivity data for each of 112 inhibitors screened in the BEAT-AML trial with the relative abundance of each cell type across individual patients. In particular, Venetoclax sensitivity correlated with the abundance of primitive cell types and anti-correlated with mature cell types, suggesting that the composition of the leukemic hierarchy in AML patients is associated with drug response. We previously demonstrated that a high LSC17 score identifies patients who do not benefit from standard chemotherapy (Ng, Nature 2016). To develop a diagnostic tool for therapy selection amongst these poor prognosis patients, we retrained the LSC17 genes against cell type abundance and derived a subscore (LSC-7) to map patients along an axis of primitive vs mature leukemic hierarchies. We show that AML samples with a high LSC-7 score (more stem-like blasts) were more sensitive to Venetoclax whereas AML patients with a low LSC-7 score (more mature blasts) benefited from treatment with Gemtuzumab-Ozogamicin (GO). Used together, the LSC17 and LSC-7 scores enable risk stratification as well as subsequent drug selection for high-risk patients, and can both be measured by a single rapid NanoString assay. To apply this approach more broadly, we identified several published studies with drug screening data from primary patient samples and accompanying RNAseq data. By correlating cell type abundance with drug response, we were able to map the critical cell types that mediate sensitivity and resistance to inhibitors of mitochondrial metabolism, histone demethylation, and the CD47-SIRPa axis, among others. Our data establish that scRNA-seq informed deconvolution of bulk expression data permits characterization of the cellular hierarchy of individual AML patients. This framework can enhance our understanding of many aspects of biological, genomic, and clinical heterogeneity in AML, and represents a powerful tool to enable personalized therapeutic decision-making in AML. Figure Disclosures Wang: Trilium Therapeutics: Patents & Royalties. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

Background: Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). From our previous in vivo miRNA enforced expression screen in human hematopoietic stem and progenitor cells (HSPC), we identified miR-130a as a regulator of self-renewal and lineage specification. Enforced expression of miR-130a in human cord blood (CB) derived HSPC caused an expansion of HSC, block in erythroid differentiation and abnormal myelopoiesis in xenografts. Thus, we examined miR-130a expression in AML and found miR-130a to be specifically upregulated in t(8,21) AML. The translocation t(8,21) is one of the most common karyotypic abnormalities in AML, accounting up to 10% of all AML cases. The consequence of this translocation is a fusion of AML1 and ETO genes, resulting in a formation of the AML1-ETO (AE) oncofusion protein, which acts as a dominant repressor of the wild type AML1/RUNX1. The ETO moiety mediates the recruitment of the nuclear corepressor (NCoR) and histone deacetylases (HDAC1-3) to block RUNX1 target gene expression. This prevents myeloid maturation, apoptosis and promotes leukemogenesis. Here we investigated the molecular mechanism of miR-130a in t(8,21) AML and how it contributes to the leukemogenesis of this AML subtype. Results: Using the TCGA dataset and our PMCC patient cohort, we identified miR-130a to be upregulated in t(8,21) AML and high miR-130a expression was associated with worse patient overall survival. To interrogate the functional significance of elevated miR-130a in t(8,21) AML, we performed knock-down (KD) experiments in the Kasumi-1 cell line, which represents a well characterized model system for t(8,21) AML. Notably, KD of miR-130a induced a significant reduction in the CD34+ cell population and an increase in differentiated CD11b+CD15+ and pro-apoptotic annexin V+ cells. We next examined the impact of miR-130a KD in CD34+ blasts from primary t(8,21) AML patient samples. In line with our findings in the Kasumi-1 cells, miR-130a KD decreased the proportion of CD34+ cells and increased the proportion of differentiated CD11b+CD15+ blasts. To investigate the effect of miR-130a KD on leukemic engraftment in vivo, we transduced CD34+ blasts from 2 patient samples and transplanted them into NSG-GF mice. miR-130a KD decreased leukemic engraftment and the proportion of transduced cells, corroborating the functional significance of high miR-130a expression in t(8,21) AML. To investigate the mechanistic action of miR-130a, we performed label-free semi-quantitative proteomics in human CB derived HSPC to uncover miR-130a targets. Surprisingly, we found the beta subunit of RUNX1, CBFb, and Transducin Beta Like 1 X-Linked Receptor 1, TBL1XR1, to be among the most repressed targets. TBL1XR1 is a component of the nuclear receptor corepressor (NCoR) complex and is involved in NCoR degradation. Thus, we performed western and immunoprecipitations (IP) assays in Flag-AE Kasumi-1 cells following miR-130a KD to examine changes in the expression of proteins associated with the AE complex. We observed increased expression of CBFβ, TBL1XR1 and CEBPA with miR-130a KD. Notably, miR-130a KD resulted in a dramatic decrease of NCoR protein levels. IP of Flag-AE showed decreased association of CBFβ and NCoR with AE, despite unaltered protein levels of AE. To investigate changes in binding occupancy of Flag-AE after miR-130a KD, we performed Cleavage Under the Targets and Release Using Nuclease (CUT&RUN) assay. Surprisingly, we observed 2-fold gain of AE sites in miR-130a KD sample compared to control. De novo motif enrichment analysis showed loss of motives for ETS and HOX transcription factors known to associate with AE following miR-130a KD. Genomic distribution of the peaks revealed a dramatic shift of AE peaks away from the promoter region to introns in miR-130a KD. Pathway enrichment analysis of the unique peaks gained in miR-130a KD showed stress responses and organelle disassembly, in line with the differentiation phenotype observed with miR-130a KD. Collectively, we uncovered a novel mechanism by which miR-130a reinforces the aberrant AE molecular program by controlling the composition and binding of the AE complex. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Introduction: Leukemia is the most common cancer in children and sequencing data suggest that the first genetic alterations often occur in utero. Children with Down syndrome (Trisomy 21, T21) have a 150-fold increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient myeloproliferative disorder (pre-leukemia) occurs, which is characterized by a clonal proliferation of immature megakaryoblasts carrying somatic mutations in the GATA1 transcription factor (GATA1s) and resolves spontaneously in most cases. In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemic clone by acquisition of additional mutations, such as in the cohesin subunit STAG2. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1s and STAG2. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the interplay between each genetic event and the cellular origin of transformation. Methods: Human long-term hematopoietic stem cells (LT-HSCs) were sorted from normal karyotype and T21 fetal livers (N-FL and T21-FL) and subsequently CRISPR/Cas9 edited to try to establish a humanized model of Down Syndrome associated pre-leukemia and AMKL. To model the initiation of the pre-leukemic state, GATA1s mutations were introduced, while additional STAG2- mutations were overlaid to model the progression to fully transformed AMKL. CRISPR/Cas9-edited control, GATA1s, STAG2- and GATA1s/STAG2- LT-HSCs were functionally interrogated in near-clonal xenograft assays, along with transcriptional and epigenetic profiling. Results: T21 status in combination with GATA1s had a profound synergistic effect on megakaryocytic lineage output in vivo compared to normal karyotype with GATA1s. Moreover, a high percentage of blasts were found in xenografts of GATA1s edited T21-FL LT-HSCs (>30%) but not in xenografts of GATA1s edited N-FL LT-HSCs. Conversely, GATA1s/STAG2- edited LT-HSCs generated grafts with >50% of blasts, regardless of T21 status. The immunophenotype of these blasts recapitulated those observed in patients diagnosed with Down Syndrome pre-leukemia and AMKL (CD117+CD34+CD41+CD71+CD33+CD4+CD7+). Thus, T21 is required for pre-leukemia development, but seems dispensable for AMKL as both N- and T21-FL LT-HSCs underwent leukemic transformation upon GATA1s/STAG2-. Serial xenotransplantation assays from primary engrafted mice were carried out to assess self-renewal properties of GATA1s-induced pre-leukemia and GATA1s/STAG2- induced AMKL. Only GATA1s/STAG2- edited N- and T21-FL grafts were able to propagate the leukemic phenotype with a high stem cell frequency, which was endowed by the additional STAG2- knock-out. ATACseq and RNAseq profiling of blast populations revealed an enrichment of GATA-binding sites with concomitant up-regulation of genes implicated in translation. To assess the role of progenitor cells in pre-leukemic initiation and leukemic progression, we CRISPR/Cas9 edited short-term HSCs, common myeloid progenitors and myelo-erythroid progenitors with GATA1s and/or STAG2- and subjected them to xenotransplantation. Strikingly, all progenitor subsets with combined GATA1s/STAG2- editing were able to drive leukemic transformation, while single GATA1s editing in the same subsets did not initiate pre-leukemia. This data strongly suggests that the initial GATA1s mutation must occur in T21 LT-HSCs, but subsequent STAG2 mutations can occur further downstream in progenitors. Lastly, to gain insight into how chromosome 21 predisposes towards pre-leukemia, three chromosome 21 miRNAs (miR-99a, -125b-2 and -155) were identified to be up-regulated in T21-FL LT-HSCs compared to N-FL LT-HSCs. Over-expression of these miRNAs in N-FL LT-HSCs induced a T21-like state with increased myeloid and megakaryocytic skewing. Dramatically, CRISPR/Cas9-edited knock-out of these miRNAs in GATA1s edited T21-FL LT-HSCs resulted in a block of pre-leukemia initiation. Conclusion: Our findings demonstrate that T21 is required for pre-leukemia initiation, which is mediated by over-expression of chromosome 21 miRNAs in LT-HSCs. Further, this data demonstrates different cell of origins between pre-leukemia initiation and AMKL progression. Ongoing studies focus on preventing the progression of pre-leukemia to AMKL by pharmacological targeting. Figure Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor survival, especially in older patients. Despite high remission rates after chemotherapy, relapse and death are frequent due to persistence of leukemia stem cells (LSCs), which possess properties linked to therapy resistance. Thus, there is an urgent need for a deeper understanding of the unique properties of LSCs. MicroRNAs (miRNAs) are non-coding RNAs that decrease expression of their target mRNAs by post-translational silencing. miRNA profiling of human AML samples fractionated based on LSC activity revealed that miR-125b is expressed at significantly higher levels on cell fractions enriched in LSCs. To evaluate the role of miR-125b in LSCs, expression of miR-125b was enforced in a hierarchical AML model cell line (OCI-AML-8227). miR-125b overexpression (OE) resulted in a significantly lower percentage of CD14+CD15+ differentiated myeloblasts (Figure 1A) and enhanced clonogenic potential in vitro (Figure 1B). Xenotransplantation of four AML patient samples with miR-125b OE revealed a significant increase in the proportion of CD117+ cells, a marker of hematopoietic and leukemic progenitors (Figure 1C). Secondary transplantation of cells harvested from primary engrafted mice at limiting dilution demonstrated a marked increase in LSC frequency with miR-125b OE compared to controls for the two AML samples tested (Figure 1D). Together, these data strongly suggest that miR-125b enhances the self-renewal of LSCs. To investigate the mechanisms by which miR-125b enhances self-renewal, proteomic analysis of miR-125b-OE Ba/F3 cells as well as in silico target prediction were performed and identified PTPN18 as a top putative target for miR-125b. PTPN18 is a tyrosine phosphatase that has been reported to dephosphorylate auto-phosphorylated kinases such as Her2 and Abl to prevent their activation. To evaluate whether PTPN18 OE can rescue the effects miR-125b on LSCs, we carried out transduction of an AML patient sample with control, miR-125b OE, PTPN18 OE, or both miR-125b and PTPN18 OE vectors followed by xenotransplantation. Similar to previous findings, miR-125b OE alone significantly reduced the frequency of CD11b+CD15+ differentiated myeloblasts. Co-transduction of miR-125b/PTPN18 OE vectors resulted in generation of significantly more CD11b+CD15+ cells compared to miR-125b OE alone (Figure 1E), suggesting that suppression of PTPN18 contributes to miR-125b-mediated enhancement of LSC self-renewal. To identify putative phosphotyrosines that might be altered through the miR-125b-PTPN18 signalling axis, we performed immunoprecipitation of phosphotyrosines followed by mass spectrometry in miR-125b-OE Ba/F3 cells and identified increased GSK3 tyrosine phosphorylation as a top target. Additionally, miR-125b OE was confirmed to enhance GSK3 tyrosine phosphorylation, whereas PTPN18 OE reduced it (Figure 1F), together strongly suggesting that miR-125b could enhance tyrosine phosphorylation of GSK3 by silencing PTPN18. GSK3A and GSK3B (GSK3A/B) are paralogous genes that share a high degree of sequence homology and belong to the glycogen synthase kinase 3 (GSK3) family. Tyrosine phosphorylation activates the kinase activity of GSK3, whereas serine phosphorylation inactivates it. We recently identified GSK inhibitors as top candidates targeting LSCs in a stemness-based drug screen using OCI-AML-8227 cells (data not shown). Treatment of OCI-AML-8227 cells with two selective inhibitors of GSK3 selectively reduced the proportion of CD34+ cells while concomitantly increasing expression of myeloid markers CD14 and CD15 (Figure 1G). Overall, our results support an important functional role for PTPN18 and GSK3 in LSC function, and present a potential novel therapeutic target against LSCs. This study highlights the importance of understanding the role of miRNAs and may identify a new druggable vulnerability in LSCs that could lead to the development of new treatment options for AML patients. Figure 1 Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium Therapeutics: Patents & Royalties.

Inflammation underlies many chronic diseases of aging, involves dysregulated myeloid cells, and has emerged as a critical process regulating both normal hematopoietic stem cell (HSC) function and myeloid malignancy. However, few of the signaling pathways that underlie these proposed linkages are known. Here, we show that bioactive lipid signaling through sphingosine 1-phosphate receptor 3 (S1PR3) regulates human myeloid lineage determination via inflammatory programs and is dysregulated in acute myeloid leukemia (AML). S1PR3 is myeloid-restricted and not expressed by quiescent HSC, but upon overexpression (OE) promoted myelopoiesis and activated inflammatory signatures. S1PR3 is deficient in primitive AML, but enriched in more mature cases. Importantly, in functionally-validated primary leukemia stem cells (LSC) sorted for high S1PR3 expression or when differentiation was induced through S1PR3OE exhibited reduced xenograft repopulating ability. Treatment of recipient mice engrafted with the S1P prodrug FTY720 reduced patient AML LSC frequency. Thus, S1PR3 marks a subset of less primitive AML cells with a distinct inflammatory signature, and activation of S1PR3 may represent a novel therapeutic approach to disrupt LSC function through induction of differentiation.

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution., Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

Blood cells are organized as a hierarchy with hematopoietic stem cells (HSCs) at the root. The advent of genomic technologies has opened the way for global characterization of the molecular landscape of HSCs and their progeny, both in mouse and human models, at the genetic, transcriptomic, epigenetic, and proteomics levels. Here, we outline our current understanding of the molecular programs that govern human HSCs and how dynamic changes occurring during HSC differentiation are necessary for well-regulated blood formation under homeostasis and upon injury. A large body of evidence is accumulating on how the programs of normal hematopoiesis are modified in acute myeloid leukemia, an aggressive adult malignancy driven by leukemic stem cells. We summarize these findings and their clinical implications.

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

Diamond-Blackfan anemia (DBA) is a hereditary bone marrow failure disorder that is characterized by erythropoiesis failure and chronic anemia and is frequently associated with physical malformations. Considered a ribosomopathy, most patients harbor pathogenic mutations in one of at least 22 large or small ribosomal protein genes in an autosomal dominant manner, however rarer cases involving mutations in GATA1, TSR2 and EPO have also been described. A recent study of the architecture of the human hematopoietic hierarchy throughout development (Notta et al, Science 2016) showed that, after birth, unipotent progenitors can derive directly from multipotent progenitors without intermediate differentiation into oligopotent progenitors. Critically, this work identified novel progenitors for which the abundance, clonal capacity and progenitor function is currently unknown in many benign and malignant hematological disorders. Specifically, multipotent, common myeloid and megakaryocyte-erythroid progenitors (MPP, CMP and MEP) were found to be functionally heterogeneous and could be subdivided (F1, F2 and F3 subtypes) based on the combination of CD71 and BAH1 expression. We hypothesized that in-depth analysis of these novel and previously reported progenitors will better elucidate hematopoiesis in DBA, providing insights in to DBA pathogenesis and erythropoietic failure. Using flow cytometry, we quantified the frequencies of 11 hematopoietic stem and progenitor cell (HSPC) populations: HSC, MPP F1-F3, CMP F1-F3, MEP F1-F3 and granulocyte-monocyte progenitors (GMP), from the BM specimens of 8 DBA patients harboring ribosomal protein mutations and 6 healthy age-matched control donors. To characterize the function of HSPCs in DBA we then utilized a highly sensitive and quantitative single-cell in-vitro stromal feeder-based differentiation assay that supports the expansion and lineage commitment of single HSCPs towards mature cell types (myeloid, erythroid and megakaryocytes) with progeny immunophenotyping and quantification by flow cytometry. Our refined quantification of HSPCs by flow cytometry showed that the defect of erythropoiesis in DBA patients starts at the CMP/MEP compartment. Namely, CMP-F2 and CMP-F3 as well as MEP-F2 and MEP-F3 sub-populations that are predicted to form erythroid cells were significantly reduced in DBA patients compared to healthy aged-matched individuals (Figure 1A-B). In contrast, CMP/MEP-F1 sub-populations that are predicted to form only myeloid cells (BAH1-/CD71-) showed no significant difference compared to healthy controls. Importantly, the multipotent compartment (HSC and MPP) did not significantly differ from that of healthy controls. Functional analysis of F2/F3 CMP/MEP HSCP sub-populations in single-cell colony assays showed that they have a high propensity to produce erythroid progeny in healthy control samples (Figure 1C). In contrast, in DBA patients these HSCPs had limited erythroid forming potential, but maintain a high propensity to develop myeloid colonies (Figure 1D). To our knowledge this is the first study that the HSCP sub-populations F1/F2/F3 HSCP fractions have been analyzed in DBA. Collectively, these data indicate that the hematopoietic defect in DBA occurs in erythroid-producing CMP/MEP populations. Our data show that these sub-populations are both reduced and dysfunctional in DBA patients and we suggest these abnormalities lead to the pure red cell aplasia seen in these patients. Figure 1: DBA patients have reduced CMP and MEP sub-populations that behave dysfunctional in-vitro. A-B) DBA patients have reduced CMP-F2/3 (A) and MEP-F2/3 (B) sub-populations in their bone marrow. C) CMP F2-F3 and MEP F2-F3 have a high propensity to produce erythroid cell types in healthy controls. D) Single-cells isolated from the CMP-F2/3 and MEP-F2 population of DBA patients produced proportionally fewer erythroid and greater myeloid colonies in in-vitro colony assays (plot shows the mean respective proportions from all healthy controls and DBA patients tested, each N=5). Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

The age-associated accumulation of somatic mutations and large-scale structural variants (SVs) in the early hematopoietic hierarchy have been linked to premalignant stages for cancer and cardiovascular disease (CVD). However, only a small proportion of individuals harboring these mutations progress to disease, and mechanisms driving the transformation to malignancy remains unclear. Hematopoietic evolution, and cancer evolution more broadly, has largely been studied through a lens of adaptive evolution and the contribution of functionally neutral or mildly damaging mutations to early disease-associated clonal expansions has not been well characterised despite comprising the majority of the mutational burden in healthy or tumoural tissues. Through combining deep learning with population genetics, we interrogate the hematopoietic system to capture signatures of selection acting in healthy and pre-cancerous blood populations. Here, we leverage high-coverage sequencing data from healthy and pre-cancerous individuals from the European Prospective Investigation into Cancer and Nutrition Study (n=477) and dense genotyping from the Canadian Partnership for Tomorrow's Health (n=5,000) to show that blood rejects the paradigm of strictly adaptive or neutral evolution and is subject to pervasive negative selection. We observe clear age associations across hematopoietic populations and the dominant class of selection driving evolutionary dynamics acting at an individual level. We find that both the location and ratio of passenger to driver mutations are critical in determining if positive selection acting on driver mutations is able to overwhelm regulated hematopoiesis and allow clones harbouring disease-predisposing mutations to rise to dominance. Certain genes are enriched for passenger mutations in healthy individuals fitting purifying models of evolution, suggesting that the presence of passenger mutations in a subset of genes might confer a protective role against disease-predisposing clonal expansions. Finally, we find that the density of gene disruption events with known pathogenic associations in somatic SVs impacts the frequency at which the SV segregates in the population with variants displaying higher gene disruption density segregating at lower frequencies. Understanding how blood evolves towards malignancy will allow us to capture cancer in its earliest stages and identify events initiating departures from healthy blood evolution. Further, as the majority of mutations are passengers, studying their contribution to tumorigenesis, will unveil novel therapeutic targets thus enabling us to better understand patterns of clonal evolution in order to diagnose and treat disease in its infancy. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

The first insight into the complexity of post-transplant in vivo dynamics of hematopoietic stem cells (HSC) in humans was only recently showcased by examining longitudinal clonal contributions in gene therapy patients. Initial blood reconstitution was achieved by short-term (ST-)HSC, but in the longer-term hematopoiesis originated from long-term (LT-)HSC that only became recruited after a latency phase of 1-2 years (Scala et al 2018). Thus, deciphering the mechanisms governing how LT-HSC might resist transplant mediated activation and/or respond to the varying hematopoietic demands that occur during homeostasis would be an important goal for improving HSC cell therapies. We previously linked INKA1(=C3orf54/FAM212A) mediated PAK4 inhibition and reduced H4K16 acetylation (H4K16ac) to quiescence in human leukemia stem cells (Kaufmann, Blood 2019). Here, we interrogate the role of this signaling axis in normal human hematopoiesis. Immunostaining revealed distinct subsets defined by the dichotomy of INKA1 and H4K16ac within cord blood ST- and LT-HSC, mechanistically supported by BioID, chromatin fiber analysis and PLA data showing INKA1 interacting with the H4K16 deacetylase, SIRT1. Among quiescent LT-HSC, we found that the INKA1high fraction (~10 % of LT-HSC) had the lowest CDK6 levels and represented LT-HSC in an alternative state of quiescence. We then used protein interaction (BioID) data to show that a shared interactor of INKA1 and PAK4, CD112 could be used to sort for the subset of CD112low LT-HSC that was in this alternatively quiescent LT-HSC state (H4K16aclow, CDK6low, CellROXlow). Compared to primary cells, culture attenuates the strict dichotomy of INKA1 and H4K16ac but only in cells in which colocalization of PAK4 with both occurred, suggesting PAK4 interferes with SIRT1-INKA1 interaction thereby permitting H4K16 acetylation. In vitro time course analysis showed that H4K16ac and PAK4 levels preceded the upregulation of the G0-exit marker CDK6 implicating a role in cell cycle priming. Pseudo-time scRNAseq analysis for INKA1high versus PAK4high, CDK6high and CD112high expression in mobilized peripheral blood (modeling in vivo HSC activation) showed enrichment of early or late diffusion indicative of quiescent versus primed cell status, respectively. Strikingly, in xenograft assays CD112low LT-HSC exhibited delayed engraftment kinetics with higher secondary repopulation capacity than faster repopulating CD112high LT-HSC reflecting the subset of LT-HSC that resist early activation. Similarly, INKA1-OE or PAK4 knock-down in vivo resulted in an early restraint in engraftment levels (@4 w), whereas 20 w engraftment reached control levels. When measured in secondary transplant assays the HSC frequency was 4 to 8-fold higher in the groups that showed this early restraint. Thus, resisting early activation (latency) preserves the regenerative potential of such HSC. In vivo 5-Fluorouracil treatment at week 4 accelerated latency exit and further increased HSC frequency of INKA1-OE cells while abolishing serial transplantation potential of controls. Hence, the induction of proliferative stress via creating acute hematopoietic demand is able to lift the INKA1-OE imposed restraint resulting in increased hematopoietic output while also promoting HSC self-renewal. Collectively, our data decipher the molecular intricacies underlying HSC heterogeneity and self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to preserve a stem cell reservoir. Disclosures Takayanagi: Kirin Holdings Company, Ltd: Current Employment. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Human long-term hematopoietic stem cells (LT-HSC) residing at the top of the hematopoietic hierarchy must meet enormous daily demand (~10e11 cells daily) while also sustaining life-long maintenance of the stem cell pool through self-renewal. This hierarchical organization is widely thought to protect LT-HSC from exhaustion by maintaining them in a quiescent and undifferentiated state, activating only in response to microenvironment signals to generate highly proliferative but more short-lived populations including short-term HSC (ST-HSC) and committed progenitors. When called upon to exit this dormant state, HSC must respond and adapt their metabolism and nutrient uptake to meet increased bioenergetic demands for cell growth and differentiation. At the same time, the events underlying cellular and metabolic activation must also be suppressed to allow LT-HSC to re-enter quiescence and ultimately maintain the LT-HSC pool through self-renewal. Thus, proper sensing of cellular output demands must be coordinated with the cell cycle and metabolic machinery of LT-HSC to balance stem cell fates and maintain hematopoietic homeostasis. However, the regulatory circuits of this demand-adapted regulation of early hematopoiesis are largely unknown. The ability of cells to receive signals or take up nutrients depends on proteins that are embedded within the plasma membrane. These proteins move to the cell's interior through endocytosis and can be degraded in the lysosomes or rerouted back to the cell surface and reused. Moreover, lysosomes are the terminal catabolic stations of the autophagy pathway that is essential for preserving stem cell function through clearance of toxic cellular components. However, little is known about the regulation and role of lysosomes in the stem cell context. Here, we describe the unexpected finding that lysosomes, whose activity is intricately balanced by TFEB and MYC, are instrumental for regulating the stemness and differentiation properties of human LT-HSC. Furthermore, we found that TFEB, which is normally implicated in stress response, induces a constitutive lysosomal flux in unperturbed LT-HSC that actively maintains quiescence, preserves self-renewal and governs lineage commitment. These effects are accompanied by endolysosomal degradation of membrane receptors, such as the transferrin receptor 1 (TfR1), pointing to a role for TFEB in coordinating how LT-HSC sense environmental changes and initiate the earliest steps of their fate transitions and lineage commitment decisions. These transitions are regulated by a TFEB/MYC dichotomy where MYC is a driver of LT-HSC anabolism and activation and counteracts TFEB function by serving as a negative transcriptional regulator of lysosomes. Moreover, our findings further suggest that active suppression of TFEB and its downstream lysosomal degradation of TfR1 within LT-HSC is required for commitment along the erythroid lineage: activation of TFEB can abolish erythroid differentiation even after lineage commitment has occurred. In summary, we uncovered a MYC-TFEB-mediated dichotomous regulation of lysosomal activity that is required to balance anabolic and catabolic processes that ultimately impact human LT-HSC fate determination. Figure Disclosures Takayanagi: Kirin Holdings Company, Ltd: Current Employment. Dick:Bristol-Myers Squibb/Celgene: Research Funding.

Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). To identify miRNA(s) that play a functional role in human hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were over-expressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. Enforced expression of miR-130a conferred a competitive repopulation advantage and induced an expansion of HSPC in xenograft assays. Secondary transplantation at limiting dilution revealed a 10-fold increase in HSC frequency, consistent with a role of miR-130a in enhancing HSC self-renewal. Label-free semi-quantitative proteomics in human HSPC combined with GSEA identified chromatin remodeling as the most significant pathway repressed by miR-130a. TBL1XR1, a component of the nuclear receptor corepressor (NCoR) complex that mediates the exchange of corepressors for coactivators, and CBFb, the β subunit of RUNX1, were among the most repressed miR-130a targets. Interestingly, interrogation of AML datasets revealed elevated expression of miR-130a in t(8,21) AML characterized by the fusion of AML1(RUNX1) and ETO genes. High miR-130a expression was correlated with poor patient overall survival. AML1-ETO recruits the aforementioned NCoR complex to repress RUNX1 target genes, thus preventing myeloid differentiation. To test whether miR-130a controls the AML1-ETO molecular program via repression of TBL1XR1, we tested the effect of miR-130a knock-down in Kasumi cell line and patient samples with the t(8,21). Notably, miR-130a KD resulted in differentiation of transduced cells and increased protein levels of TBL1XR1. Immunoprecipitation of AML1-ETO following miR-130a KD revealed loss of NCoR and CBFβ. Thus, we have uncovered a novel mechanism whereby elevated expression of miR-130a in t(8,21) AML endows enhanced self-renewal and perpetuates the aberrant molecular programs driven by AML1-ETO through inhibition of TBL1XR1.

Inherited genetic variants substantially contribute to the risk of myeloproliferative neoplasms (MPNs), a type of blood cancer that arise in the hematopoietic stem cell (HSC) compartment. To identify the germline genetic factors and underlying mechanisms predisposing to MPNs, we conducted a genome-wide association study involving 3,797 MPN cases and 1,152,977 controls and identified 17 genome-wide significant loci. We find an enrichment for risk variants mapping to accessible chromatin in HSCs, an association between MPN risk and multi-lineage blood cell traits, an association with longer leukocyte telomere length, and a significant genetic correlation with predisposition to clonal hematopoiesis of indeterminate potential. Collectively, all of these findings implicate HSC function and self-renewal. In addition to these global studies, we performed in depth mechanistic studies at one locus located ∼12kb downstream of the transcription factor GFI1B. Fine-mapping analyses and reporter assays identified rs524137 as the likely causal variant with the risk allele decreasing hematopoietic reporter activity. Endogenous deletion of the 1.6 kb enhancer region harboring this variant with Cas9 ribonucleoproteins in adult human hematopoietic stem and progenitor cells (HSPCs) resulted in ∼65% editing with a 36% reduction in GFI1B expression. Furthermore, deletion of GFI1B enhancer improved the maintenance of phenotypic HSCs during a 7-day HSPC culture and increased progenitor self-renewal capacity in colony replating assays. Interestingly, deletion of this enhancer only affected HSPC function without disruption of erythropoiesis, an independent effect observed with GFI1B coding perturbation. These results and our ongoing studies demonstrate that the GFI1B MPN risk locus expands HSPCs and reinforces this general mechanism as a risk factor for MPN acquisition.

The mechanisms governing how human hematopoietic stem cells (HSC) balance their capacity for maintaining long-term (LT) regeneration with meeting daily and/or stress demands are unknown. We previously linked INKA1-mediated PAK4 inhibition along with reduction of H4K16 acetylation (H4K16ac) to quiescence in leukemia stem cells. Here, we uncover mutually exclusive immunostaining for INKA1 and H4K16ac in cord blood HSC; supported by BioID and PLA data showing INKA1 interacts with the H4K16 deacetylase SIRT1. The proteomic data also pointed us to CD112, that enabled isolation of LT-HSC into transcriptionally distinct subsets that respond differently to regenerative stress: a CD112low subset (H4K16aclow, CDK6low, ROSlow, INKA1high) exhibited a transient restraint before contributing to hematopoietic reconstitution and a primed CD112high subset (H4K16achigh, CDK6high, ROShigh, INKA1low) that responded rapidly. Protein and scRNAseq pseudotime analysis of in vitro and in vivo (G-CSF-) activated HSC, respectively, confirmed upregulation of H4K16ac, PAK4, CDK6 and CD112 coinciding with cell-cycle priming. INKA1-overexpression (OE) or PAK4 knock-down (KD) induced transient restraint in reconstitution (@4w) with 20w engraftment reaching control levels; HSC frequency increased 4-8 fold as measures in secondary transplants. Thus, INKA1-OE or PAK4-KD enables HSC to resist early activation, thereby protracting their regenerative potential (a phenomenon we term latency). By elevating hematopoietic demand with 5-FU, the INKA1-OE imposed restraint was released engendering even further increased HSC frequency; in controls regenerative capacity was abolished. Collectively, our data uncover a new molecular axis for HSC self-renewal regulation and point to latency as an orchestrated physiological response that integrates quiescence control with HSC fate choices to maintain LT-HSC pool size.

Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

Genetic analyses have shaped much of our understanding of cancer. However, it is becoming increasingly clear that cancer cells display features of normal tissue organization, where cancer stem cells (CSCs) can drive tumor growth. Although often considered as mutually exclusive models to describe tumor heterogeneity, we propose that the genetic and CSC models of cancer can be harmonized by considering the role of genetic diversity and nongenetic influences in contributing to tumor heterogeneity. We offer an approach to integrating CSCs and cancer genetic data that will guide the field in interpreting past observations and designing future studies.