LSK Population Research Articles

Synergy of Stag2-Cohesin Loss Results in Expansion of Npm1c-Mutant Hematopoietic Stem and Progenitor Cells

STAG2 is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3D genome partitioning structure known as topologically structural domains (TADs). Our previous work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor population (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impaired sub-TADs and affects key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. Yet, the extent and cooperative transformational role of Stag2-cohesin with leukemia driver mutations, such as Npm1c, remains unexplored. To determine whether Stag2-cohesin regulates leukemic 3D chromatin organization, we generated dual Stag2 Δ Npm1 c/+ murine models with tamoxifen inducible UbcCreER T2. After 4 weeks, HSPCs (LSK cells; Lin-Sca1+Kit+) are increased in Stag2 Δ Npm1 c/+ double mutant mice ( Figure 1A). Within LSK, Stag2 Δ Npm1 c/+ but not Npm1 c/+ has marked expansion of the myeloid bias MPP2 (LSK+Flk2-Cd150+Cd48+) and MPP3 (LSK+Flk2-Cd150-Cd48+) compartment. Interestingly, Stag2 Δ single mutant exhibit LSK and MPP3 expansion at 16 weeks, suggesting the early expansion of HSPC compartment is the result of Stag2 ΔNpm1 c/+ synergistic acceleration. No difference in serial replating of LSK and GMP (LSK-Cd34+FcyRII+) cells were observed between Npm1 c/+ and Stag2 Δ Npm1 c/+. However, Stag2 Δ Npm1 c/+ LSK cells had impaired reconstitution capacity and myeloid biased output in primary and secondary transplantation. To determine if the blockage is specific to Stag2-cohesin loss, we generated the Smc3/Npm1c double mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in LSK or MPP3 population in Smc3 +/- Npm1 c/+ mice, suggesting it is specific to Stag2-cohesin loss which obstructs differentiation in Npm1 c/+ HSPC. Mechanistically, we performed bulk ATAC-seq on sorted HSC LT (LSK+Flk2-Cd150+Cd48-) and MPP3 population, as poor long-term reconstitution suggests functional alteration in HSC LT. Comparing to WT, both Stag2 Δ and Stag2 Δ Npm1 c/+ HSC LT have increased chromatin accessibility, while only Stag2 Δ Npm1 c/+ MPP3 have persistent opening of chromatin. Motif analysis of increased accessible peaks identified RUNX2, GATA3 and MEIS1 are among the increased accessible region in Stag2 Δ Npm1 c/+ MPP3, which reflects the increased myeloid output in the transplantation. Recently, various group have suggested that Npm1c directly binds to chromatin and drives the overexpression of genes, especially at HOX clusters and MEIS1. This suggests a possible synergistic mechanism which Stag2-cohesin regulates chromatin accessibility of key TF loci, which influences normal and leukemic stem and progenitor differentiation. To comprehensively profile the LSK population, we performed scRNAseq at 4 weeks post mutation activation, results showed a marked immune pathway activation in Stag2 Δ Npm1 c/+ LSK cells. To inter cell fate transition, we employed CellDancer, an RNA velocity based algorithm and found that Stag2 Δ Npm1 c/+ MPP cells have altered differentiation trajectory and delayed kinetics at MPP3 stages, suggesting a differentiation block ( Figure 1B). In conjunction with our results, we postulate that Stag2-cohesin regulates TF interactions with regulatory elements during cell fate transitions. We are currently investigating the direct interaction of Stag2-cohesin with regulatory elements in Npm1 c/+ cells and compare to WT and Stag2 Δ Npm1 c/+ cells. We will investigate the 3D genome organization of Npm1 c/+ and Stag2 Δ Npm1 c/+ cells to determine whether Stag2 loss induces the phenotypic changes via Ctcf-dependent or independent manner.

Compound Loss of Dnmt3a and Kmt2c in Myeloid Malignancies

Monosomy 7, or loss of 7q (-7/del(7q)), is one the most common chromosomal anomalies in myelodysplastic syndromes (MDS, ~10% of patients), a malignant disorder of the blood system with few treatment options. There are currently no targeted therapies available for -7/del(7q) MDS, and patients progress to a deadly acute myeloid leukemia (AML). The 7q segment is rich in genes that are implicated in MDS pathophysiology, including KMT2C encoding a histone H3 lysine 4 methyltransferase (depositing H3K4me1) that marks active gene enhancers during myeloid differentiation. Co-occurring DNA methyltransferase DNMT3A mutations are significantly overrepresented in -7/del(7q) MDS, especially in the absence of other chromosomal aberrations (23%). Individually, loss in DNMT3A activity in hematopoietic stem cells (HSCs) has shown bias towards self-renewal due to a failure to shut off stem-cell-related genes, whereas a loss of KMT2C leads to aberrant myelopoiesis. Generally, only one allele of KMT2C and DNMT3A is lost in MDS, suggesting that these haploinsufficient tumor suppressors may need to cooperate to drive disease. We hypothesized that disruption of Dnmt3a cooperated with loss of Kmt2c creating an aberrant epigenetic state that may accelerate development of MDS and progression to AML. To address this, we generated mice that lacked one copy of both Kmt2c and Dnmt3a in the bone marrow by conditional deletion with a hematopoietic-specific inducible Mx1-driven Cre recombinase. After inducing Cre-mediated recombination by poly(I:C) treatment creating Kmt2c(+/del):Dnmt3a(+/del) animals, the hematopoietic stem and progenitor compartment was subjected to comprehensive immunophenotypic and functional characterization. In serial competitive bone marrow repopulation assays, cells with combined heterozygous loss of both Dnmt3a and Kmt2c demonstrated increased competitive advantage in secondary transplants and retained ability to reconstitute tri-lineage hematopoiesis in tertiary transplants, unlike single gene deletion and wild-type controls. Consistently, immunophenotypic analysis of the bone marrow compartment 16 weeks after the third transplant demonstrated persistence of the primitive long-term hematopoietic stem cells (LT-HSCs) defined as Lineage -Sca1 +cKit + (LSK) CD150 +CD48 -, which were absent in other genotypes. Further, we found expansion of the LT-HSCs in aged 80-week old Kmt2c(+/del):Dnmt3a(+/del) animals that were also characterized by a gain in self-renewal as seen by their ability to serially replate in semisolid media (MethoCult GM3434). To test if combined loss of Kmt2c and Dnmt3a led to disease development, next we established a large cohort of animals non-competitively transplanted with Kmt2c(+/del):Dnmt3a(+/del), single gene deletion, and wild-type control bone marrow, and initiated a tumor watch by monthly monitoring the composition of their peripheral blood by CBCs and flow cytometry. Sixty weeks after transplantation, over a half of double knock-out animals developed myeloid disease characterized by neutrophilia and/or monocytosis accompanied by anemia, occasional thrombocytopenia, and splenomegaly most consistent with myelodysplasia. Immunophenotypically, we found expanded LSK populations (LT-HSCs specifically) in the bone marrow and evidence of extramedullary hematopoiesis in the spleen. At the same time, the majority of mice with single gene deletion and all wild-type controls remained healthy. Overall, our studies demonstrate that combined loss of Kmt2c and Dnmt3a results in the development of myelodysplastic disease with long latency through endowing the HSCs with enhanced self-renewal capacity. We are currently interrogating the epigenetic profiles and gene expression patterns resulting from combined disruption of Dnmt3a and Kmt2c aiming to identify gene targets and signaling networks for future therapeutic intervention. Better understanding of the molecular pathogenesis of the -7/del(7q) MDS with concurrent DNMT3A mutations is critical to develop more effective treatment approaches and to improve outcomes for MDS patients.

TMI-Based Dose-Escalated Bone Marrow Transplantation Can Help Preserve the Bone Marrow Microenvironment and Reduce Cellular Senescence in Old Mice

Introduction:Senescence is one of the hallmarks of aging and age-related conditions, including but not limited to, inflammaging, frailty, and various age-related diseases. It has been well-established that radiation and chemotherapy can accelerate senescence. While myeloablative conditioning-based allogeneic hematopoietic cell transplant (HCT) is a curative treatment for patients with leukemia. The dose of total body irradiation (TBI) conditioning has to be reduced in older patients to reduce potential high treatment-related mortality, which may still cause accelerated senescence in aged individuals, resulting in poor survival outcomes. We initiated the testing of a novel total marrow irradiation (TMI) based myeloablative conditioning in older patients to enhance the anti-leukemia effect and overall survival. We hypothesize that this new radiation preconditioning strategy may also reduce radiation-induced senescence in aged individuals compared to TBI to reduce treatment-associated side effects and improve the outcomes of allogeneic HCT. We tested this hypothesis in the present study and further investigated the mechanisms of the effects of TMI-based myeloablative and dose-escalated bone marrow transplantation (BMT) on old mice by examining the changes in expression of various senescence markers and senescence-associated secretory phenotype (SASP) in the bone marrow (BM) and selected organs. Method: The radiation treatment regimens were: TMI (12:4), TMI (16:4) and TBI (12:12), and TBI (16:16), were developed as described (Zuro et al., 2021); i.e. (12:4) means the radiation dose to the BM was 12 Gy and the dose to the organs was 4 Gy. The young (8 weeks old) and aged mice (90 weeks old) of host C57BL/6j (JAX000664, CD45.2) were irradiated in 2 fractions 24h apart, and 25 million whole BM cells from donor (JAX002014, CD45.1) were transplanted by i.v. at 24 h after irradiation. For the hematopoietic stem and progenitor cell (HSPC) population study, more mature hematopoietic progenitors (Lin − Sca-1 −c-Kit + or LK cells) and HSPCs (Lin − Sca-1 + c-Kit + or LSK cells) were examined in BM by flow analysis. Cultured bone marrow mesenchymal stem cells (MSCs) were analyzed for the gene expression of the senescence marker CDNK1a (or p21) and SASP factors MMP13 and CXCL12 by qPCR. For senescence study in organs such as the liver, gut, and spleen, senescence-associated beta-galactosidase (SA-β-gal) staining was performed and expression of the CDNK2a (or p16) was analyzed by qPCR. Results: TMI or TBI treatment with young donor BMT repopulated the BM HSPC population without significant bone damage in aged mice (data not shown). While the BM LK and LSK populations significantly reduced in aged mice after TMI or TBI with young donor BMT compared to aged control mice ( Fig 1A), these populations were not different from young mice groups ( Fig 1B). In addition, TMI or TBI treatment with young donor BMT reduced MSC senescence. Cultured MSCs analysis showed the reduction of p21, MMP13, and CXCL12 expression ( Fig 1C-E) after TMI or TBI with young donor BMT in aged compared to aged control without radiation and BMT. However, while TMI treatment and BMT reduced senescence in the organs, mice receiving TBI treatment and BMT showed a higher senescence burden. In the liver, the aged control mice showed a significant increase in SA-β-gal staining, which was decreased in TMI (12:4) and TMI (16:4) mice ( Fig 1F). In contrast, this effect was not observed in aged mice after TBI (12:12) or (16:16) ( Fig 1G). Moreover, the senescence marker p16 expression in the liver and gut was reduced only in TMI (12:4) and TMI (16:4), not in TBI (12:12) or (16:16) ( Fig 1H, I), whereas the p16 expression in the hematopoietic tissue, spleen was decreased in all treatment groups TMI vs. TBI ( Fig 1J). Conclusion: TMI-based dose escalates with young donor BMT in aged mice preserved BM microenvironment and reduced senescence of organs while TBI treatment causes senescence of the organs. Young donor BMT, which combined with TMI or TBI treatment, preserved bone structure and reduced senescence of HSPC and MSC. However, TMI treatment decreased the SA-β-gal staining and p16 expression significantly in organs such as the liver and gut, but not by TBI treatment. This study showed the senescence reduction in both bone marrow and organs after young donor BMT and TMI treatment. For further study, we have a plan to study the mechanism and signaling pathway of senescence reduction.

Adipocytes control hematopoiesis and inflammation through CD40 signaling.

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with aging. Although CD40 is mainly expressed by immune cells, CD40 is also present on adipocytes. We aimed to delineate the role of adipocyte CD40 in the aging hematopoietic system and evaluated the effects of adipocyte CD40 deficiency on cardiometabolic diseases. Adult adipocyte CD40-deficient mice (AdiCD40KO) mice had a decrease in bone marrow hematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased bone marrow adiposity and T-cell activation, along with elevated plasma corticosterone levels, a phenotype that became more pronounced with age. Atherosclerotic AdiCD40koApoE-/- (CD40AKO) mice also displayed changes in the LSK population, showing increased myeloid and lymphoid multipotent progenitors, and augmented corticosterone levels. Increased T-cell activation could be observed in bone marrow, spleen, and adipose tissue, while the numbers of B cells were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T cells and larger necrotic cores. Analysis of peripheral adipose tissue in a diet-induced model of obesity revealed that obese AdiCD40KO mice had increased T-cell activation in adipose tissue and lymphoid organs, but decreased weight gain and improved insulin sensitivity, along with increased fat oxidation. In conclusion, adipocyte CD40 plays an important role in maintaining immune cell homeostasis in bone marrow during aging and chronic inflammatory diseases, particularly of the lymphoid populations. Although adipocyte CD40 deficiency reduces atherosclerosis burden and ameliorates diet-induced obesity, the accompanying T-cell activation may eventually aggravate cardiometabolic diseases.

Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

Inhibition of MDM2 Improves the Therapeutic Effect of Hypomethylating Agents in Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML)

Hypomethylating agents (HMA) are the current standard care for MDS and CMML. However, a large proportion of patients experience HMA treatment failure that is associated with poor prognosis. Novel treatments are urgently needed for these diseases. We performed next generation sequencing (NGS) based mutation profiling that targeted the 81 most frequently mutated genes in baseline bone marrow (BM) mononuclear cells (MNCs) from patients with MDS and CMML (N=83) prior to HMA treatment. Analysis of impact of the mutations on outcomes of HMA treatment indicated that patients with TP53 mutations (N=22) had significantly worse overall survival (HR = 3.29; P < 0.05; q < 0.05). This result is consistent with previous reports and suggests that strengthening the activity of wild type (WT) TP53, through strategies such as inhibition of TP53 negative regulator MDM2, may improve therapeutic efficacy of HMAs in patients with MDS and CMML.To test this hypothesis, we evaluated in vitro the effects of the combination of the MDM2 inhibitor DS-3032b and azacytidine (AZA). Due to the dearth of MDS or CMML cell lines, initial analysis was performed in the TP53 WT AML cell line MOLM13. Results indicated that DS-3032b and AZA combination induced a synergistic decrease in cell survival (coefficient of drug interaction=0.65), accompanied with increased apoptosis and CDKN1A1 (P21) RNA expression. No combination survival effect was observed in the TP53 mutant AML cell lines SKM1 or TF1.We next evaluated in vivo combination therapeutic effects of the MDM2 inhibitor DS-5272 and AZA in a Tet2-knockout (KO) (Tet2flox/flox/Vav-Cre) mouse model. Prior work indicated that Tet2-KO mice had an MDS/CMML-like phenotype and a persistent long-term (LT) BM repopulating activity that was resistant to AZA. DS-5272 and AZA were administered to mice sequentially: DS-5272 (50 mg/kg) was administered orally for 5 days, followed by daily AZA (2.5 mg/kg, i.p.) for a 7 days. DS-5272 was also administered on the 1st and 3rd days during AZA treatment. After a 2-week gap, the treatment cycle was repeated once. Following the combination treatment, Tet2-KO mice showed significantly reduced monocytosis (P<0.05) and increased platelet counts (P<0.001) in peripheral blood (PB), and significant reductions in Gr1+/CD11b1+ myeloid cells (P<0.001), common myeloid progenitors (CMPs) (P<0.005), and Lin-/Sca1+/c-Kit1+ cells (LSKs) (P<0.01) in BM. Next, we evaluated the impact of treatment on LT BM repopulation activity. CD45.2+ BM cells isolated from drug-treated Tet2-KO mice were mixed with BM cells from CD45.1 WT mice and then transplanted into lethally irradiated CD45.1 WT recipient mice. Significantly lower (P<0.05) CD45.2 chimerism was observed in PB of the recipients transplanted by BM cells from DS-5272 and AZA combination treated Tet2-KO mice compared to recipients that received cells from vehicle control and mono-agent treated Tet2-KO mice, starting from 4 months post-transplantation (Fig.1A). Lower CD45.2 chimerism was also observed in the BM LSK population (P<0.001) of the same recipients at the endpoint of 6 months post-transplantation.To study the molecular mechanisms underlying the combinational therapeutic effects of the MDM2 inhibitor and AZA observed in the Tet2-KO mice, we performed RNA-seq on BM LSK cells isolated from drug-treated mice. Compared to LSKs from vehicle control mice, over 800 genes with significantly altered RNA expression (>1.5 fold; FDR<0.05) were identified in the LSKs from DS-5272 and AZA combination treated Tet2-KO mice, whereas 110 and 476 genes with altered RNA expression were identified in the LSKs of DS-5272 or AZA treated Tet2-KO mice respectively. REACTOME pathway analysis identified 263 pathways that were significantly (FDR<0.25) and uniquely activated in the BM LSKs from mice treated with DS-5272 and AZA combination. Many of these pathways were associated with TP53 stability, TP53 activity, and TP53 regulated apoptosis and cell cycle (Fig.1B), suggesting a coordination between MDM2 inhibition and HMA treatment to activate TP53 in BM HSPCs.In conclusion, our work provides proof-of-concept evidence that combining an MDM2 inhibitor with AZA can improve the therapeutic efficacy of AZA in MDS and CMML, through mechanisms including synergistic activation of TP53 in BM HSPCs and inhibition of LT BM repopulating activity. This concept should be further evaluated through pre-clinical studies and clinical trials. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Daver: Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Hanmi: Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Genentech: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding.

Activation of p53 Enhances Antileukemia Activity of Bcr-Abl Tyrosine Kinase Inhibitors in a Murine Model of CML

Although CML patients under Bcr-Abl tyrosine kinase inhibitor (TKI) therapy have experienced high response rates and long-time survival, 50% or more patients relapse upon therapy cessation, largely because TKIs do not eliminate CML stem cells. We previously reported that inhibition of antiapoptotic Bcl-2 proteins synergized with TKIs to eradicate CML stem/progenitor cells. The tumor suppressor p53 induces apoptosis in part by modulating Bcl-2 family proteins. Although rarely mutated in CML, p53 activity is antagonized by Bcr-Abl tyrosine kinase-regulated MDM2. We hypothesize that activation of p53 by MDM2 inhibition will synergize with TKIs to eliminate CML cells. Using an inducible transgenic Scl-tTa- BCR-ABL mouse model, we first determined the expression of p53 and its target genes in bone marrow (BM) cells of Tet-on (control) and Tet-off (CML) mice. We found markedly increased RNA levels of p53 and its targets Bax, Noxa, and MDM2 in CML as compared to control mice. Using CyTOF mass cytometry and cytofkid automatic subset detection/clustering based on the expression of cell surface markers, we then determined p53 and its target proteins in bulk and Lin-Sca-1+cKit+ (LSK) populations at single cell levels and found overexpression of p53 and its targets such as Noxa in CML BM cells (CD45+) compared to controls. The differential expression was most pronounced in the LSK populations. For both control and CML mice, LSK cells expressed significantly higher p53 protein than their respective bulk cell population. We next evaluated the sensitivity of these cells to BH3 peptide priming. CML BM cells and more so LSK populations were more sensitive to BH3 peptides, especially to Noxa peptide than cells from control mice (P=0.044 in CD45+ and P=0.0005 in LSK populations) suggesting that CML cells are more primed to apoptosis stimuli. Finally, we tested the antileukemia activity of combined MDM2 and Bcr-Abl inhibition in mice engrafted with BM cells from Scl-tTa- BCR-ABL /GFP mice. Combined inhibition of Bcr-Abl tyrosine kinase (imatinib) and MDM2 (DS-5272) strongly reduced BM leukemic populations and LSK cells, increased Noxa, decreased leukemia burden, and significantly prolonged survival (P=0.0021 vs. control). Median survival days for control, imatinib, DS-5272, and the combination groups were 85, 110, 155, and 248; respectively.Our results demonstrate that CML cells/stem cells have increased p53 signaling and are highly primed to undergo apoptosis. Combined p53 activation and Bcr-Abl tyrosine kinase inhibition targets CML stem/progenitor cells and has the potential to significantly improve response and cure rates of CML. DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding. Seki:Daiichi Sankyo: Employment. Andreeff:Daiichi Sankyo: Consultancy.

MiR-199b-5p Differentially Modulates Hematopoietic Stem Cell Self Renewal in Steady State and Stress Hematopoiesis

The emergence of microRNAs (miRNAs) is providing novel insights into the molecular control networks underlying hematopoiesis and diseases that stem from aberrations in hematopoiesis. We, amongst others, have shown that miR-199b is significantly downregulated in acute myeloid leukemia (AML) and is a promising prognostic factor for FAB-M5 subtype. Herein, to determine miR-199b's role in steady state and stress hematopoiesis, we utilized our recently generated miR-199b knockout (KO) mice (CRISPR approach) to assess frequencies of hematopoietic stem progenitor cells (HSPCs) in WT and miR-199b KO mice through flow cytometry. Under steady state conditions, deletion of miR-199b resulted in significant reduction of Lin- Sca-1+ c-Kit+ (LSK), long-term hematopoietic stem cells (LT-HSCs) (Lin− CD117+ Sca1+ CD150+ CD34low/− CD48−) and increases in multipotent progenitors (MPPs) (Lin− CD117+ Sca1+ CD150− CD34+). However, 5FU induced stress hematopoiesis resulted in significant increases in frequencies of LSK cells, LT-HSCs, and Lin- Sca-1- c-Kit+ (KL) cells compared to wild-type (WT) litter mates. Furthermore, silencing of miR-199b resulted in a significant increase of granulocyte monocyte progenitors (GMP), which was reversed during stress hematopoiesis. The highest expression levels of miR-199b were restricted to the LT-HSC fraction. Mir-199b levels remained moderately high in MPPs and LSK cells. Primarily within the LSK population, miR-199b expression was most pronounced in LT-HSCs and to a lesser extent, MPPs, indicating that miR-199b may play a functional role in LT-HSC activity. Gene profiling studies in HSPCs identified Integrin alpha 8 (Itga8) as a conserved target of miR-199b. We then examined Itga8 transcript levels in HSCs from miR-199b knockout (-/-) mice and found significant elevation of Itga8 transcripts compared to WT mice. One possible mechanism for the differential expansion of miR-199b-/- HSPCs under steady state and stress is an increased or decreased rate of cell cycling. To test this, we examined cell proliferation using Ki67 and Hoechst 33342 markers. MiR-199b-/- LT-HSC at steady state showed significantly decreased proliferation whereas miR-199b-/- LT-HSCs under 5FU induced stress showed significant expansion when compared to wild-type. Next, we tested the adhesion abilities of LT-HSCs from miR-199b KO mice under both steady state and stress hematopoiesis. At steady state, miR-199b-/- LT-HSCs significantly elevated adhesion to vitronectin and fibronectin. Interestingly, like HSPC frequencies, stress hematopoietic-derived miR-199b-/- LT-HSCs showed significantly decreased adhesion to both vitronectin and fibronectin. These findings imply that miR-199b intrinsically regulates HSC behavior, including cell adhesion. To elucidate whether miR-199b's effects on LT-HSCs are mediated by Itga8, we performed an Itga8 knockdown in WT and miR-199b-/- LT-HSCs. Cell proliferation assessed by Ki67 positive cells revealed that downregulation of Itga8 in miR-199b KO LT-HSCs reverts to a miR-199b-induced decrease of LT-HSC proliferation. Intriguingly, we observed that silencing of Itga8 reversed the miR-199b-induced increase of cell adhesion. To corroborate that miR-199b-/- LT-HSCs are fully functional, we performed a competitive repopulation assay. BM derived lineage negative hematopoietic progenitor cells were further purified via immunomagnetic selection for SLAM marker purified (CD150+, CD244+ and CD48-) to enrich the LT-HSC fraction. MiR-199b-/- or miR-199b+/+ LT-HSCs (n=100 LT-HSCs) were transplanted intravenously together with 2.5 X 105 competitor recipient cells. Overall donor BM cellularity was markedly increased in LT-HSCs derived from 5FU treated miR-199 KO mice and overall donor BM cellularity was markedly decreased in LT-HSCs derived from steady state miR-199 KO mice. This suggested that under stress, miR-199b-/- LT-HSCs self-renew extensively after transplantation to repopulate the recipient. The analyses of HSPCs in BM of primary recipients revealed a significant increase in donor LT-HSCs in recipients that were transplanted with LT-HSCs derived from the 5FU induced stress model. Intriguingly, this didn't translate into subsequent increases in MPP, GMP or MEPs, suggesting that miR-199b may govern the self renewal of LT-HSCs and also have progenitor cell specific roles as evident from its expression during early hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epigenetic and Single Cell Analyses Reveal Dual Fate Decision Points in Pre-Primed Hematopoietic Stem Cells during Erythro-Megakaryopoiesis

Recent single cell analyses have shown that hematopoietic differentiation is more complex than the traditional hierarchical model suggests, and that lineage commitment may occur earlier in the hierarchy than previously thought (Notta, 2016; Psaila, 2017). In particular, the relationship between bipotential megakaryocyte-erythroid progenitors (MEP) and common myeloid progenitors (CMP) is unclear in erythro-megakaryopoiesis. While it has been established that mouse MEP and CMP populations contain different proportions of megakaryocytic and erythroid lineage-committed cells, our aim was to determine when lineage-commitment occurs and whether a ‘default’ differentiation pathway exists.We established the transcriptional and epigenetic profiles of 14 flow cytometry-enriched primary murine hematopoietic populations, including hematopoietic stem/progenitor cells (HSPC) and lineage-committed populations. We performed transcriptome profiling (RNASeq), chromatin accessibility profiling (Assay for Transposable-Accessible Chromatin; ATACSeq), and DNA methylation profiling (MethylCSeq). We also performed ChIPSeq of H3K27ac on erythroblasts (EB) and immature megakaryocytes (Mk) to identify putative active enhancer elements.Principle component analysis (PCA) of the RNASeq data demonstrated variation between lineages. Primary cells and cell lines were separated by PC1 (20% of variance) while PC2 (14% of variance) separated HSPC and Mk-committed cells from EB, monocytes and granulocytes. Likewise, PCA of ATACSeq data also indicated that HSPC and Mk shared more similarity than other populations: PC1 (19% of variance) separated EB and non-EB populations while PC2 (12% of variance) separated HSPC and Mk-committed cells from EB, monocytes, granulocytes and lymphocytes (Figure 1). These data demonstrate that the transcriptional and chromatin accessibility profiles of Mk are more similar to HSPC than to other populations.We defined putative active enhancers (pAE) as the intersection of ATACSeq and H3K27ac ChIPSeq peaks in EB and Mk. We identified 2098 EB- and 6386 Mk-specific pAE, and 5989 shared pAE. To predict pAE in progenitors we overlaid EB and Mk pAE with successive ATACSeq peaks in lineage-restricted progenitors (CFU-E, CFU-Mk), followed by CMP and the Lin-Sca+Kit+ population (LSK). 97% and 89% of Mk pAE were detected in CMP and LSK, whereas only 84% and 64% of EB pAE were detected. To validate which EB and Mk pAE in LSK were active (as opposed to poised), we compared our LSK pAE with the indexing-first ChIP histone marks identified by Lara-Astiaso et al., (Science , 2014). We found that 31% of Mk-, but only 13% of EB pAE, were active in the LSK. These data indicate that the MK enhancer profile is established earlier than that of EB.Our comparison of the DNA methylation (CH3) profiles showed that, unlike the RNASeq, ATACSeq, and H3K27ac profiles, erythropoiesis was associated with an 87% decrease in CH3 from CMP and LSK sets; 15% of EB CH3 peaks were established de novo. In contrast, megakaryopoiesis was associated with a 33% increase in CH3 peaks from CMP and LSK sets; 50% of Mk CH3 peaks were established de novo. Many of these de novo methylation sites were associated with genes directing non-Mk lineages that are downregulated compared to CMP (e.g., Dach2, Sox5, Kit, Klf1, Tet1, Fog1).To determine when lineage-commitment occurs we performed single cell RNASeq (10X Genomics platform) on ~9000 cells from each of LSK, CMP, MEP, and GMP (granulocyte-macrophage progenitors) populations. The Seurat software (Sajita, 2015) was used to ‘cluster’ cells with similar expression profiles. MEP contained 5 erythroid clusters of different maturation stages (57% of cells), and 1 cluster with an Mk gene signature (5% of cells). CMP contained, among others, 8 Mk clusters (60% of cells) and 1 cluster (2% of cells) co-expressing Mk and EB genes. LSK contained 4 Mk clusters (26% of cells) but no populations expressing EB-associated genes.We conclude that the transcriptional and epigenetic Mk profile is detectable at the earliest stages of hematopoiesis. The requirement of de novo methylation and silencing of other lineage-associated factors prevents the classification of Mk as a default pathway. In contrast, the EB profile is established after most megakaryocyte progenitor cells have become committed and extensive transcriptional and epigenetic reprogramming is required to permit erythropoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enhanced Free Fatty Acid Uptake Via CD36 Promotes a Metabolic Switch to B-Oxidation within Hematopoietic Stem Cells in Response to Acute Infection

Acute infection is known to promote rapid differentiation of hematopoietic stem cells (HSC) and expansion of leukocytes. Metabolic changes in the HSC underpin the mammalian response to pathogenic stimuli however, knowledge of how this occurs is not fully understood. Here we investigate the immunometabolic changes which facilitate HSC expansion in response to acute infection. We created a novel in vivo transplant model to understand if the immune response to infection involves the acquisition of free fatty acids (FFA) by HSCs in the bone marrow (BM). We transduced CD45.1 lineage negative, c-kit positive cells with firefly luciferase and transplanted into CD45.2 animals. Post engraftment animals were treated with lipopolysaccharide (LPS). Live animal imaging confirmed activation of luciferase in the BM, demonstrating in vivo, uptake of long chain FFA by haematopoietic cells in response to LPS. To confirm the specific haematopoietic stem and/or progenitor cells (HSPC) with increased lipids during infection, mice were treated with S.typhimurium for 72h then sacrificed Analysis of LSK, MPP and HSC populations (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) showed increased intracellular BODIPY 493/503 neutral lipid dye and uptake of Bodipy FL-C12 (FFA linked to bodipy) at 72 h compared to control non-infected animals. Together, these experiments show that HSC, multipotent progenitor (MPP) and LSK (LN-, CD117+, Sca1+) cells all acquire FFA in response to bacterial infection. Seahorse mitochondrial stress test confirmed increased oxygen consumption levels in HSC from LPS (16 hours) and S.typhimurium (72 hours) treated C57BL/6. LSK metabolic activity for different substrates was assessed LSKs from LPS treated animals had an increased dependency on β-oxidation when compared to control cells. Moreover, the β-oxidation inhibitor etomoxir blocks LSK basal metabolism and reduced HSC expansion as measured by Ki-67 staining in LPS and S.typhimurium treated animals. To understand the importance of β-oxidation in HSC expansion in response to infection we first measured CPT1A expression in HSC. RNA analysis confirmed CPT1A was increased in sorted HSCs in response to LPS. Next we transplanted WTor CPT1A KD cells into WT animals, and treated with LPS. HSC expansion as measured by Ki-67 staining was inhibited in the CPT1A KD cells in response to LPS, thus confirming the importance of β-oxidation based metabolism in HSC expansion. To determine how FFA are transported into the HSC we analysed the expression of known genes to be involved in lipid uptake. HSC sorted from LPS and S.typhimurium treated animals had increased CD36 mRNA expression compared to control. C57BL/6 mice were pre-treated with the CD36 inhibitor sulfosuccinimidyl oleate (SSO) before injection with LPS. HSCs had had less lipids, lower maximal respiration and reduced cycling when compared to animals treated with LPS alone. In a similar way CD36 KO animals were treated with LPS for 16 h, no increased FFA uptake or increased lipid content or HSC cycling was observed. Seahorse mitochondrial stress test confirmed CD36-/- LSK have increased basal ECAR but no change in basal OCR in response to S.typhimurium infection. Moreover, CD36-/- LSK have reduced dependency on β-oxidation. To determine if the uptake of FFA response is specific to HSC we transplanted CD36+/+ (CD45.1) LK into CD36-/- (CD45.2) animals. We found that CD36 expression was elevated in CD36+/+ HSC transplanted into CD36-/- treated with LPS, normal uptake of FFA, an increase in lipid content and cycling of HSCs. In addition, transplanted WT CD36+/+ (CD45.1) LSK into CD36-/- (CD45.2) animals reversed the metabolic phenotype observed in CD36-/- in response to LPS. We transplanted WT(CD36+/+) or CD36KO (WT(CD36-/-)) LSK into WT animals infected with S. typhimurium for 4 days. WT(CD36-/-) transplanted animals showed enhanced mortality, increased weight loss and increased liver injury. We also observed less HSC cycling and a reduced lipid content in HSC from WT(CD36-/-) compared to WT(CD36+/+). These findings expand the mechanistic understanding of the interplay between HSC and the bone marrow microenvironment, and in doing so explains how immunometabolic reprogramming of HSCs during infection supports the metabolic demands of HSCs in response to a pathogenic challenge without which results in increased susceptibility to infection. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:AbbVie: Research Funding; Janssen: Research Funding.

P2Y12-dependent regulation of emergency hematopoiesis after myocardial infarction

Abstract Introduction Inflammation is essential for wound healing after myocardial infarction (MI). Leukocytes, especially neutrophils and monocytes, orchestrate removal of necrosis and regulation of tissue remodeling. Beside local recruitment from the blood, leukocyte supply via increased hematopoiesis is of major relevance for post-ischemic myocardial inflammation. Little is known about the pathways that carry the signals for increased demand for leukocytes from the site of injury to upstream hematopoietic stem cells (LSK) in the bone marrow (BM). In this study, we investigate the role of the P2Y12-ADP-receptor mediated regulation for emergency hematopoiesis after MI. Methods For standardized MI, a model of permanent coronary ligation was used in C57/Bl6 and P2Y12−/− mice. Changes in plasmatic ADP levels after MI were screened using ELISA, whereas the expression of the P2Y12-ADP-receptor in cell populations isolated from the BM was investigated by qPCR. CFU assays added further functional insight on hematopoietic proliferation in vitro. The effect of P2Y12 on the hematopoietic system after MI was investigated by inhibiting the P2Y12-receptor via prasugrel and compared to inhibition of the thromboxane pathway via acetylsalicylic acid (ASA). Proliferation of LSK in BM and leukocyte composition in blood, BM and infarct tissue after MI were assessed via FACS. Leukocyte composition in the infarcted myocardium was validated by immunohistochemistry. Finally, left ventricular function (LV-EF) and remodeling were investigated by echocardiography. Results 24 h after MI, we found a peak of plasmatic ADP levels. LSK as upstream hematopoietic progenitors in the BM express a P2Y12-receptor, which was validated on transcriptional and protein level. Whereas ADP stimulation of LSK led to significantly larger colony growth in vitro on the one hand, percentage of cycling LSK were significantly reduced 48 h after MI in P2Y12−/− mice compared to WT mice, assessed by Ki67/DAPI cell cycle analysis. Prasugrel treatment showed similar effects, translating into reduced numbers of downstream hematopoietic progenitors GMP and MDP 72 h after MI. Treatment with ASA however had no significant effect neither on cycling LSK nor progenitor populations. Consequently, decreased medullary hematopoiesis under P2Y12-inhibition led to reduced infiltration of inflammatory cells in the infarct tissue 7 days after MI, finally resulting in significantly improved outcome in terms of LV-EF 3 weeks after MI. Conclusion In this study, we demonstrate that P2Y12-mediated signaling is involved in emergency hematopoiesis after MI and advocates post-MI inflammation. In turn, inhibition of P2Y12-mediated signaling contributes to improved wound healing and prevention of adverse cardiac remodeling after MI, which adds a yet unknown mechanism to the success story of modern P2Y12-receptor blockers. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Bacteria-Induced Acute Inflammation Does Not Reduce the Long-Term Reconstitution Capacity of Bone Marrow Hematopoietic Stem Cells.

Pathogen-initiated chronic inflammation or autoimmune diseases accelerate proliferation and promote differentiation of hematopoietic stem cells (HSCs) but simultaneously reduce reconstitution capacity. Nevertheless, the effect of acute infection and inflammation on functional HSCs is still largely unknown. Here we found that acute infection elicited by heat-inactivated Escherichia coli (HIEC) expanded bone marrow lineage-negative (Lin)− stem-cell antigen 1 (Sca-1)+cKit+ (LSK) cell population, leading to reduced frequency of functional HSCs in LSK population. However, the total number of BM phenotypic HSCs (Flk2−CD48−CD150+ LSK cells) was not altered in HIEC-challenged mice. Additionally, the reconstitution capacity of the total BM between infected and uninfected mice was similar by both the competitive repopulation assay and measurement of functional HSCs by limiting dilution. Thus, occasionally occurring acute inflammation, which is critical for host defenses, is unlikely to affect HSC self-renewal and maintenance of long-term reconstitution capacity. During acute bacterial infection and inflammation, the hematopoietic system can replenish hematopoietic cells consumed in the innate inflammatory response by accelerating hematopoietic stem and progenitor cell proliferation, but preserving functional HSCs in the BM.

Free Fatty Acid Uptake By Hematopoietic Stem and Progenitor Cells Drives Immune Cell Expansion in Response to Salmonella Typhimurium infection

Haematopoietic stem and progenitor cells (HSPC) are tightly regulated in a balance between self-renewal and differentiation and undergo rapid expansion in response to infection. In the context of acute bacterial infection, the hematopoietic system needs energy to drive the immune response necessary for host survival. In an adult human, bone marrow adipose tissue accounts for up to 70% of bone marrow and we have previously shown that free fatty acids are transferred from adipocytes to AML blasts providing the AML blasts more energy for proliferation and survival (Shafat, Blood 2017). Therefore, we hypothesis that during an acute bacterial infection, the HSPC population uptakes free fatty acids (FFA) to support and sustain the rapid response to bacterial infection. To determine if HSPC acquired FFA we infected C57BL/6 with Salmonella Typhimurium (S.typhimurium) or treated C57BL/6 with lipopolysaccharide (LPS) for 2 and 16 hours. Using both models we show that the HSPC populations (HSC (haematopoietic stem cell), MPP (multipotent progenitor) and LSK (LN-, CD117+, Sca1+)) all accumulate FFA as measured by staining with neutral lipid-specific BODIPY 493/503 dye. To determine if this was uptake or accumulation of fatty acid within the HSPC of animals infected with S.typhimurium we used BODIPY®-dodecanoic acid fluorescent fatty acid to trace FFA uptake. HSPC populations from animals treated with LPS had a higher uptake of BODIPY®-dodecanoic acid when compared to the control treated mice. To assess metabolic changes within HSC population in response to infection, cells were sorted (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) and analysed for gene expression changes. In HSC, increases were observed in several metabolic genes including FABP4, CD36 and CPT1 in response to S.typhimurium as well as LPS at 16 hours post stimulation. Moreover, following cell sorting, the LSK (LN-, CD117+, Sca1+) population were assessed for metabolic activity for different substrates. The LSK population from LPS treated animals had an increased dependency on beta oxidation when compared to the control. Moreover, etoximir (a beta-oxidation inhibitor) inhibited LSK basal metabolism and reduced LSK expansion (as measured by colony forming cell assay and Ki-67 staining) in animals treated with LPS. Our data indicates that infection drives uptake of FFA in different HSPC populations. The FFA are metabolised by beta oxidation which provides the energy for the expansion of leukocytes. Disclosures Bowles: Abbvie: Research Funding; Janssen: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Functional Complementation between β-Catenin and Hoxa9 Sustains HSC Self-Renewal in a Prmt1-Dependent Manner

Self-renewal is a fundamental property essential for both normal and malignant hematopoietic stem cells (HSCs). While we and others have previously shown that activation of Hoxa9 or β-catenin enhances HSC self-renewal, its inactivation has modest impacts on adult HSCs and their malignant counterparts (Cobas et al., 2004; Lawrence et al., 2005; Siriboonpiputtana et al., 2017; Smith et al., 2011; So et al., 2004; Zhao et al., 2007), suggesting the presence of complementary pathways capable of mediating self-renewal in the absence of either protein. However, simultaneous inactivation of other key Hox genes involved in hematopoietic self-renewal including Hoxb3/b4 did not yield additional hematopoietic phenotypes in the Hoxa9 knockout (KO) background (Magnusson et al., 2007). Similarly, suppression of γ-catenin in β-catenin KO mice did not result in additional hematopoietic defects, and exhibited largely normal hematopoietic functions (Koch et al., 2008). Therefore, the alternative pathways that support hematopoietic self-renewal upon inactivation of Hoxa9 or β-catenin remain largely unknown. Our recent studies examining the regulation of posterior Hoxa loci reveals a key function of long non-coding RNA, HOTTIP in protecting Hoxa9 gene expression and uncovers a co-regulation of canonical Wnt signalling pathways by HOTTIP in HSCs, providing a molecular link between these two previously unrelated pathways. This finding is also consistent with our recent report demonstrating their functional complementation in AML stem cells, where suppression of Hoxa9 sensitizes HSC-derived AML stem cells to β-catenin inhibition and ablates their transformation ability, suggesting a novel crosstalk between β-catenin and Hoxa9in mediating hematopoietic self-renewal. To this end, the current study developed and characterized a novel β-cateninfl/fl Hoxa9-/-Rosa-CreER mouse model. In contrast to single Hoxa9 or β-catenin inactivation where a mild hematopoietic phenotype has been reported in adult HSCs, we found that double inactivation of Hoxa9 and β-catenin resulted in severe hematopoietic defects. In vitroclonogenic assays revealed that bone marrow cells harbouring combined inactivation of Hoxa9/β-catenin generated markedly reduced myeloid colony numbers (&amp;gt;9-fold reduction compared to either single KO alone) of which mature CFU-G (50%) and CFU-E (50%) were the predominant composition. Colonies generated from isolated LSK (Lin-c-kit+Sca1+) populations were equivalent in quantity but devoid of multipotential progenitors (CFU-GEMM) in contrast to the diverse colony composition of control and single KO cells. Transplantation of β-cateninfl/flHoxa9-/-bone marrow to lethally irradiated recipient mice followed by β-catenin inactivation (4 days tamoxifen treatment) resulted in defects in all HSC and progenitor compartments compared to single KO alone at all time points measured (3wk, 6wk and 12 wk post tamoxifen treatment). Immunophenotypic analysis revealed that the defect originated as early as the LT-HSC stage with a drastic 7-fold reduction in LT-HSCs (Lin-c-kit+Sca1+CD150+CD48-) and 3-fold reduction in ST-HSCs (Lin-c-kit+Sca1+CD150-CD48-). Consistent with a stem cell defect, we also observed significant reductions in downstream myeloid progenitor populations including common myeloid progenitor (CMP) (2-fold), granulocyte-monocyte progenitor (GMP) (2-fold) and megakaryocyte and erythrocyte progenitors (MEP) (7-fold). Mechanistically, Hoxa9 and β-catenin co-regulate expression of Prmt1, a key epigenetic regulator with multifaceted functions in mediating RNA splicing and DNA damage response in hematopoietic cells. To further investigate the role of Prmt1 in hematopoietic development, we generated a novel Prmt1 KO model where Prmt1 can be conditionally inactivated in HSCs. Deletion of Prmt1 alone phenocopied simultaneous inactivation of β-catenin and Hoxa9, resulting in severe reductions in LT-HSC (3.5-fold), ST-HSC (4-fold) and downstream hematopoietic progenitor populations (CMP 4.5-fold, GMP 3.5-fold and MEP 4-fold). Together these data suggest that the posterior Hoxa loci and canonical Wnt pathways are developmentally regulated in a complementary manner as a safeguard mechanism to allow efficient hematopoietic self-renewal, which is largely dependent on intact Prmt1 functions. Disclosures No relevant conflicts of interest to declare.

Disruption of the Normal Hematopoietic Hierarchy Leading to Stem Cell Exhaustion in an Animal Model of Myelodysplastic Syndrome

The ineffective hematopoiesis that is characteristic of myelodysplastic syndrome (MDS) suggests functional defects of hematopoietic stem and progenitor cells (HSPC). NUP98-HOXD13 (NHD13) transgenic mice recapitulate many features of human MDS such as ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and transformation to acute myeloid leukemia (AML), and have been used as a pre-clinical model for human MDS. NHD13 mice universally develop signs of MDS (e.g., peripheral blood cytopenia, macrocytosis, dysplasia) at approximately 5 months of age, with median survival of 10 months. Two month old NHD13 mice do not show clear evidence of MDS such as peripheral blood cytopenia, dysplasia, or transformation to AML. Bone marrow nucleated cells (BMNC) from two month old NHD13 mice have a modest 1.3-fold increase of lineage negative (LN) BMNCs compared to age matched WT mice. The increased number of LN BMNCs appeared to be primarily due to a 3.4-fold increase of the LN Sca-1+cKit-(LS+Kˉ) cells, an early lymphoid-committed precursor. Lineage negative Sca-1+ c-Kit+ (LSK) cells, which include the most immature, undifferentiated cells, can be divided into five sub populations, based on expression of Flk2, CD150, and CD48. These populations have been designated Long-Term Hematopoietic Stem Cell (LT-HSC), Short-Term HSC, (ST-HSC), and Multi-Potent Progenitor 2, 3, and 4 (MPP2, MPP3, and MPP4) based on functional assays. Two-month old NHD13 mice had decreased MPP4 (5-fold), decreased LT-HSC (3.6-fold) and increased ST-HSC (2.3-fold) compared with the age matched WT mice. The expansion of ST-HSC two-month old NHD13 mice was associated with increased cell proliferation of ST- HSC, as assessed by bromo-deoxy-uridine (BRDU) incorporation. We next studied LSK subsets from NHD13 mice aged seven months, which coincided with peripheral blood findings consistent with MDS (e.g. anemia, thrombocytopenia, macrocytosis), BM from seven month old NHD13 mice showed significant reductions of all LSK population subsets. LT-HSCs show differential expression of the CD41 antigen, and CD41ˉ LT-HSCs are more quiescent than CD41+ LT-HSCs and are thought to reside at the apex of the hematopoietic differentiation hierarchy. Although there was no difference in the absolute number of quiescent CD41ˉ LT-HSC between two and six month old WT mice, six month old NHD13 mice show a marked decrease (4.2 fold) in CD41ˉ LT-HSCs, suggesting exhaustion of LT-HSC in NHD13 mice. Colony forming assays were used to assess function of the five LSK sub-populations in vitro. LT-HSC and ST- HSC from NHD13 BMNC did not produce any colonies in two independent experiments, whereas MPP2 and MPP3 from NHD13 BMNC produced a similar number and lineage distribution of colonies compared to WT BMNC. This result suggested that HSCs from NHD13 BMNC may be functionally impaired, and that NHD13 hematopoietic progenitor cells may instead be derived primarily from MPP2 and MPP3. To evaluate HSC self-renewal activity, the five LSK subsets from NHD13 BMNC were transplanted to lethally irradiated mice together with 5 x 105 WT BMNC competitor cells. None of the NHD13 LSK sub-populations showed evidence of engraftment. Since NHD13 LN BMNC have previously been shown to be more prone to apoptosis than their WT counterpart, it is possible that lack of engraftment of NHD13 LSK subsets was due to the ex vivo sorting procedure. However, we also considered the possibility that NHD13 lineage positive (LP) BMNC had acquired self-renewal potential, and were contributing to long term hematopoiesis in the NHD13 BM. Therefore, we transplanted LP and LN BMNC from NHD13 or WT mice into WT recipients, again with WT competitor BMNC. Almost half of the NHD13 LP recipients showed long-term (&gt;26 weeks) myeloid engraftment, whereas none of the WT LP recipients showed long term myeloid engraftment. Taken together, these findings suggest that the primitive LT-HSC (LSK Flk2ˉ CD150+CD48ˉ CD41ˉ) from NHD13 BM become exhausted with age, corresponding to the presentation of findings consistent with MDS (peripheral blood cytopenia, macrocytosis). Furthermore, self-renewal activity of NHD13 LP BMNCs suggest the existence of a compensatory mechanism for the homeostasis of hematopoiesis in MDS. Disclosures Aplan: NIH: Patents &amp; Royalties: royalties for the invention of NUP98-HOXD13.

CD9 Marks Flt3+ Multipotent Hematopoietic Progenitors within Lsk Cells

Hematopoietic Stem Cells (HSCs) that give rise to all lineages of the blood are defined as a lineage-negative (Lin-), c-Kit+, Sca-1+(LSK) population in mouse. The expression of Flt3 on LSK cells is accompanied by loss of self-renewal capacity, and transition to lymphoid-primed multipotent progenitors (LMPPs). The magnitude of Flt3 surface abundance has been shown to be correlated with lineage potential, where megakaryocyte/erythroid (MegE) potential is incompatible with high Flt3 levels (Buza-Vidas et al. 2011). However, it is unclear whether MegE capacity is marked by Flt3 itself or restricted to a specific population within the Flt3 spectrum. Interestingly, it has been shown that commitment to MegE lineages is an early event in HSC differentiation and that MegE progenitors share several cell-surface markers with HSCs. One such marker is CD9, which is highly expressed on megakaryocyte progenitors (Nakorn, Miyamoto, and Weissman 2003)as well as on the most primitive stem cells in mouse (Karlsson et al. 2013). In fact, we have recently shown that CD9 captures all long-term reconstitution activity of the murine LSK population, even in cell fractions that lacks some of the most established HSC markers .To compare CD9- and Flt3 expression we performed FACS analysis of the LSK compartment and plotted CD9 high- and low cells on top of the CD34/Flt3 expression profile. Interestingly we found that even if CD9 expression were predominantly high in LT-(CD34-Flt3-) and ST-HSCs (CD34+Flt3-), CD9highcells reached all the way into the Flt3+population but were lost in LMPPs, as defined by the 25% highest Flt3 expressing cells. To addresswhether CD9highcells represent a distinct subpopulation within Flt3intcells, LSKCD34+Flt3int(Flt3int) and LSKCD34+FLT3intCD9high(CD9high) populations, were subjected to single-cell RNA-Sequencing analysis. Interestingly,our results demonstratethatCD9highcells are enriched in the most primitive cluster of the Flt3intpopulation. This was further validated in transplantation assays where only CD9highFlt3intcells reconstituted irradiated recipients. However, long-term reconstitution was limited to lymphoid lineages, while myeloid engraftment was only observed at early time points and self-renewal activity was absent. To address the clonal capacity of CD9highcells, we designed a protocol to assess the potential of single-cells to generate B-, Myeloid (My)- and Erythroid (Er) cells in vitro.In these conditions 68% of clones generated from CD9highcells were bipotent (55% My/B and 13% My/Er). In contrast, the majority of CD9lowcells were unipotent (32% My- and 52% B cells).Moreover,the colony forming capacity for the CD9highpopulation was significantly higher compared to CD9lowcells and when assayed for colony-forming unit-megakaryocyte (CFU-MK) capacity, Meg potential was exclusively observed in LT-HSCs, ST-HSCs as well as the CD9highFlt3intpopulation, while neither CD9lownor LMPPs generated any megakaryocyte colonies. In an attempt to improve the efficiency of MegE differentiation, single-cell clonogenic assays were performed in suspension cultures. Here,multipotency including MegE potential was gradually down regulated with increasing Flt3 cell surface expression, and decreasing CD9 expression. Together our results shows thatCD9 captures the most primitive cells within the LSKFlt3+population, including multipotent cells with MegE differentiation potential, thus resolving the heterogeneity of Flt3+hematopoietic progenitors and identifying a multipotent LSKCD9+Flt3+population distinct from LMPPs. Disclosures No relevant conflicts of interest to declare.

Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

Diabetes induces myeloid bias in bone marrow progenitors associated with enhanced wound macrophage accumulation and impaired healing.

Diabetes induces dysregulation throughout the spectrum of myeloid lineage cells from progenitors to terminally differentiated cells. Another complication of diabetes is persistent inflammation, including prolonged accumulation of macrophages, which contributes to impaired wound healing. However, it remains unclear whether diabetes disrupts the response of bone marrow progenitors to peripheral injury and whether such dysregulation leads to sustained inflammation and impaired healing. Here, we demonstrated that diabetic mice (db/db, referred to here as DB) exhibit myeloid lineage bias during homeostasis and following injury. In addition, cells in the LSK (Lin- Sca-1+ cKit+ ) population of DB mice are preprogrammed towards myeloid commitment at the transcriptional level, and cultured myeloid progenitors from DB mice produce more monocytes ex vivo than their non-diabetic counterparts. We also show via bone marrow transfer between interleukin-1 receptor 1 KO (Il1r1-/- ) and DB mice that IL-1R1 signaling is likely not involved in myeloid skewing in DB mice. Furthermore, in vitro experiments indicated that macrophage colony-stimulating factor receptor signaling is not likely involved in enhanced myeloid transcription factor expression in LSK cells of DB mice. Our findings indicate that myeloid lineage commitment in bone marrow may contribute to increased macrophage numbers observed in diabetic skin wounds, and that strategies to regulate monopoiesis during homeostasis or post-wounding may improve diabetic wound healing. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nucleosome Binding Protein HMGN1 Promotes HSPC Competitive Advantage with Decreased Quiescence and Cooperates with AML Oncogenes to Block Differentiation and Increase Clonogenicity

Acute myeloid leukemia (AML) is characterized by rapid growth and differentiation arrest of immature myeloblasts. The AML blast is defined by having “open” chromatin, thus, we hypothesized that alterations of chromatin compaction may promote AML. The nucleosome binding protein HMGN1 is known to decompact chromatin and is located on chr21q22 in one of the most recurrently amplified regions in AML. We previously presented in vitro data showing that HMGN1 overexpression delayed myeloid differentiation in association with an increase in global levels of Histone H3K27 acetylation downstream of the histone acetyltransferases (HATs) CBP and p300. The goal of this study was to define the effect of HMGN1 overexpression on hematopoiesis in vivo and to identify leukemia oncogenes that cooperate with HMGN1 in primary hematopoietic cells.We analyzed native WT and OE-HMGN1 (transgenic overexpressing HMGN1) hematopoiesis at steady state in young and older mice. OE-HMGN1 transgenic mice had a significant increase in lineage-negative cells with age (Fig A, *p=0.02). We next performed competitive bone marrow transplantation with CD45.1 (WT) vs CD45.2 (WT or OE-HMGN1) donors measuring the relative contribution of CD45.1 and 45.2 cells to hematopoiesis over time after 3 consecutive serial transplants, each at 16 weeks. OE-HMGN1 stem and progenitor cells (HSPCs) had a progressive clonal advantage during serial transplantation (Fig B, *p<0.0002). This clonal advantage was observed across multiple HSPC populations and in all mature lineages. Lin-Sca1+Kit+ (LSK) and Lin-Kit+ (LK) HSPCs were sorted 4 months after transplantation for cell cycle analysis, ex vivo differentiation assays, and RNA-sequencing. OE-HMGN1 HSPCs were less quiescent in vivo, with fewer cells in G0 (Fig C, LK p= 0.0007, LSK p=0.01), and had a relative defect in differentiation capacity in vitro compared to WT. Gene set enrichment analysis was consistent with these phenotypes, showing enrichment of senescence and cell cycle regulatory gene expression programs in WT compared to OE-HMGN1 HSPCs.HMGN1 overexpression alone did not cause leukemia in mice. To screen for synergy between HMGN1 and AML oncogenes, we immortalized myeloid progenitors from WT or OE-HMGN1 mice with a HoxB8 transgene and measured growth properties after transduction with retroviruses expressing one of several AML-associated fusion proteins (BCR-ABL, MLL-AF9, PML-RAR, AML-ETO9a, MOZ-TIF2) or mutated oncogenes (FLT3, NPM1, NRAS, GATA2) with GFP. AML-ETO9a (AE9a) and MOZ-TIF2 had the most significant cooperation with HMGN1, most notably by enhancing their ability to block myeloid differentiation in vitro. This is of interest because, like we found for HMGN1, AE9a and MT2 are known promote leukemia via chromatin effects dependent on CBP/p300 and/or acetyltransferase activity. In confirmatory experiments, combination of AE9a and HMGN1 exacerbated the delay in myeloid differentiation induced by either gene alone (p<0.05), and HMGN1 enhanced serial colony formation capacity of AE9a in semi-solid media (p<0.05).In vivo transplantation of WT or OE-HMGN1 c-Kit+ HSPCs transduced with AE9a led to development of AML ~110 days after injection in both groups. However, subpopulation analysis of AE9a-expressing leukemias showed that LSK and MPP-like populations were expanded in the context of OE-HMGN1 compared to WT (p<0.05). Remarkably, global H3K27ac levels were significantly higher in LT-HSC-like HMGN1-OE AE9a leukemia cells using single cell subpopulation analysis by intracellular flow cytometry compared to WT (Fig D p=0.008), similar to what we observed in HSPCs after competitive transplant. Ex vivo clonogenic capacity was increased in AE9a leukemias that overexpressed HMGN1 in an LTC-IC assay (Fig E *p<0.05). Limiting dilution secondary transplants are ongoing to test if these differences correlate with increased leukemia-initiating capacity per cell in vivo in leukemias that overexpress HMGN1.Our study suggests that overexpression of HMGN1 provides a clonal advantage to hematopoietic populations including long-term stem cells. HMGN1 also cooperates with specific AML oncogenes, possibly those associated with H3K27 acetylation/HAT activity, to enhance leukemic phenotypes. Further study is needed to determine if HMGN1 overexpression confers specific therapeutic vulnerabilities, such as to HAT inhibitors or other chromatin-targeting agents. [Display omitted] DisclosuresLane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy.

Leukemia Risk Gene ARID5B is a Crucial Regulator of B-Cell Development

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Genome wide association studies by us and others have identified germline genetic variants at the ARID5B locus that strongly influence susceptibility to ALL (Nat Genet 2009, J Clin Oncol 2012). Despite compelling results from these genetic association studies, the molecular mechanisms by which ARID5B contributes to ALL pathogenesis remains largely unknown. ARID5B is a member of the ARID transcription family characterized by a conservative AT rich binding domain, but the physiological functions of ARID5B are poorly understood. Therefore we sought to develop mouse models to comprehensively characterize the roles of ARID5B in normal and malignant hematopoiesis. To this end, we first established a Vav-1 specific Arid5b overexpression (AOE) mouse model. Mice were designed with a tetO element knocked in before the start codon of Arid5b to create an Arid5b tetO mouse. Arid5b tetO mice were then crossed with Vav-1-tTA mice and, overexpression of Arid5b confirmed in the hematopoietic system of progenies with the desired genotype.At 6-8 weeks old complete blood counts (CBC) analysis revealed that AOE mice had a large decrease in white blood cells (WBCs) and a slight reduction in red blood cells (RBCs) and hemoglobin (Hb). We also noted a significant reduction in the bone marrow cellularity of our Arid5b overexpressing mice. To further characterize our AOE mouse model we used flow cytometry to quantify the proportions of mature as well as various stem and progenitor cell populations during hematopoiesis. We found that AOE mice showed an increase in the MegE biased MPP2 (LSK flt3−/CD150+/CD48+) population and a decrease in the lymphoid biased MPP4 (LSK (Flt3+/CD150−/CD48+/−) population. Overexpression of Arid5b resulted in a significant reduction in the proportion of all bone marrow B cell populations (Hardy Fraction A-F) (Figure 1A) and B220+ cells in the spleen. In vitro methylcellulose colony forming assays further revealed a loss of functional pre B lymphoid progenitor in AOE bone marrow. To determine if the hematopoietic defects seen in younger AOE mice was due to a delay in hematopoiesis we aged AOE mice. We found that as we aged AOE mice there was still a reduction in bone marrow cellularity and all bone marrow B cell populations. Surprisingly, as mice were aged we found that AOE mice were prone to sudden death and displayed a dramatic reduction in overall survival when compared to wildtype littermates with a mean survival age of 8 months (Figure 1B). At the time of death, we found that AOE mice presented with severely enlarged spleens. To predict the death of AOE mice we performed ultrasounds to track spleen volume. Based on ultrasounds performed on AOE mice and wildtype littermates, AOE mice spleens become larger than those of their littermates as early as 6 months old, however spleen volume did not predict sudden death. In aged AOE mice, while the spleens were grossly enlarged and contained larger amounts of red pulp and increased Ter119+ cells, there was still a reduction in the proportion of B cells and no increase in the proportions of other white blood cells (Figure 1D). Older AOE mice exhibited anemia, hypergammaglobulinemia, and splenomegaly. By 30 weeks, defects in B cell proportions were seen in the lymph nodes of AOE mice via immunohistochemistry (Figure 1C). Compared to wildtype littermates, AOE mice displayed reduced white blood cells, red blood cells, and platelets (Figure 1D). The anemia was associated with higher reticulocyte numbers and increased serum erythropoietin concentration. The life span of erythrocytes from AOE mice was less than that of wildtype littermates. Together, these results indicated that Arid5b plays an important role in B lymphopoiesis and erythropoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.