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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.

Myeloproliferative neoplasms (MPNs) are blood cancers characterized by excessive production of mature myeloid cells that result from the acquisition of somatic driver mutations in hematopoietic stem cells (HSCs)1. While substantial progress has been made to define the causal somatic mutation profile for MPNs2, epidemiologic studies indicate a significant heritable component for the disease that is among the highest known for all cancers3. However, only a limited set of genetic risk loci have been identified, and the underlying biological mechanisms leading to MPN acquisition remain unexplained. Here, to define the inherited risk profile, we conducted the largest genome-wide association study of MPNs to date (978,913 individuals with 3,224 cases) and identified 14 genome-wide significant loci, as well as a polygenic signature that increases the odds for disease acquisition by nearly 3-fold between the top and median deciles. Interestingly, we find a shared genetic architecture between MPN risk and several hematopoietic traits spanning distinct lineages, as well as an association between increased MPN risk and longer leukocyte telomere length, collectively implicating HSC function and self-renewal. Strikingly, we find a significant enrichment for risk variants mapping to accessible chromatin in HSCs compared with other hematopoietic populations. Finally, gene mapping identifies modulators of HSC biology and targeted variant-to-function analyses suggest likely roles for CHEK2 and GFI1B in altering HSC function to confer disease risk. Overall, we demonstrate the power of human genetic studies to illuminate a previously unappreciated mechanism for MPN risk through modulation of HSC function.

Myeloproliferative neoplasms (MPNs) are blood cancers that are characterized by the excessive production of mature myeloid cells and arise from the acquisition of somatic driver mutations in haematopoietic stem cells (HSCs). Epidemiological studies indicate a substantial heritable component of MPNs that is among the highest known for cancers1. However, only a limited number of genetic risk loci have been identified, and the underlying biological mechanisms that lead to the acquisition of MPNs remain unclear. Here, by conducting a large-scale genome-wide association study (3,797 cases and 1,152,977 controls), we identify 17 MPN risk loci (P

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. Significance: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse. See related video: https://vimeo.com/442838617 See related article by E. Waanders et al .

Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world., Single-cell proteomics can provide insights into the molecular basis for cellular heterogeneity. Here, the authors develop a multiplexed single-cell proteomics and computational workflow, and show that their strategy captures the cellular hierarchies in an Acute Myeloid Leukemia culture model.

Detection of cancer-associated somatic mutations has broad applications for oncology and precision medicine. However, this becomes challenging when cancer-derived DNA is in low abundance, such as in impure tissue specimens or in circulating cell-free DNA. Next-generation sequencing (NGS) is particularly prone to technical artefacts that can limit the accuracy for calling low-allele-frequency mutations. State-of-the-art methods to improve detection of low-frequency mutations often employ unique molecular identifiers (UMIs) for error suppression; however, these methods are highly inefficient as they depend on redundant sequencing to assemble consensus sequences. Here, we present a novel strategy to enhance the efficiency of UMI-based error suppression by retaining single reads (singletons) that can participate in consensus assembly. This ‘Singleton Correction’ methodology outperformed other UMI-based strategies in efficiency, leading to greater sensitivity with high specificity in a cell line dilution series. Significant benefits were seen with Singleton Correction at sequencing depths ≤16 000×. We validated the utility and generalizability of this approach in a cohort of >300 individuals whose peripheral blood DNA was subjected to hybrid capture sequencing at ∼5000× depth. Singleton Correction can be incorporated into existing UMI-based error suppression workflows to boost mutation detection accuracy, thus improving the cost-effectiveness and clinical impact of NGS.

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.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours. Funding sources for this study include grants to the Pancreatic Cancer Sequencing Initiative program from the Ontario Institute for Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation, the Canada Foundation for Innovation; research award to F.N. from the OICR and the Canadian Institutes for Health Research (CIHR); Canadian Friends of the Hebrew University, the SMGS Family Foundation, NCI grant P50 CA102701 (Mayo Clinic SPORE in Pancreatic Cancer) and NCI grant R01 CA97075 (Pancreatic Cancer Genetic Epidemiology Consortium). F.N. is supported by a fellowship award from CIHR and is a recipient of a scholar’s research award from the Ontario Institute of Cancer Research (OICR), through support from the Ontario Ministry of Research and Innovation. G.Z. is a Clinician–Scientist of the Fonds de la Recherche en Sante du Quebec. P.J.C. is a Wellcome Trust Senior Clinical Fellow. T.J.H., L.D.S., J.D.M. and S.G. are recipients of Senior or Clinician–Scientist Awards from the Ontario Institute for Cancer Research

A deep proteomics analysis was conducted on a primary acute myeloid leukemia culture system to identify potential protein targets regulated by miR-126. Leukemia cells were transduced either with an empty control lentivirus or one containing the sequence for miR-126, and resulting cells were analyzed using ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD001994. The proteomics data and statistical analysis described in this article is associated with a research article, “miR-126 regulates distinct self-renewal outcomes in normal and malignant hematopoietic stem cells” (Lechman et al., 2016) [1], and serves as a resource for researchers working in the field of microRNAs and their regulation of protein levels.

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.

Individual cancer cells exhibit functional heterogeneity of many cancer hallmarks including the capacity for sustaining long-term clonal maintenance, a stemness property involving self-renewal. Our studies established that only rare AML cells possessed such leukemia stem cell (LSC) properties and that AML is a cellular hierarchy. LSC were found to be highly relevant to human disease as gene signatures specific to LSC were much more predictive of patient response to therapy and overall survival compared to the bulk non-LSC AML cells. We have now provided insights into how LSC develop during leukemogenesis. Through sequencing of purified populations of normal blood cells, AML cells, and xenografts from paired diagnosis/relapse AML samples, we tracked the full arc of leukemia development in humans: from the cell of origin (an HSC) that acquires the first genetic mutation; to pre-leukemic clonal expansion of HSC; the generation of genetically diverse LSC; and finally to the cellular origin of relapse (rare LSC subclones) within the diagnosis sample. Similar studies have been undertaken in B-ALL where relapse-fated subclones were found within the diagnosis sample before exposure to chemotherapy. These clones possess distinct epigenetic and metabolic properties that underlie their partially resistance to chemotherapy and they possess stemness signatures that drive their ability to regenerate relapse disease. These studies resulted in identification of novel therapeutic targets of relapse-fated subclones to begin envisioning approaches to eradicate relapse before it develops. Finally, the identification of clonally expanded pre-leukemic HSPC in the diagnosis blood sample of many AML samples predicted that it should be possible to identify individuals who are at risk for progressing to AML long before AML develops. We genotyped individuals from the general population who were enrolled in the large cohort (EPIC) who eventually developed AML and compared them to the enrollment sample of others who never progressed to AML. We have found a clear signature that is able to predict with high accuracy those individuals who have age related clonal hematopoiesis who progress to AML, almost 10 years prior to AML development. This is distinct from individuals who have benign clonal hematopoiesis who never progress. These new findings offer the potential for future intervention to permit AML prevention trials. Citation Format: John E. Dick. Stem cells play a role in human leukemia from origin to relapse [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA21.

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.

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

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the

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 incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure(1). The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion(2,3). However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)(4-8). Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

In the originally published version of this Letter, ref. 43 was erroneously provided twice. In the 'Estimation of relative cell-type-specific composition of AML samples' section in the Methods, the citation to ref. 43 after the GEO dataset GSE24759 is correct. However, in the 'Mice' section of the Methods, the citation to ref. 43 after 'TAMERE' should have been associated with a new reference1. The original Letter has been corrected online (with the new reference included as ref. 49).

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.

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase-mediated (DNA-PK-mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK-deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK-deficient quiescent leukemia cells and BRCA/DNA-PK-deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients.

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.

Item does not contain fulltext In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

Hematopoietic cells are arranged in a hierarchy where mature blood cells arise from stem and progenitor precursors. AML is also hierarchical with differentiated blasts arising from leukemic stem/progenitor cells. Recent studies show that metabolites can affect epigenetic marks; however, it is unknown whether metabolic enzymes can directly localize to the nucleus to regulate stemness in AML and normal hematopoietic cells. Here, we discovered that the mitochondrial enzyme, Hexokinase 2, localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stemness. Metabolic enzymes that localize to nucleus of stem cells were identified by evaluating stem and bulk fractions of OCI-AML-8227 leukemia cells, which are arranged in a hierarchy with functionally defined stem cells. We separated OCI-AML-8227 cells into CD34+38- and CD34-38+ populations by FACS and prepared nuclear and cytoplasmic lysates. Immunoblotting of the lysates revealed that the metabolic enzyme Hexokinase 2 (HK2) was increased in the nuclear fraction of 8227 stem cells compared to bulk cells. In contrast, other mitochondrial enzymes such as Enolase1, Aconitase2, and Succinate Dehydrogenase A & B, were not detected in the nuclear lysates. HK2 is an outer mitochondrial membrane protein that phosphorylates glucose to glucose-6-phosphate, initiating glycolysis. We confirmed nuclear HK2 in OCI-AML-8227 stem cells by confocal microscopy and also demonstrated nuclear HK2 in AML cell lines (OCI-AML2, NB4, K563, and MV411) and in 7 of 9 primary AML samples. We FACS sorted normal cord blood into populations of stem/progenitor (HSC, MPP, MLP, CMP, GMP and MEP) and differentiated (Monocytes, Granulocytes, B, T, and NK) cells. The localization of HK2 in these cells was analysed and quantified by immunofluorescence. Nuclear HK2 was detected in the stem/progenitor cells and progressively declined to minimal levels as cells matured. Next, we explored mechanisms that regulate nuclear localization of HK2. AKT-mediated phosphorylation of HK2 promoted localization to mitochondria while inhibition of phosphorylation increased its nuclear levels. Moreover, the nuclear import of HK2 was dependent on IPO5, a member of b-importin family that imports protein to the nucleus; CRM1 was responsible for HK2 nuclear export. We tested whether the nuclear localization of HK2 was functionally important to maintain stemness. We overexpressed HK2 tagged with nuclear localizing signals (PKKKRKV or PAAKRVKLD) in 8227 and NB4 leukemia cells. Selective overexpression of HK2 in the nucleus did not alter the rate of proliferation of the cells, however there was enhanced clonogenic growth and inhibition of retinoic acid-mediated cell differentiation. Conversely, we selectively reduced nuclear HK2 by expressing HK2 with an outer mitochondrial localization signal while knocking down endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 in AML cells did not alter growth rate, but did reduce clonogenic growth and increased differentiation after treatment with retinoic acid. 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 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. A top hit in our screen was Exonuclease 3'-5' domain containing 2 (EXD2), involved in DNA repair. Of note, DNA damage induces differentiation of AML cells. In 8227 cells, nuclear EXD2 was higher in the stem cell fraction compared to the bulk fraction. Moreover, knockdown of EXD2 reduced AML growth, clonogenic growth and decreased nuclear HK2 levels. Finally, nuclear HK2 overexpression conferred resistance to the PARP inhibitor, olaparib. In summary, we discovered that unphosphorylated HK2 localizes to the nucleus in malignant and normal hematopoietic stem cells. Through mechanisms independent of its kinase function, nuclear HK2 maintains AML cells in their stem/progenitor state potentially by regulating DNA damage and repair. Thus, we define a new role for a mitochondrial enzyme in the regulation of stemness and differentiation. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Schimmer:Medivir Pharmaceuticals: Research Funding; Otsuka Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy.

Leukemia is the most common cancer in children. Sequencing data from identical twins suggests that the first genetic alterations in childhood leukemia occur in utero. Children with Down syndrome (Trisomy 21, T21) have an increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient pre-leukemia disease occurs, which is characterized by a clonal proliferation of immature megakaryocytes carrying somatic mutations in the GATA1 transcription factor. These acquired GATA1 mutations lead to the expression of an N-terminal truncated protein (GATA1-Short). In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemia by acquisition of additional genetic mutations in the transient leukemia clone, predominantly in genes of the cohesin complex. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1-Short and additional cohesin mutations. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the mechanisms of leukemic transformation and the interplay between each genetic component. Therefore, we wanted to establish a tractable human model system to investigate the initiation and evolution of transient leukemia and AMKL using CRISPR/Cas9 genome editing in primary human hematopoietic stem cells (HSCs). To model the initiation of Down syndrome associated pre-leukemia, we utilized both neonatal cord blood and fetal liver derived LT-HSCs and other progenitor populations to express either the short or long isoform of GATA1 (GATA1-Short or GATA1-Long). This was carried out using an improved methodology that permits the in vitro and in vivo functional interrogation of CRISPR/Cas9 edited human LT-HSCs at the single cell level (Wagenblast et al., bioRxiv 609040). Importantly, in this case, expression of either GATA1 isoform remained under the regulatory control of the endogenous promoter. Culture of single LT-HSC, short-term (ST-HSC) and myelo-erythroid progenitors (MEP) revealed a drastic shift towards megakaryocytic lineage output upon exclusive expression of GATA1-Short compared to control or GATA1-Long, regardless of the developmental source of the derived cells. To investigate the functional consequences of exclusive GATA1-Short expression in LT-HSCs in vivo, we performed near-clonal xenotransplantation assays in NSG and NSGW41 mice. Strikingly, GATA1-Short edited LT-HSCs injected mice displayed a higher percentage of human CD41+CD45- megakaryocytic lineage derived cells and a decrease in human GlyA+CD45- erythroid cells compared to control. Morphological analysis revealed more immature forms of erythroid cells and fewer enucleated erythrocytes in GATA1-Short edited LT-HSCs injected mice. In order to add an additional genetic determinant to our model, we utilized T21 fetal liver derived LT-HSCs. Un-manipulated T21 LT-HSCs and other progenitor populations showed a bias towards erythroid, myeloid and megakaryocytic lineages at the expense of lymphoid fates. In vitro, the combination of T21 and CRISPR/Cas9-mediated GATA1-Short in LT-HSCs led to an increase in megakaryocytic lineage output, while decreasing erythroid output. This phenotype was similar to what was observed in normal karyotype fetal liver derived LT-HSCs. However, near clonal transplantation of GATA1-Short edited T21 LT-HSCs in NSG mice generated exclusive CD33+ myeloid grafts with disproportionate high levels of CD41+CD45- megakaryocytic lineage derived cells compared to T21 control. In addition a distinct CD34+CD41+CD71+CD45+ population was present. Thus, this phenotype is reminiscent of Down Syndrome associated transient leukemia. In summary, by using an improved CRISPR/Cas9 single cell methodology we show how GATA1 regulates lineage fate in normal and T21 LT-HSCs and other progenitor populations. Importantly, we show for the first time a humanized mouse model of Down syndrome associated transient leukemia, which was induced from T21 human fetal liver derived LT-HSCs engineered to express GATA1-Short. Current studies focus on adding additional mutations of the cohesin complex to progress transient leukemia to AMKL. Disclosures No relevant conflicts of interest to declare.

Summary Cellular stress responses serve as crucial decision points balancing persistence or culling of hematopoietic stem cells (HSCs) for lifelong blood production. Although strong stressors cull HSCs, the linkage between stress programs and self-renewal properties that underlie human HSC maintenance remains unknown, particularly at quiescence exit when HSCs must also dynamically shift metabolic state. Here, we demonstrate distinct wiring of the sphingolipidome across the human hematopoietic hierarchy and find that genetic or pharmacologic modulation of the sphingolipid enzyme DEGS1 regulates lineage differentiation. Inhibition of DEGS1 in hematopoietic stem and progenitor cells during the transition from quiescence to cellular activation with N-(4-hydroxyphenyl) retinamide activates coordinated stress pathways that coalesce on endoplasmic reticulum stress and autophagy programs to maintain immunophenotypic and functional HSCs. Thus, our work identifies a linkage between sphingolipid metabolism, proteostatic quality control systems, and HSC self-renewal and provides therapeutic targets for improving HSC-based cellular therapeutics., Graphical Abstract, Highlights • Sphingolipid composition is diverse across the human hematopoietic hierarchy • DEGS1 is a sphingolipid enzyme required for hematopoietic stem cell (HSC) function • Modulating DEGS1 function activates autophagy and the unfolded protein response • Variations in sphingolipid homeostasis serve to regulate HSC fate, Lipid metabolism is distinctly regulated in human hematopoietic stem cells (HSCs) versus progenitors. Xie et al. profiled the sphingolipidome in human cord blood and show that modulating sphingolipids during the transition from quiescence to cellular activation in ex vivo culture induced proteostatic cellular stress programs to maintain HSC self-renewal.

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to the insufficiently characterized subpopulations of Leukemia Stem Cells (LSCs) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter which enables stemness assessment to enrich and functionally characterize LSCs. We revealed heterogeneous activity of the ERG+85 enhancer based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemo-resistance genes, demonstrated increased clonogenicity and resistance to chemo- and radio- therapy as compared to their tagBFPNeg counterparts. Moreover, tagBFPHigh enriched fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasion ability. Most importantly, tagBFPHigh fraction was enriched for leukemia initiating cells in a xenograft assay. We also identified USP9X deubiquitinase enzyme as a novel ERG transcriptional target that sustains ERG+85 positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to the preferential inhibition of the ERG-dependent leukemias. In summary, we have developed a new strategy to characterize LSCs and propose ERG targeting via USP9X inhibition as a potential anti-leukemia treatment.

Summary Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function., Graphical Abstract Highlights ► miR-126 is a novel regulator of the HSC quiescence/proliferation equilibrium ► Reduction in miR-126 induces an expansion of long-term HSC without exhaustion ► Constitutive miR-126 expression promotes HSC quiescence and progenitor proliferation ► miR-126 attenuates PI3K/AKT activation in response to cytokine stimulation, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway to promote HSC quiescence and progenitor proliferation.

// Adi Zipin-Roitman 1 , Nasma Aqaqe 1 , Muhammad Yassin 1 , Shahar Biechonski 1 , Mariam Amer 1 , Mark F. van Delft 2, 3 , Olga I. Gan 2, 8 , Sean P. McDermott 2, 4 , Alla Buzina 6, 8 , Troy Ketela 6, 8 , Liran Shlush 2, 5 , Stephanie Xie 2, 8 , Veronique Voisin 6, 8 , Jason Moffat 6, 8 , Mark D. Minden 7, 9 , John E. Dick 2, 8 , Michael Milyavsky 1 1 Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada 3 Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia 4 Leidos Biomedical Research, Washington D.C., USA 5 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel 6 Donnelly Centre for Cellular and Biomedical Research, University of Toronto, Toronto, Canada 7 Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Canada 8 Department of Molecular Genetics, University of Toronto, Toronto, Canada 9 Department of Medicine, University of Toronto, Toronto, Canada Correspondence to: Michael Milyavsky, email: mmilyavsky@post.tau.ac.il Keywords: SMYD2 lysine methyltransferase, AML, quiescence, chemotherapy, DNA damage Received: November 23, 2016 Accepted: January 24, 2017 Published: February 06, 2017 ABSTRACT The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2. Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the selfrenewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell–derived inflammatory signals. Donor T cell–derived tumor necrosis factor–α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell–derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell–derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL invivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Adjusting hematopoietic hierarchy In adults, more than 300 billion blood cells are replenished daily. This output arises from a cellular hierarchy where stem cells differentiate into a series of multilineage progenitors, culminating in unilineage progenitors that generate over 10 different mature blood cell types. Notta et al. mapped the lineage potential of nearly 3000 single cells from 33 different cell populations of stem and progenitor cells from fetal liver, cord blood, and adult bone marrow (see the Perspective by Cabezas-Wallscheid and Trumpp). Prenatally, stem cell and progenitor populations were multilineage with few unilineage progenitors. In adults, multilineage cell potential was only seen in stem cell populations. Science , this issue p. 10.1126/science.aab2116 ; see also p. 126

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.