Experimental Hematology

Progressively acquired somatic mutations in hematopoietic stem cells are central to pathogenesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). They can lead to proliferative advantages, impaired differentiation and progressive cytopenias. MDS or CMML patients with high-risk disease are treated with hypomethylating agents including 5-azacytidine (AZA). Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. To address this, we characterised the somatic mutations of individual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naive B cell) haematopoietic cells in treatment naive and AZA-treated MDS and CMML via high-throughput single cell genotyping. The mutational burden was similar across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo. Our data show that even highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. Key points* Highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment. * The mutational burden in matched bone marrow and peripheral blood cells in MDS and CMML was similar throughout myelopoiesis.

STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern as Stat3 deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediated Stat3 deletion in 20% of the hematopoietic compartment. Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast to Stat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells revealed altered transcriptional responses in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient Lin-ckit+Sca1+ cells accumulated {gamma}H2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs. Key PointsO_LISTAT3 is critical for hematopoietic activity and hematopoietic stem cell maintenance in non-inflammatory conditions C_LIO_LISTAT3 has a cell-intrinsic role in the suppression of interferon signaling and myeloid-skewed transcription in hematopoietic stem cells C_LI

Acute megakaryoblastic leukemia (AMKL) is a rare and aggressive pediatric acute myeloid leukemia subtype, frequently driven by the ETO2::GLIS2 (EG) gene fusion in de novo AMKL patients. The EG fusion confers poor prognosis and high relapse risk. In a previous study, we demonstrated that human fetal hematopoietic cells are intrinsically permissive to EG-driven transformation and that microenvironmental cues critically enhance leukemogenesis in less permissive human cord blood (CB) cells. However, most pediatric AML models are established in adult recipient mice, potentially overlooking developmental niche-specific effects. Here, we investigated whether mouse neonates provide a more permissive environment for EG-driven leukemogenesis from human CB hematopoietic stem and progenitor cells (HSPCs). Comparison of xenotransplants of EG-transduced CB CD34+ cells into sublethally irradiated NSG neonate or adult mice show that neonatal recipients are more supportive of EG-driven leukemia when cell doses are adjusted for recipient size. Neonates exhibited a higher frequency of leukemia-initiating cells (LIC), enhanced expansion of EG/GFP+ leukemic cells, and increased disease penetrance compared with adults at similar dose/size ratio, despite similar overall human hematopoietic engraftment. Leukemic cells arising in neonates and adults displayed comparable phenotypes and secondary transplantation capacities, indicating functional equivalence. Both CD34+CD38- and CD34+CD38+ CB subpopulations were sensitive to EG-driven transformation, with neonates consistently showing higher LIC frequencies than adults. Together, our findings demonstrate that the developmental stage of the host microenvironment is a critical extrinsic determinant of EG-mediated leukemogenesis. Neonatal recipients constitute a physiologically relevant and highly sensitive model for studying pediatric AMKL initiation with low cell inputs, enabling refined investigation of microenvironmental signals that promote leukemia development and may reveal novel therapeutic vulnerabilities

Tyrosine kinase inhibitors (TKI) only rarely eradicate leukemic stem cells (LSC) in chronic myeloid leukemia (CML) which commonly necessitates life-long therapy and monitoring of patients. Understanding details of leukemic hematopoiesis in CML may identify targetable pathways for sustained LSC elimination. This study utilized multiomic single-cell characterization of the CD14-CD34+ hematopoietic stem and progenitor cell (HSPC) compartment in CML. Combined proteo-transcriptomic profiling of 597 genes and 51 proteins (CITE-seq) was performed along with parallel detection of BCR-ABL1 transcripts in 70,000 HSPC from 16 chronic phase patients and five healthy controls. CD14-CD34+ HSPC from diagnosis samples displayed distinct myeloid cell bias with cells mainly annotated as LSC, lympho-myeloid progenitors (LMP)-II, erythrocyte and megakaryocyte progenitors, while few hematopoietic stem cells (HSC), LMP-I, dendritic cell or B cell progenitors were detected. In-depth analysis of the immature CD14-CD34+CD38-/low compartment revealed two distinct populations of BCR-ABL1-expressing CML LSC (denoted LSC-I and LSC-II), where LSC-I showed features of quiescence and CD45RA-cKIT-CD26+ TKI therapy-resistant phenotype. These subtypes of immature LSC showed high surface expression of TIM3 and transcription of the von Willebrand factor gene (VWF). Our findings imply that expression of VWF and TIM3 distinguish LSC from HSC and may be linked to aberrant myeloid-biased hematopoiesis in CML. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML. Key pointsO_LIWe present a method to detect BCR-ABL1 expression at the single-cell level that is compatible with high-throughput CITE-seq C_LIO_LIThe most immature BCR-ABL1-expressing LSC population in primary CML shows enhanced expression of von Willebrand factor and TIM3 C_LI

JAK2-V617F is the most frequent somatic mutation causing myeloproliferative neoplasm (MPN). However, JAK2-V617F can also be found in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) with a frequency much higher than the prevalence of MPN. The factors controlling the conversion of JAK2-V617F CHIP to MPN are largely unknown. We hypothesized that IL-1{beta} mediated inflammation is one of the factors that favors this progression. We examined mono- or oligoclonal evolution of MPN by performing bone marrow transplantations at limiting dilutions with only 1-3 JAK2-mutant HSCs per recipient. Genetic loss of IL-1{beta} in JAK2-mutant hematopoietic cells or inhibition by a neutralizing anti-IL-1{beta} antibody restricted the early clonal expansion of these JAK2-mutant HSCs resulting in a reduced frequency of a CHIP-like state and a lower rate of conversion to MPN. The MPN disease-promoting effects of IL-1{beta} were associated with damage to sympathetic innervation leading to loss of nestin-positive mesenchymal stromal cells and required the presence of IL-1R1 on bone marrow stromal cells. The anti-IL-1{beta} antibody protected these mesenchymal stromal cells from IL-1{beta} mediated damage and limited the expansion of the JAK2-mutant clone. Our results identify IL-1{beta} as a potential therapeutic target for preventing the transition from JAK2-V617F CHIP to MPN. Brief summaryIn a mouse model of oligo-clonal myeloproliferative neoplasm (MPN), IL-1{beta} produced by JAK2-mutant cells favored expansion of sub-clinical JAK2-V617F clones and initiation of MPN disease.

The mechanisms underlying myeloid malignancies deletions are not well understood, nor is it clear why specific genomic hotspots are predisposed to particular deletions. In the current study we inspected the genomic regions around recurrent deletions in myeloid malignancies, and identified microhomology-mediated end-joining (MMEJ) signatures in recurrent deletions in CALR, ASXL1 and SRSF2 loci. Since MMEJ deletions are the result of DNA double-strand breaks (DSBs), we introduced CRISPR Cas9 DSBs into exon 12 of ASXL1, successfully generating recurrent ASXL1 deletion in human hematopoietic stem and progenitor cells (HSPCs). A systematic search of COSMIC dataset for MMEJ deletions in all cancers revealed that recurrent deletions enrich myeloid malignancies. Despite this myeloid predominance, we provide evidence that MMEJ deletions occur in multipotent HSCs. An analysis of DNA repair pathway gene expression in single human adult bone marrow HSPCs could not identify a subpopulation of multipotent HSPCs with increased MMEJ expression, however exposed differences between myeloid and lymphoid biased progenitors. Our data indicate an association between MMEJ-repaired DSBs and recurrent MMEJ deletions in human HSCs and in myeloid leukemia. A better understanding of the source of these DSBs and the regulation of the HSC MMEJ repair pathway might aid with preventing recurrent deletions in human pre-leukemia.

Interactions with the bone marrow (BM) niche are crucial for promoting self-renewal and survival of acute myeloid leukemia (AML) cells. Consequently, AML cells express a variety of surface receptors to engage with BM niche cells and extracellular matrix proteins, including laminins. Despite the association of laminin receptor expression with stemness in healthy hematopoiesis, the role of laminin receptors in AML remains poorly understood. In this study, we present a comprehensive examination of the laminin receptors integrin 3{beta}1, 6{beta}1, 7{beta}1 and basal cell adhesion molecule (BCAM) in AML. We demonstrate that high mRNA expression of all four laminin receptors correlates with poor overall survival. Notably, integrin 6 and 7 display the highest cell surface presentation among the examined laminin receptors and are higher expressed on AML cells compared to healthy controls. Moreover, our results indicate that integrin 7 expression allows to distinguish between leukemic stem cells (LSC) and non-LSC populations. Specifically, integrin 7 appears to mark non-LSC with enhanced migratory potential. Together, our results confirm the association of high laminin receptor expression with poor prognosis and establish integrin 7 as marker of high migratory non-LSC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/587290v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@bdcc20org.highwire.dtl.DTLVardef@a45fd2org.highwire.dtl.DTLVardef@18f058forg.highwire.dtl.DTLVardef@b613fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease produced by a unique oncogenic event involving the constitutively active tyrosine kinase (TK) BCR/ABL1. TK activity explains most features of CML, such as tumor development and maintenance. TK-inhibitory (TKI) drugs have changed its prognosis and natural history. Unfortunately, the ABL1 gene persists unaffected by TKIs, leukemic stem cells (LSCs) remains, resistant mutations arise and adverse effects may occur during treatment. To address this problem, we have designed a potential therapeutic alternative with CRISPR/Cas9 genome editing nucleases that target LSCs. The strategy was successfully developed in murine and human cell lines and finally was evaluated in primary LSCs isolated from CML transgenic mice and from CML patients. Mouse CML-LSCs edited were orthotopic transplanted in immunodeficient NSG niches where restored the normal hematopoiesis. Importantly, patient-derived xenografts with CD34+-LSCs edited, repopulated and restored the normal hematopoiesis in immunodeficient NSG niches. We show, for the first time, how CRISPR technology efficiently interrupts the BCR/ABL1 oncogene in murine and human LSCs to provide a significant therapeutic benefit. We propose human CML as a potential candidate for CRISPR therapy, providing proof-of-principle for genome editing in CML patients, and open new avenues for the application of this technique in other fusion genes. Key pointsCRISPR system destroys BCR/ABL oncogene and induces a therapeutic benefit in a CML mouse model and CML patient derived xenografts.

Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells (HSC) that are most frequently caused by acquired somatic mutations in JAK2. A number of conditional mouse models of JAK2-V617F-driven MPN have been generated that rely on Cre-LoxP mediated activation, resulting in polyclonal disease. To more closely mimic the monoclonal origin of human MPN, transplantations of single purified JAK2-mutant HSCs or bone marrow (BM) at limiting dilutions into lethally irradiated recipient mice have been previously performed. However, irradiation is known to alter the BM microenvironment and also to induce transient aplasia accompanied by elevated cytokine levels that promotes the expansion of the mutant clone. To overcome these limitations, we examined whether JAK2-V617F-mutant HSCs are able to engraft and initiate MPN in non-conditioned recipients. We found that BM from two different MPN models, one expressing the human JAK2-V617F, and another expressing the mouse Jak2-V617F, efficiently engrafted and initiated MPN in non-irradiated immunocompromised Rag2-/- recipients. MPN evolved even in transplantations at limiting dilutions, showing high competitiveness of single JAK2-mutant HSCs. Thus, JAK2-V617F mutant HSCs can outcompete resident non-mutated HSCs in the absence of elevated cytokine levels and without the need of emptying stem cell niches by irradiation. However, only BM from mice expressing the mouse Jak2-V617F engrafted and initiated disease in non-conditioned C57BL/6 mice, while BM from mice expressing the human JAK2-V617F was rejected, indicating that mouse Jak2-V617F is ignored by the immune surveillance. These results provide a possible explanation why JAK2-V617F is so frequently found in healthy individuals with clonal hematopoiesis.

Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking is a more common and overlooked mechanism for structural rearrangement-mediated gene activation in AML. Key pointsO_LIExpression analysis of over 1500 pediatric AML samples demonstrates MNX1 expression as a universal feature of t(7;12)(q36;p13) AML as well as in rare cases without t(7;12)(q36;p13) C_LIO_LIMNX1 is activated by an enhancer-hijacking event in t(7;12)(q36;p13) AML and not, as previously postulated, by the creation of a MNX1::ETV6 oncofusion gene. C_LI

TET2 inactivating mutations serve as initiating genetic lesions in the transformation of hematopoietic stem and progenitor cells (HSPCs). In this study, we analyzed known drugs in zebrafish embryos for their abilities to selectively kill tet2-mutant HSPCs in vivo, and we found that the exportin 1 (XPO1) inhibitors, selinexor and eltanexor, selectively kill tet2-mutant HSPCs. In serial replating colony assays, these small molecules were selectively active in killing murine Tet2-deficient Lineage-, Sca1+, Kit+ (LSK) cells, and also TET2-inactivated human acute myeloid leukemia (AML) cells. Selective killing of TET2-mutant HSPCs and human AML cells by these inhibitors was due to increased levels of apoptosis, without evidence of DNA damage based on increased {gamma}H2AX expression. The finding that TET2 loss renders HSPCs and AML cells selectively susceptible to cell death induced by XPO1 inhibitors provides preclinical evidence of selective activity of these drugs, justifying further clinical studies of these small molecules for the treatment of TET2-mutant hematopoietic malignancies and to suppress clonal expansion in age-related TET2-mutant clonal hematopoiesis.

Inversion of chromosome 16 [inv(16)] generates the fusion gene CBFB::MYH11 (CM) and is one of the most common chromosomal rearrangements in Acute Myeloid Leukemia (AML). Expression of CM is required for leukemia initiation. Patients with inv(16) at diagnosis invariably have the rearrangement at relapse, leading to the assumption that CM is also required after leukemic transformation. However, a role for CM in leukemia maintenance has yet to be shown experimentally. To address this, we used an inducible CM knockdown (KD) mouse model and found that decreased CM eliminated leukemia cells from the peripheral blood and spleen, but not the bone marrow, despite all populations exhibiting significantly decreased CM mRNA and protein. The surviving CM KD cells in the bone marrow showed decreased apoptosis and proliferation, and increased expression of autophagy related genes. Surprisingly, with prolonged KD of CM, [~]40% of mice re-established disease despite maintaining decreased CM. Our work indicates that CM is required for leukemia survival in the spleen and peripheral blood, but in the bone marrow CM KD leukemia cells can survive and re-establish disease independent of the fusion protein. These findings imply that targeting CM alone has potential to reduce leukemic burden but not cure the disease.

Germline RUNX1 mutations lead to familial platelet disorder with associated myeloid malignancies (FPDMM), which is characterized by thrombocytopenia and a life-long risk (35-45%) of hematological malignancies. We recently launched a longitudinal natural history study for patients with FPDMM at the NIH Clinical Center. Among 29 families with research genomic data, 28 different germline RUNX1 variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 24 of 54 (44.4%) non-malignant patients, somatic mutations were detected in at least one of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes. BCOR was the most frequently mutated gene (in 9 patients), and multiple BCOR mutations were identified in 4 patients. Mutations in 7 other CHIP or AML driver genes (DNMT3A, TET2, NRAS, SETBP1, SF3B1, KMT2C, and LRP1B) were also found in more than one non-malignant patient. Moreover, three unrelated patients (one with myeloid malignancy) carried somatic mutations in NFE2, which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in elderly patients. In summary, there are diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring dynamic changes of somatic mutations prospectively will benefit patients clinical management and reveal mechanisms for progression to myeloid malignancies. Key PointsO_LIComprehensive genomic profile of patients with FPDMM with germline RUNX1 mutations. C_LIO_LIRising clonal hematopoiesis related secondary mutations that may lead to myeloid malignancies. C_LI

The homeobox gene, Hoxa1, has two different isoforms generated by alternative splicing: a full-length homeodomain-containing Hoxa1 (Hoxa1-FL), and a truncated Hoxa1 (Hoxa1-T), that lacks the homeodomain. Oncoretroviral overexpression of wildtype Hoxa1 cDNA (WT-Hoxa1), which generates both Hoxa1 isoforms, in murine hematopoietic stem and progenitor cells (HSPCs) perturbed hematopoiesis, resulting in myelodysplastic syndromes (MDS) in mice. Overexpression of a mutated Hoxa1 cDNA (MUT-Hoxa1) that generates Hoxa1-FL but not Hoxa1-T led to a more severe MDS capable of transforming to secondary acute myeloid leukemia (sAML). Similar to human MDS, DNA damage repair pathways were downregulated in Hoxa1-overexpressing hematopoietic progenitor cells. Conditional knock-in mouse models revealed a Hoxa1-FL dosage-dependent effect on MDS disease severity. Our data reveal that increased expression of Hoxa1-FL in HSPCs is sufficient to initiate MDS in mice. CD34+ cells from up to 50% of patients with MDS had elevated HOXA1-FL expression, highlighting the clinical relevance of our mouse models. Statement of SignificanceOur study demonstrates that Hoxa1 is a key regulator of HSPCs and that increased expression of the transcriptionally active Hoxa1-FL can initiate MDS in mice. Furthermore, HOXA1-FL expression is upregulated in a significant proportion of human MDS patients and likely contributes to the disease in these patients.

BackgroundMixed phenotype acute leukemia (MPAL) is a rare subgroup of leukemia characterized by blast cells that display both myeloid and lymphoid lineage features, making this cancer difficult to diagnose and treat. A deeper characterization of MPAL at the molecular level is essential to better understand similarities/differences to the more common and better-studied leukemias, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on MPAL bone marrow (BM) samples in an attempt to develop a more granular map of the MPAL microenvironment landscape. MethodsWe analyzed [~]16,000 cells from five pediatric MPAL BM samples collected at diagnosis to generate a single-cell transcriptomic landscape of B/Myeloid (B/My) and T/Myeloid (T/My) MPAL blasts and associated microenvironment cells. Cell clusters were identified using principal component analysis and uniform manifold approximation and projection (UMAP). Unsupervised analysis was performed to determine the overall relationship among B/My MPAL, T/My MPAL, and other acute leukemias - B-ALL, T-ALL, and AML. Supervised differentially expressed gene (DEG) analysis was performed to identify B/My and T/My MPAL blast-specific signatures. MPAL sample transcriptome profiles were compared with normal BM stem and immune cells to identify MPAL-specific dysregulation. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. Comparative analysis was performed on diagnostic samples based on their future minimal residual disease (MRD) and relapse status. ResultsB/My MPAL and T/My MPAL blasts displayed distinct subtype-specific blast signatures. UMAP analysis revealed that B/My MPAL samples had greater overlap with B-ALL samples, while T/My MPAL samples clustered separately from other acute leukemia subtypes. Genes overexpressed in both MPAL subtypes blasts compared to other leukemias and healthy controls included PLIN2, CD81, and UBE2S. B/My MPAL blast-specific genes included IRS2, SMIM3, and HBEGF, whereas T/My MPAL blast-overexpressed genes included IER5, BOD1L1, and HPGD. Sirtuin signaling, p38 MPAK signaling, and PI3K signaling pathways were upregulated in B/My MPAL blasts while oxidative phosphorylation and Rho family GTPases signaling pathways were upregulated in T/My MPAL blasts. Transcriptomic, pathways, and cell communication level differences were observed in the MPAL samples based on future MRD and clinical outcome status. ConclusionsWe have for the first time described the single-cell landscape of pediatric MPAL and demonstrate that B/My and T/My MPAL have unique scRNAseq profiles distinct from each other as well as from ALL and AML.

While del(5q) MDS patients comprise a well-defined hematological subgroup, the molecular basis underlying its origin, and the reason behind the relapse after lenalidomide remains unknown. Using scRNA-seq on CD34+ progenitor cells from patients with del(5q) MDS we were able to identify cells harboring the deletion, enabling us to deeply characterize the transcriptional impact of this genetic insult on disease pathogenesis and treatment response. We found, across all patients, an enrichment of del(5q) cells in GMP and megakaryocyte-erythroid progenitors not described to date. Interestingly, both del(5q) and non-del(5q) cells presented similar transcriptional lesions when compared to progenitors from healthy individuals, indicating that all cells, and not only those harboring the deletion, are altered in these patients and may contribute to aberrant hematopoietic differentiation. However, GRN analysis revealed a group of regulons with aberrant activity in del(5q) cells that could be responsible for triggering altered hematopoiesis, pointing to a more prominent role of these cells in the phenotype of these patients. An analysis of del(5q) MDS patients achieving hematological response upon lenalidomide treatment showed that the drug reverted several transcriptional alterations in both del(5q) and non-del(5q) cells, but other lesions remained, which may be responsible for potential future relapses. Moreover, lack of hematological response was associated with the inability of lenalidomide to reverse transcriptional alterations. Collectively, this study provides a deep characterization of del(5q) and non-del(5q) cells at single-cell resolution, revealing previously unknown transcriptional alterations that could contribute to disease pathogenesis, or lack of responsiveness to lenalidomide. KEY POINTS- Del(5q) and non-del(5q) CD34+ cells share similar transcriptional alterations, with del(5q) cells presenting additional lesions. - Hematological response to lenalidomide is associated with the reversal of some transcriptional lesions in del(5q) and non-del(5q) cells

Ascorbate (vitamin C) limits hematopoietic stem cell (HSC) function and suppresses leukemia development by promoting the function of the Tet2 tumor suppressor. In humans, ascorbate is obtained from the diet while in mice it is synthesized in the liver. In this study, we show that deletion of the Slc23a2 ascorbate transporter severely depleted ascorbate from hematopoietic cells. Slc23a2 deficiency increased HSC reconstituting potential and self-renewal potential upon transplantation into irradiated mice. Slc23a2 deficiency also increased the reconstituting and self-renewal potential of multipotent hematopoietic progenitors (MPPs), conferring the ability to long-term reconstitute irradiated mice. Slc23a2-deficient HSCs and MPPs divided much less frequently than control HSCs and MPPs. Increased self-renewal and reconstituting potential were observed particularly in quiescent Slc23a2-deficient HSCs and MPPs. The effect of Slc23a2 deficiency on MPP self-renewal was not mediated by reduced Tet2 function. Ascorbate thus regulates quiescence and restricts self-renewal potential in HSCs and MPPs such that ascorbate depletion confers MPPs with long-term self-renewal potential. KEY POINTSO_LIDeletion of the ascorbate transporter, Slc23a2, increases quiescence and self-renewal potential in HSCs and multipotent progenitors C_LIO_LIAscorbate depletion is sufficient to confer long-term self-renewal potential upon multipotent hematopoietic progenitors C_LI

Initial clinical trials with drugs targeting epigenetic modulators - such as bromodomain and extraterminal (BET) inhibitors - demonstrate modest results in acute myeloid leukemia (AML). The main reason for this involves an increased transcriptional plasticity within AML, which allows cells to escape the therapeutic pressure. To study mechanisms of resistance, we investigated immediate epigenetic and transcriptional responses following BET inhibition, and could demonstrate that BET inhibitor-mediated release of BRD4 from chromatin is accompanied by an acute compensatory feedback loop that attenuates inhibition, or even increases expression, of specific transcriptional modules. This adaptation is most marked at key AML maintenance genes and is mediated by p300, suggesting a rational therapeutic opportunity by combining BET- and p300- inhibition. p300 activity is required during all steps of adaptation. However, the transcriptional programs that p300 regulates to induce resistance to BETi differ between AML subtypes. Remarkably, in some AMLs, p300 regulates a series of transitional transcriptional patterns that allow homeostatic adjustments during earlier stages of resistance to BET-inhibitors. In consequence, p300 remains crucial throughout all stages of resistance in sensitive AML-subtypes, although its importance declines following the development of chronic resistance to BET inhibitors in some other AMLs. Altogether, our study elucidates the mechanisms that underlie an "acute" state of resistance to BET inhibition, achieved through p300 activity, and how these mechanisms remodel to become "chronic". Importantly, however, our data also suggest that a sequential treatment with BET- and p300 inhibition may prevent resistance development, thereby improving outcomes. Key pointsO_LIA mechanistic feedback to p300 enables acute tolerance to BET inhibition. C_LIO_LIp300 regulates transcriptional networks that lead to chronic resistance to BET inhibition. C_LIO_LISequential BET-, followed by p300-inhibition, is synthetically lethal in AML, and is optimally deployed during earlier stages of resistance to BET inhibitors. C_LI

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (lin-, Sca-1+, c-Kit+) cells which closely resemble MPP1 cells. HPCLSK reconstitute hematopoiesis in lethally irradiated recipient mice over more than eight months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD or MLL-AF9 their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSK can be applied to transgenic mice and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, Cdk6-/- HPCLSKs induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells. Key pointsO_LIWe describe the generation of murine cell lines (HPCLSK) which reliably mimic hematopoietic/leukemic progenitor cells. C_LIO_LICdk6-/- BCR/ABLp210 HPCLSKs uncover a novel role for CDK6 in homing. C_LI

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) originates from the bone marrow, which besides hematopoietic cells also contains different (supportive) cell types, including mesenchymal stromal cells (MSCs), osteocytes, chondrocytes, fibroblasts, and adipocytes. These (supportive) cell types create a bone marrow microenvironment that facilitates leukemogenesis and provide a survival benefit to leukemic cells that also affects the response to chemotherapeutic drugs. We here show that the survival benefit provided by supportive tissues does not depend on the type of supportive cells and does not differ between supportive cells collected at the time of full leukemia (diagnosis), at end of consolidation therapy or from healthy controls. The need for supportive cells, however, clearly differed between subtypes of BCP-ALL, with BCR-ABL1-positive and TCF3-PBX1-positive subtypes being the most dependent on this support. Various supportive cell types provided a survival benefit to BCP-ALL cells, with a median benefit of 18% (chondrocytes) and 30-36% (MSCs, osteocytes, and fibroblasts), which was not observed for mature adipocytes. This benefit was direct cell-cell contact dependent and decreased upon physical separation of cell populations in a transwell setting. BCP-ALL cells in contrast to MSCs and the other supportive tissue types hardly produce cyto-/chemokines. The secretome of MSCs changed upon co-culture with BCP-ALL cells resulting in 1.2-fold to 1.4-fold (median) higher levels for the cyto-/chemokines IL6, CCL22, CXCL10, and CXCL5. Together, these data suggest that BCP-ALL cells manipulate different components of the bone marrow supportive tissues via direct cell-cell contact which favors the survival of leukemic cells. This strengthens our earlier observation that BCP-ALL cells hijack the bone marrow microenvironment and offers the perspective that interference with this stromal interaction and/or released cyto-/chemokines may be of additive value in the treatment of BCP-ALL.