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Despite the inclusion of inherited myeloid malignancies as a separate entity in the World Health Organization Classification, many established predisposing loci continue to lack functional characterization. While germline mutations in the DNA repair factor ERCC excision repair 6 like 2 (ERCC6L2) give rise to bone marrow failure and acute myeloid leukaemia, their consequences on normal haematopoiesis remain unclear. To functionally characterise the dual impact of germline ERCC6L2 loss on human primary haematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs), we challenged ERCC6L2-silenced and patient-derived cells ex vivo. Here, we show for the first time that ERCC6L2-deficiency in HSPCs significantly impedes their clonogenic potential and leads to delayed erythroid differentiation. This observation was confirmed by CIBERSORTx RNA-sequencing deconvolution performed on ERCC6L2-silenced erythroid-committed cells, which demonstrated higher proportions of polychromatic erythroblasts and reduced orthochromatic erythroblasts versus controls. In parallel, we demonstrate that the consequences of ERCC6L2-deficiency are not limited to HSPCs, as we observe a striking phenotype in patient-derived and ERCC6L2-silenced MSCs, which exhibit enhanced osteogenesis and suppressed adipogenesis. Altogether, our study introduces a valuable surrogate model to study the impact of inherited myeloid mutations and highlights the importance of accounting for the influence of germline mutations in HSPCs and their microenvironment.

The DExD/H-box RNA helicase DHX34 is a Nonsense-mediated decay (NMD) factor that together with core NMD factors co-regulates NMD targets in nematodes and in vertebrates. Here, we show that DHX34 is also associated with the human spliceosomal catalytic C complex. Mapping of DHX34 endogenous binding sites using Cross-Linking Immunoprecipitation (CLIP) revealed that DHX34 is preferentially associated with pre-mRNAs and locates at exon-intron boundaries. Accordingly, we observed that DHX34 regulates a large number of alternative splicing (AS) events in mammalian cells in culture, establishing a dual role for DHX34 in both NMD and pre-mRNA splicing. We previously showed that germline DHX34 mutations associated to familial Myelodysplasia (MDS)/Acute Myeloid Leukemia (AML) predisposition abrogate its activity in NMD. Interestingly, we observe now that DHX34 regulates the splicing of pre-mRNAs that have been linked to AML/MDS predisposition. This is consistent with silencing experiments in hematopoietic stem/progenitor cells (HSPCs) showing that loss of DHX34 results in differentiation blockade of both erythroid and myeloid lineages, which is a hallmark of AML development. Altogether, these data unveil new cellular functions of DHX34 and suggests that alterations in the levels and/or activity of DHX34 could contribute to human disease.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase ( COASY ), induced by mutations in SF3B1 , affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1 mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

HNRNPA2B1 is associated with prostate cancer (PC) disease aggressiveness and underlies pro-tumourigenic cellular stress responses. By analysing >500 PC transcriptomes, we reveal that HNRNPA2B1 over-expression is associated with poor patient prognosis and stress response pathways. These include the “protein processing in the endoplasmic reticulum” (ER) pathway, which incorporates the unfolded protein response (UPR). By RNA-sequencing of HNRNPA2B1-depleted cells PC cells, we identified HNRNPA2B1-mediated down-regulation of UPR genes including the master ER-stress sensor IRE1, which induces ER proteostasis. Consistent with IRE1 down-regulation in HNRNPA2B1-depleted cells, we observed reduced splicing of the IRE1-target and key UPR effector XBP1s. Furthermore, HNRNPA2B1 depletion up-regulates expression of the IRE1-dependent decay (RIDD) target gene BLOC1S1, which is degraded by activated IRE1. We identify a HNRNPA2B1-IRE1-XBP1-controlled four gene prognostic biomarker signature (HIX) which classifies a subgroup of primary PC patients at high risk of disease relapse. Pharmacological targeting of IRE1 attenuated HNRNAPA2-driven PC cell growth. Taken together, our data reveal a putative novel mechanism of UPR activation in PC by HNRNPA2B1, which may promote an IRE1-dependent yet potentially-targetable recurrent disease phenotype.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a novel connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.

Myelodysplastic syndromes (MDS) are clonal stem cell diseases characterized mainly by ineffective hematopoiesis. Here, we present an approach that enables robust long-term engraftment of primary MDS stem cells (MDS-SC) in mice by implantation of human mesenchymal cell–seeded scaffolds. Critically for modeling MDS, where patient sample material is limiting, mononuclear bone marrow cells containing as few as 104 CD34+ cells can be engrafted and expanded by this approach with the maintenance of the genetic make-up seen in the patients. Noninvasive high-resolution ultrasound imaging shows that these scaffolds are fully perfused. Our data show that the human microenvironment but not mouse is essential to MDS-SC homing and engraftment. Notably, the alternative niche provided by healthy donor mesenchymal stromal cells enhances engraftment of MDS-SCs. This study characterizes a new tool to model MDS human disease with the level of engraftment previously unattainable in mice and offers insights into human-specific determinants of the MDS-SC microenvironment. Significance: These findings are significant for understanding the niche dependence of MDS. This report provides the evidence of the migratory behavior of hematopoietic stem cells in myeloid cancers. Our model offers a unique opportunity to study the clonal behavior of the myeloid/lymphoid cancers and delineate how cancer cells interact with different niches.

International audience; The unfolded protein response (UPR) is an evolutionarily conserved adaptive signaling pathway triggered by a stress of the endoplasmic reticulum (ER) lumen compartment, which is initiated by the accumulation of unfolded proteins. This response, mediated by three sensors-Inositol Requiring Enzyme 1 (IRE1), Activating Transcription Factor 6 (ATF6), and Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK)-allows restoring protein homeostasis and maintaining cell survival. UPR represents a major cytoprotective signaling network for cancer cells, which frequently experience disturbed proteostasis owing to their rapid proliferation in an usually unfavorable microenvironment. Increased basal UPR also participates in the resistance of tumor cells against chemotherapy. UPR activation also occurs during hematopoiesis, and growing evidence supports the critical cytoprotective role played by ER stress in the emergence and proliferation of leukemic cells. In case of severe or prolonged stress, pro-survival UPR may however evolve into a cell death program called terminal UPR. Interestingly, a large number of studies have revealed that the induction of proapoptotic UPR can also strongly contribute to the sensitization of leukemic cells to chemotherapy. Here, we review the current knowledge on the consequences of the deregulation of UPR signaling in leukemias and their implications for the treatment of these diseases.

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis with increased incidence in elderly individuals. Genetic alterations do not fully explain the molecular pathogenesis of the disease, indicating that other types of lesions may play a role in its development. In this work, we analyzed the transcriptional lesions of human HSCs, demonstrating how aging and MDS are characterized by a complex transcriptional rewiring that manifests as diverse linear and non-linear transcriptional dynamisms. While aging-associated lesions seemed to predispose elderly HSCs to myeloid transformation, disease-specific alterations may be involved in triggering MDS development. Among MDS-specific lesions, we detected the overexpression of the transcription factor DDIT3. Exogenous upregulation of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis, with an accumulation of cells in early stages of erythroid differentiation, as determined by single-cell RNA-sequencing. Increased DDIT3 expression was associated with downregulation of transcription factors required for normal erythropoiesis, such as KLF1, TAL1 or SOX6, and with a failure in the activation of their erythroid transcriptional programs. Finally, DDIT3 knockdown in CD34+ cells from MDS patients was able to restore erythropoiesis, as demonstrated by immunophenotypic and transcriptional profiling. These results demonstrate that DDIT3 may be a driver of MDS transformation, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing these patients. KEY POINTSO_LIHuman HSCs undergo a complex transcriptional rewiring in aging and MDS that may contribute to myeloid transformation. C_LIO_LIDDIT3 overexpression induces a failure in the activation of erythroid transcriptional programs, leading to inefficient erythropoiesis. C_LI

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

Abarrategi et al. summarize the methods used for human hematopoietic cell xenotransplantation and their milestones. They also discuss the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis., Xenotransplantation of patient-derived samples in mouse models has been instrumental in depicting the role of hematopoietic stem and progenitor cells in the establishment as well as progression of hematological malignancies. The foundations for this field of research have been based on the development of immunodeficient mouse models, which provide normal and malignant human hematopoietic cells with a supportive microenvironment. Immunosuppressed and genetically modified mice expressing human growth factors were key milestones in patient-derived xenograft (PDX) models, highlighting the importance of developing humanized microenvironments. The latest major improvement has been the use of human bone marrow (BM) niche–forming cells to generate human–mouse chimeric BM tissues in PDXs, which can shed light on the interactions between human stroma and hematopoietic cells. Here, we summarize the methods used for human hematopoietic cell xenotransplantation and their milestones and review the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis.

Myelodysplastic syndrome (MDS) is a hematological clonal stem cell disease. Recurrent splicing factors mutations are reported in 50% of MDS. Interestingly, mutations in the splicing factor gene SF3B1 are over-represented in MDS with ring sideroblasts (MDS-RS), co-occurring in up to 90% of patients. In MDS-RS, anemia is the major clinical manifestation. Erythropoiesis stimulating agents (ESAs) are used to treat anemia; however, the overall response rates are 20% to 40% with a duration of response of 18-24 months. New therapeutic options are needed to improve response to ESAs treatment and delay red blood cell transfusion, which are associated with acute myeloid leukemia progression and increase in morbidity. Mutations in SF3B1 modify the recognition pattern of the 3' splice site and lead to subsequent mis-splicing of its targets. To identify critical mis-splicing events involved in the erythroid differentiation blockage, we performed splicing analysis on RNA sequencing generated from hematopoietic stem/progenitor cells undergoing differentiation. Three MDS primary samples harboring SF3B1 mutations and three age-matched healthy donors cultured under normoxia and hypoxia conditions were initially used for the analysis. High depth RNA sequencing and differential splicing analyses using rMATS identified 2,845 mis-spliced events including 200 shared between hypoxia and normoxia conditions. Here, using a cohort of 42 MDS samples, we report the mis-splicing of the coenzyme A synthase (COASY) transcript. Heme synthesis relies on succinyl-CoA synthesis, and its production itself depends on the availability of cellular CoA. We thus hypothesised that COASY mis-splicing is a key driver of ineffective erythropoiesis in MDS-RS patients. In primary hematopoietic cells, COASY is upregulated during erythroid differentiation and its silencing in CD34 + cells severely impedes the generation of mature erythroid cells CD71 - CD235a + and causes disruption in heme production. Functional characterisations of the CRISPR-CAS9 edited K562 SF3B1K700E and the SF3B1-mutated HNT-34 cell lines confirmed that COASY mis-splicing impairs COASY protein synthesis that ultimately results in 60% loss of the protein. Metabolomic analysis showed that COASY mis-splicing depletes cells in CoA and succinyl-CoA metabolites, however this phenotype can be rescued by supplementation with vitamin B5, a CoA precursor. Consequently, we showed in vitro that saturating the 40% of remaining COASY enzyme with vitamin B5 or supplementing medium with its downstream by-product, succinyl-CoA, improved erythropoietic differentiation in MDS SF3B1mut patients. In summary, our results for the first time show that SF3B1 mutations induce coenzyme A synthase (COASY) transcript mis-splicing, that consequently leads to measurable defects in metabolites essential for heme biosynthesis. Our report reveals a novel critical role of COASY in regulating normal bone marrow erythropoiesis through control of succinyl-coA during human erythroid differentiation. Remarkably, partial loss of the coenzyme A synthase in MDS-RS patients leads to disruption in the erythroid lineage as well as heme deficiency, that can be rescued by exogenous treatment with vitamin B5 or succinyl-CoA. Therefore, vitamin B5 could represent a very attractive agent to combine with existing treatments in order to increase erythroid maturation and delay red blood cell transfusion dependency in MDS-RS patients. Graphical representation: SF3B1 mutant causes mis-splicing in COASY that results in loss of protein. Deficiency in COASY triggers a downregulation of succinyl-CoA that is involved in the rate limiting step of heme synthesis. Heme deficiency subsequently impairs erythroid differentiation. Treatment of MDS SF3B1 mutant cells with vitamin B5 (precursor of CoA), or succinyl-CoA, rescues erythroid differentiation. Figure 1 Figure 1. Disclosures Platzbecker: Geron: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; AbbVie: Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; StemLine: Consultancy; Takeda: Consultancy; Astex: Research Funding. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Introduction: Transformation of chronic myelomonocytic leukaemia (CMML) to secondary acute myeloid leukaemia (sAML) is frequent and invariably fatal, but poorly characterized. Understanding the transcriptional programs driving progression could reveal therapeutic strategies to prevent/delay transformation. We integrated serial single-cell CITE-seq on CD34+ cells from CMML patients before and after sAML, to evaluate transcriptional and hierarchical dynamics in the hematopoietic stem and progenitor (HSPC) compartment. Methods: Four CMML patients with matched bone marrow (BM) from CMML and sAML (± interim remission after azacitidine) were included. Three age-matched healthy controls were sourced from elective hip arthroplasties. Total 13 samples were processed. BM mononuclear cells were thawed and double MACS/FACS-enriched for CD34+ HSPCs. Cells were incubated with 14 HSPC/lineage-defining TotalSeq-A oligonucleotide-conjugated antibodies before parallel analysis of transcriptome and surface protein expression by 10X (Fig 1A). Results: Total 68,244 HSPCs were analyzed. Controls displayed near-identical UMAP distributions so were pooled for analyses. Optimal UMAP embeddings were generated for each CMML series independently (Fig 1B). UMAP clearly separated populations unique to CMML and sAML samples. Cell identities were inferred by unsupervised annotation from public datasets. CMML samples displayed loss of lymphoid progenitors, but also near-total absence of transcriptionally normal apical HSCs. All patients displayed aberrant expansions of progenitor populations, variously dominated by MPPs, CMPs or GMPs, as validated by antibody-derived tag (ADT) expression patterns. Pseudotime resolved patient-specific transformation trajectories, with blast expansions emerging from GMP (n=2), CMP/LMPP (n=1) or HSC/MPP (n=1). However, blasts consistently upregulated HSC-like transcriptional programs, in some cases with clear stepwise reversion from committed to stem-like transcriptional states along the computed trajectory (Fig 1B). Accordingly, blasts expressed classical HSC marker genes (ID2, DUSP1, CD52) alongside surface expression of private lineage markers but lacking ADT profiles of apical HSCs (Fig 1C). This decoupling, together with the pseudotime profiles, suggested progressive restoration of an HSC-like signature in downstream progenitors as a consistent feature. Post azacitidine there was marked expansion of megakaryocyte-erythroid progenitors, but despite recovery of normal hematopoiesis no restoration of the depleted apical HSCs. Subsequent sAML represented expansion of pre-existing (treatment-resistant) blasts in 3 case, but emergence of a new, transcriptionally-distinct blast population in the other. To characterise populations emergent at sAML we evaluated differentially expressed genes (DEG) vs respective CMML counterparts, then intersected gene lists across series. We found 319 genes upregulated in sAML conserved across ≥3 series. Among the most consistently upregulated genes at sAML were DUSP2 and all subunits of ITM2 (notably ITM2A, recently linked to AML). Pathways enriched in sAML included mTOR/PI3K/AKT and VEGF signalling (Fig 1D-E). Also overexpressed were several surface proteins (CD63, CD74, CD82, CD99) also aberrantly upregulated at earlier CMML, suggesting potential as biomarkers to predict/track transformation. Of the sAML signature genes 68 are considered 'druggable', including PIK3R1, NFKBIA and CXCL8. To identify early drivers of transformation we performed unsupervised clustering for each pooled series, comparing DEGs for successive intermediate CMML-to-sAML clusters along the computed trajectories. Common upregulated pathways included VEGF, MAPK and IL18 signaling, with 4 genes, notably the AP1 component JUNB, progressively upregulated during early transformation in all series (Fig 1F-G). Conclusions: We characterize the repartitioning of HSPC subsets in CMML, including loss of transcriptionally-normal apical HSCs, not restored upon azacitidine response; this may partially explain the transient nature of responses. Cell-of-origin and paths to acute transformation were heterogeneous, but with consistent co-opting of HSC-like programs in lineage-committed blasts. PIK3R1 and JUNB, and the PI3K/AKT/mTOR and VEGF pathways, emerge as candidate mediators of CMML transformation. Figure 1 Figure 1. Disclosures Somervaille: Novartis: Consultancy, Honoraria. Wiseman: Novartis: Consultancy; StemLine: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; Astex: Research Funding.

Cytarabine (AraC) represents the most effective single agent treatment for AML. Nevertheless, overriding AraC resistance in AML remains an unmet medical need. Here we show that the CHK1 inhibitor (CHK1i) GDC-0575 enhances AraC-mediated killing of AML cells both in vitro and in vivo, thus abrogating any potential chemoresistance mechanisms involving DNA repair. Importantly, this combination of drugs does not affect normal long-term hematopoietic stem/progenitors. Moreover, the addition of CHK1i to AraC does not generate de novo mutations and in patients’ samples where AraC is mutagenic, addition of CHK1i appears to eliminate the generation of mutant clones. Finally, we observe that persistent residual leukemic cells are quiescent and can become responsive to the treatment when forced into cycle via granulocyte colony-stimulating factor (G-CSF) administration. This drug combination (AraC+CHK1i+G-CSF) will open the doors for a more efficient treatment of AML in the clinic., Overriding cytarabine resistance in AML remains an unmet medical need. Here, the authors show that the CHK1 inhibitor GDC-0575 in combination with cytarabine and G-CSF has a significant anti-leukemic effect without toxicity to normal marrow stem and progenitor cells.

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with >6,000 proteins and > 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

Summary Despite advances in our understanding of interactions between mouse hematopoietic stem cells (HSCs) and their niche, little is known about communication between human HSCs and the microenvironment. Using a xenotransplantation model and intravital imaging, we demonstrate that human HSCs display distinct motile behaviors to their hematopoietic progenitor cell (HPC) counterparts, and the same pattern can be found between mouse HSCs and HPCs. HSCs become significantly less motile after transplantation, while progenitor cells remain motile. We show that human HSCs take longer to find their niche than previously expected and suggest that the niche be defined as the position where HSCs stop moving. Intravital imaging is the only technique to determine where in the bone marrow stem cells stop moving, and future analyses should focus on the environment surrounding the HSC at this point., Highlights • HSC niche should be defined by the position where HSCs stop moving • HSC anchorage to the niche can be studies using time-lapse intravital imaging, In this article, Bonnet and colleagues dissect the early stages of human HSC anchorage to the niche using time-lapse imaging and demonstrate that HSCs show differential motile behaviors compared with their HPC counterparts when arriving in their niche.

Mesenchymal progenitor cells (MPCs) have been hypothesized as cells of origin for sarcomas, and c-Fos transcription factor has been showed to act as an oncogene in bone tumors. In this study, we show c-Fos is present in most sarcomas with chondral phenotype, while multiple other genes are related to c-Fos expression pattern. To further define the role of c-Fos in sarcomagenesis, we expressed it in primary human MPCs (hMPCs), immortalized hMPCs and transformed murine MPCs (mMPCs). In immortalized hMPCs, c-Fos expression generated morphological changes, reduced mobility capacity and impaired adipogenic- and osteogenic-differentiation potentials. Remarkably, immortalized hMPCs or mMPCs expressing c-Fos generated tumors harboring a chondrogenic phenotype and morphology. Thus, here we show that c-Fos protein has a key role in sarcomas and that c-Fos expression in immortalized MPCs yields cell transformation and chondrogenic tumor formation. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS: PI11/00377 to J.G.-C.; and RTICC: RD12/0036/0027 to J.G-C, RD12/0036/0020 to S.M.) and the Madrid Regional Government (CellCAM; P2010/BMD-2420 to J.G.-C) in Spain. A.A. was supported by Juan de la Cierva program of the Spanish Plan Nacional (MINECO) and Sara Borrell program of the ISCIII/FEDER. A.Al. was supported by the “Miguel Servet” program of the ISCIII/FEDER. We gratefully acknowledge support from Asociación Pablo Ugarte (CIF G86121019) and AFANION (CIF G02223733). The experiments were approved by the appropriate committees. Sí

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological clonal disorders. Here, we have tested the bone marrow (BM) cells from 38 MDS patients covering all risk groups in two immunodeficient mouse models: NSG and NSG-S. Our data show comparable level of engraftment in both models. The level of engraftment was patient specific with no correlation to any specific MDS risk group. Furthermore, the co-injection of mesenchymal stromal cells (MSCs) did not improve the level of engraftment. Finally, we have developed an in vitro two-dimensional co-culture system as an alternative tool to in vivo. Using our in vitro system, we have been able to co-culture CD34+ cells from MDS patient BM on auto- and/or allogeneic MSCs over 4 weeks with a fold expansion of up to 600 times. More importantly, these expanded cells conserved their MDS clonal architecture as well as genomic integrity.

Juvenile myelomonocytic leukemia (JMML) is a rare aggressive myelodysplastic/myeloproliferative neoplasm of early childhood, initiated by RAS-activating mutations. Genomic analyses have recently described JMML mutational landscape; however, the nature of JMML-propagating cells (JMML-PCs) and the clonal architecture of the disease remained until now elusive. Combining genomic (exome, RNA-seq), Colony forming assay and xenograft studies, we detect the presence of JMML-PCs that faithfully reproduce JMML features including the complex/nonlinear organization of dominant/minor clones, both at diagnosis and relapse. Further integrated analysis also reveals that although the mutations are acquired in hematopoietic stem cells, JMML-PCs are not always restricted to this compartment, highlighting the heterogeneity of the disease during the initiation steps. We show that the hematopoietic stem/progenitor cell phenotype is globally maintained in JMML despite overexpression of CD90/THY-1 in a subset of patients. This study shed new lights into the ontogeny of JMML, and the identity of JMML-PCs, and provides robust models to monitor the disease and test novel therapeutic approaches.

Introduction The epigenome is significantly perturbed in hematological malignancies with global DNA hypomethylation and localized hypermethylation of gene promoter CpG islands. Whether specific gene promoter hypermethylation can contribute to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) in humans by affecting HSPC biology, independently of genetic mutations, has not previously been investigated due to the lack of appropriate tools. We show for the first time that it is possible to target de novo DNA methylation using CRISPR/Cas9 in human CD34+ cells isolated from cord blood (CB). DNA methylation targeted to key cell cycle control gene promoters, INK4b (p15) and ARF (p14), is permanently maintained after dCas9 3A3L degradation and inherited as cells differentiate; inhibiting gene expression and affecting the colony forming potential of CD34+ cells. This demonstrates that specific DNA hypermethylation events can permanently change HSPC biology and impact differentiation, potentially contributing to pre-malignant processes. Methods Human CD34+ HSPCs were isolated from human CB and maintained in liquid culture for 24 hours before nucleofection with mRNA encoding an adapted form of CRISPR/Cas9 which has no nuclease activity (dCas9) and is fused to the catalytic domain of DNA methyltransferase 3A (DNMT3A) and 3L (3A3L). The nucleofection cocktail contained dCas9 3A3L or dCas9 3A3L-mut (lacks methyltransferase activity) and 1 to 3 guide RNAs to target DNA methylation to combinations of the INK4a-ARF-INK4b locus. Cells were then seeded into methylcellulose for a primary colony forming assay (CFU). Colonies were scored after 14 days and cells were either harvested and pooled or individual colonies were picked for single-colony molecular analyses. The DNA was extracted and methylation at the INK4a-ARF-INK4b promoters was quantified using targeted bisulfite sequencing; target gene expression was measured using qPCR. The remaining cells from the primary CFU were re-plated a second (secondary CFU) and third (tertiary CFU) time and colonies were again scored after 14 days. Results and Conclusions Targeting DNA methylation to the INK4a-ARF-INK4b locus or INK4b individually in human CD34+ cells resulted in maintenance of hypermethylation at ARF and/or INK4b gene promoters in individual BFU-E (burst-forming unit-erythroid) and CFU-GM (granulocyte, macrophage) colonies as measured by single-colony targeted bisulfite sequencing after the primary CFU; causing heritable repression of INK4b gene expression in the differentiated cells. Some CpGs were up to 90% methylated, indicating that DNA methylation added at these gene promoters is highly stable as cells differentiate. Hypermethylation of ARF and INK4b was found in some colonies even after the tertiary CFU, demonstrating long-term maintenance of promoter hypermethylation. Unexpectedly, no DNA hypermethylation was detected at INK4a in differentiated cells, but whether this is the case for all subpopulations of HSPCs (i.e. HSCs or lymphoid progenitors) is under investigation. Hypermethylation of INK4b and ARF increased the colony forming potential of CD34+ cells in primary, secondary and tertiary CFUs, compared to the control. Conversely, methylation targeted to INK4b alone did not significantly affect the number of colonies in the first CFU, and decreased the number of colonies in the secondary CFU. This suggests a complex interplay between key cell cycle regulators ARF and INK4b in CD34+ cells and during differentiation which can be disrupted by DNA hypermethylation and gene repression. These findings demonstrate the novel insights we can gain by using CRISPR/Cas9 tools to target DNA methylation and these investigations will reveal how gene promoter hypermethylation can impact HSPC function. Furthermore, studying this locus may uncover an important role for DNA hypermethylation in the development of myeloid malignancies, since INK4b is frequently hypermethylated, but rarely mutated, in myeloid dysplastic/proliferative neoplasms and acute myeloid leukemia. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

As significant numbers of acute myeloid leukemia (AML) patients are still refractory to conventional therapies or experience relapse, immunotherapy using T-cells expressing chimeric antigen receptors (CARs) might represent a valid treatment option. AML cells frequently overexpress the myeloid antigens CD33 and CD123, for which specific CARs can be generated. However, CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs), and its targeting could potentially impair normal hematopoiesis. In contrast, CD123 is widely expressed by AML, while low expression is detected on HSPCs, making it a much more attractive target. In this study we describe the in vivo efficacy and safety of using cytokine-induced-killer (CIK) cells genetically modified to express anti-CD33 or anti-CD123 CAR to target AML. We show that both these modified T-cells are very efficient in reducing leukemia burden in vivo, but only the anti-CD123 CAR has limited killing on normal HSPCs, thus making it a very attractive immunotherapeutic tool for AML treatment.Leukemia accepted article preview online, 7 February 2014; doi:10.1038/leu.2014.62.

Evidence of the presence of abnormalities in the primitive hematopoietic stem cells, as well as functional analysis, has demonstrated that Myelodysplastic syndrome (MDS) is propagated by rare and distinct human MDS propagating cells (MDS-PCs) in 5q- and in refractory anemia with ring sideroblastic (

Introduction: Hypoxia inducible factors (HIF-1α and HIF-2α) are the main mediators of hypoxic responses that operate in both normal and pathological conditions. Recent evidence indicates that HIF-1α and HIF-2α could have overlapping, unique and even sometimes opposing activities in both normal physiology and disease. Despite an increase in our understanding of the different pathways regulated by HIF-1α and HIF-2α, the role played by each factor in HSC maintenance and leukemogenesis is still controversial.Areas covered: This review summarizes our current understanding of HIF-1α and HIF-2α activities and discusses the implications and challenges of using HIF inhibitors therapeutically in blood malignancies.Expert opinion: As HIF inhibitors are currently under clinical evaluation in different cancers, including hematological malignancies, a more thorough understanding of the unique roles performed by HIF-1α and HIF-2α in human neoplasia is warranted.

Despite the recent evidence of the existence of myelodysplastic syndrome (MDS) stem cells in 5q-MDS patients, it is unclear whether haematopoietic stem cells (HSCs) could also be the initiating cells in other MDS subgroups. Here we demonstrate that SF3B1 mutation(s) in our cohort of MDS patients with ring sideroblasts can arise from CD34+CD38−CD45RA−CD90+CD49f+ HSCs and is an initiating event in disease pathogenesis. Xenotransplantation of SF3B1 mutant HSCs leads to persistent long-term engraftment restricted to myeloid lineage. Moreover, genetically diverse evolving subclones of mutant SF3B1 exist in mice, indicating a branching multi-clonal as well as ancestral evolutionary paradigm. Subclonal evolution in mice is also seen in the clinical evolution in patients. Sequential sample analysis shows clonal evolution and selection of the malignant driving clone leading to AML transformation. In conclusion, our data show SF3B1 mutations can propagate from HSCs to myeloid progeny, therefore providing a therapeutic target., Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders with diverse phenotypes and can derive from hematopietic stem cells after the acquisition of specific somatic aberrations. In this study, the authors show that MDS initiating cells in some cases of sideroblastic anemia with SF3B1 mutations, can arise from hematopoietic stem cells.

Acute myeloid leukemia-initiating cells (LICs) are responsible for the emergence of leukemia and relapse after chemotherapy. Despite their identification more than 15 years ago, our understanding of the mechanisms responsible for their self-renewal activity and their chemoresistance remains poor. The slow progress in this area is partly due to the difficulty of studying these cells ex vivo. Indeed, current studies are reliant on xenotransplantation assays in immunodeficient mice. In this paper, we report that by modeling key elements of the bone marrow niche using different stromal feeder layers and hypoxic culture conditions, we can maintain LICs over at least 3 weeks and support their self-renewal properties demonstrated through primary and secondary successful xenograft. We provide a proof of principle that this niche-like culture system can be used to study LIC chemoresistance following in vitro cytarabine treatment similarly to the xenograft chemotherapy model. We found that although LICs are believed to be more chemoresistant than non-LICs, functionally defined LICs are not enriched after cytarabine treatment, and heterogeneity in their resistance to treatment can be seen between patients and even within the same patient. We present a culture system that can be used as an in vitro surrogate for xenotransplantation and that has the potential to dramatically increase the throughput of the investigation of LICs. This would further provide the means by which to identify and target the functionality of the different signaling pathways involved in the maintenance and resistance of LICs to improve acute myeloid leukemia treatments.

The biological and clinical behavior of hematological malignancies are not only determined by the properties of the leukemic cells themselves, but are also highly affected by interaction with the microenvironment, pointing to the existence of an active crosstalk between the two compartments. Previous studies showed that acute myeloid leukemia (AML) cells actively modify endothelial cells ex vivovia several pathways, mainly mediated by VEGF. However, as anti-VEGF therapies haven't produced successful results in clinical trials, an extensive study of the crosstalk between AML and the vascular niche in the bone marrow (BM) is required to provide new therapeutic strategies. In the present study we combined the use of mouse models of AML, human AML patient-derived xenografts (PDX) and direct analysis on patient-derived BM biopsies to provide a global, reliable picture of the bone marrow vasculature in AML disease. We found several abnormalities in the vascular architecture and function in PDX, such as increased number of endothelial cells, increased microvascular density (MVD), decreased vascular mean diameter and increased hypoxia. Furthermore, using two-photon confocal intravital imaging we witnessed increased vascular permeability upon AML engraftment, observed homogeneously among different PDX. Interestingly, induction chemotherapy failed to normalize the vascular permeability in the BM, despite significant reduction in AML engraftment. We identified increased nitric oxide (NO) as a major mediator of the AML-induced vascular leakiness in the BM. Increased levels of NO and activated NOS3 were found in PDX and in an independent cohort of patient-derived BM biopsies. Strikingly, inhibition of NO production using genetic and pharmacological approaches reduced the vascular permeability, potentiated the normal HSC function and significantly improved treatment response in PDX. These results strongly support the notion of a primary function of the vascular permeability in AML progression, drug response and in affecting normal stem cell function, and they call for clinical trials incorporating NOS inhibitors during the remission phase to target the abnormal vascular niche and improve the treatment response. Disclosures No relevant conflicts of interest to declare.

Introduction Defects in pre-mRNA splicing have recently been implicated in the pathogenesis of myelodysplastic syndrome, particularly RARS where SF3B1, a key component of the spliceosome machinery is mutated in up to 83% of the cases. Moreover, as homozygosity is the exception to the rule for these mutations and SF3B1 homozygous systems have been previously reported as lethal, then such aberrations are most certainly not benign bystanders and are likely to play a significant role in pathogenesis of the disease. Besides the strong correlation of SF3B1 mutations and the presence of ringed sideroblasts, the mechanism through which these mutations affect their target cells, distort the RNA splicing process and subsequently lead to disease still remains unclear. Methodology In order to gain insight into the effects of SF3B1 mutations on the engraftment kinetics of haemopoietic stem cells, bone marrow CD34+ cells from RARS patients (n=4) with SF3B1 mutations (with mutant allele burden 20% to 48%) were transplanted in NOD/SCID/interleukin-2 receptor γ chain–null (NOD/SCID/IL2rγ−/−) mice (1-3 mice/patient), and were also plated in committed progenitor (colony-forming cell, or CFC) and primitive progenitor (long-term bone marrow culture-initiating cell, or LTC-IC) cultures. Following on, whole-exome sequencing (Illumina) was performed on pre-transplant (bone marrow CD34+/TNC), post-transplant week-24 (human CD45+CD33+) and LTC-IC week-5 samples. In addition, we also performed whole-exome sequencing on FACS-purified HSCs (Lin- CD45+CD34+CD38- CD45RA-CD90+CD49f+), MPPs (Lin- CD45+CD34+CD38-CD45RA-CD90-CD49f-), GMPs (Lin- CD45+CD34+CD38+CD135+ CD45RA+) and MEPs (Lin- CD45+CD34+CD38+CD135-CD45RA-) from 3 of these patients. Results Our data demonstrates that the SF3B1 mutations arise from the early HSCs and are propagated without diminution in the mutant allele burden from HSC to more committed myeloid progenitors (i.e. MPPs, GMPs and MEPs). We also show, that the transplantation of bone marrow CD34+ cells derived from SF3B1 mutant RARS patients into mice leads to persistent long-term engraftment (0.01% - 4%, up to 24 weeks) of only myeloid cells but not lymphoid cells, irrespective of the type of SF3B1 mutation. The RARS-originated cells recovered from the human cell-engrafted mice maintained its genotypic characteristics, as demonstrated by the presence of SF3B1 mutations which remained largely unchanged between the pre- and post-transplant samples. This phenomenon was also observed in LTC-IC and CFC culture experiments where the SF3B1 mutant allele burden was maintained at the same level. In all analysed samples, there was no difference in the SF3B1 mutation burden between the LTC-IC with cytokines or without cytokines, and CFC under normoxic or hypoxic conditions. Whole-exome sequencing also revealed additional 20-60 known and/or novel candidate somatic mutations which were present concurrently in mice, LTC-IC and/or CD34+ (pre-transplant) cells for each RARS experiment. These include genes involved in epigenetic modifications, cell signalling/transcription regulation, DNA maintenance and cytoskeletal organization. These candidate mutations were either maintained at the same mutant allele burden or reduced/increased between the pre-transplant, post-transplant and LTC-IC, and are likely to be the sub-clones of SF3B1 clone. For example, in one RARS case JAK2 V617F mutation changed from 20% at pre-transplant to 9% (average mutant allele burden in mice, n=3) at post-transplant and 10% in LTC-IC. However, DNMT3A W581S mutation present in another RARS case was largely maintained i.e. 44% at pre-transplant, 38% at post-transplant (n=1) and 43% in LTC-IC. Conclusion These results demonstrate that SF3B1 mutations arise from the early HSCs, persist in the MEPs/GMPs, and retain their engraftment capacity in-vivo as well as their long-term proliferation potential in-vitro. Our murine xenograft RARS model comprised of human myeloid progenitor and mature cells, the majority of these cells, if not all, originated from the patients original clonal HSC pool as exhibited by its aberrant genotypic characteristics. Furthermore, this study also suggests that the cells carrying the SF3B1 mutations have a clonal growth advantage as compared to normal cells present in these RARS patients, and is therefore likely to contribute to the RARS disease phenotype. Disclosures: Bonnet: Janssen and Janssen: Collaborative Project and Consultant Other; Genetech: Collaborative project, Collaborative project Other.

Abstract 4225 Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after conventional chemotherapy regimens. Therefore it is necessary to develop novel alternative approaches. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial T-cell receptors constituted by a specific antigen-binding domain, and a signaling region, that, upon antigen recognition, leads to T-cell activation, and lysis of the target cells. AML is a potential optimal target for CAR strategy because of the over-expression of a number of surface antigens like CD33, CD123. Since CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently emerged as new potential attractive molecules based on its differential expression pattern, being still wildly overexpressed by AML population, and at the same time less expressed on HSPCs. Here we describe the in vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK) cells genetically modified to express CAR molecules specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted NSG mice (median of hCD45+CD33+ 0.6% before treatment), genetically modify T cells had a potent antitumor effect. Indeed, the bone marrow of control untreated animals or mice treated with un-manipulated CIK cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-ζ and 8/10 anti-CD123-CD28-OX40-ζ treated mice we couldn't detect any AML cells. Similar results have been obtained when the treatment via T cell injection start when high AML burden has been obtained (median of hCD45+CD33+ 70% before treatment). One week after the last CIK's injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK cells and with anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells respectively. We performed secondary transplantation on the residual AML cells present in these animals and mice were treated again with transduced CIK cells. Residual AML cells were still sensitive to CARs approach, leading once again to an almost complete eradication of the disease (median level of hCD45+CD33+ engraftment was 98%, 0.02% and 0.04% respectively for untreated mice, anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells). Furthermore, a fundamental issue was to determine the safety profile of such approach against normal hematopoietic precursors. In untreated mice injected with primary cord blood derived CD34+ cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with un-manipulated, anti-CD33-CD28-OX40-ζ or anti-CD123-CD28-OX40-ζ transduced CIK-cells the levels of human compartment was 40%, 11.7% and 26.3% respectively. Moreover when we consider specifically the CD34+CD38- compartment, enriched in HSC, the level of engraftment was 1.92%, 1.02%, 0.55% and 0.83%. Secondary transplantations are now ongoing to give a more complete profile about the remaining HSC repopulating capability after treatment. To more closely mimic a physiological context, similar experiments are ongoing using mice engrafted with normal adult bone marrow instead of umbilical cord blood. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy particularly in the context of minimal residual disease in high-risk transplanted AML patients. Moreover CAR approach could be potentially used to treat patients resistant to conventional chemotherapeutic approaches or for whom high dose chemotherapy treatment could not be proposed. Disclosures: No relevant conflicts of interest to declare.

Summary Hematopoietic stem and progenitor cells (HSPCs) are exposed to low levels of oxygen in the bone marrow niche, and hypoxia-inducible factors (HIFs) are the main regulators of cellular responses to oxygen variation. Recent studies using conditional knockout mouse models have unveiled a major role for HIF-1α in the maintenance of murine HSCs; however, the role of HIF-2α is still unclear. Here, we show that knockdown of HIF-2α, and to a much lesser extent HIF-1α, impedes the long-term repopulating ability of human CD34 + umbilical cord blood cells. HIF-2α-deficient HSPCs display increased production of reactive oxygen species (ROS), which subsequently stimulates endoplasmic reticulum (ER) stress and triggers apoptosis by activation of the unfolded-protein-response (UPR) pathway. HIF-2α deregulation also significantly decreased engraftment ability of human acute myeloid leukemia (AML) cells. Overall, our data demonstrate a key role for HIF-2α in the maintenance of human HSPCs and in the survival of primary AML cells.