Hematopoietic Stem Cell Activity Research Articles

Hematopoietic Stem Cells and Their Niche in Bone Marrow.

Extensive research has explored the functional correlation between stem cells and progenitor cells, particularly in blood. Hematopoietic stem cells (HSCs) can self-renew and regenerate tissues within the bone marrow, while stromal cells regulate tissue function. Recent studies have validated the role of mammalian stem cells within specific environments, providing initial empirical proof of this functional phenomenon. The interaction between bone and blood has always been vital to the function of the human body. It was initially proposed that during evolution, mammalian stem cells formed a complex relationship with the surrounding microenvironment, known as the niche. Researchers are currently debating the significance of molecular-level data to identify individual stromal cell types due to incomplete stromal cell mapping. Obtaining these data can help determine the specific activities of HSCs in bone marrow. This review summarizes key topics from previous studies on HSCs and their environment, discussing current and developing concepts related to HSCs and their niche in the bone marrow.

Variation in mesenchymal KITL/SCF and IGF1 expression in middle age underlies steady-state hematopoietic stem cell aging

AbstractIntrinsic molecular programs and extrinsic factors including proinflammatory molecules are understood to regulate hematopoietic aging. This is based on foundational studies using genetic perturbation to evaluate causality. However, individual organisms exhibit natural variation in the hematopoietic aging phenotypes and the molecular basis of this heterogeneity is poorly understood. Here, we generated individual single-cell transcriptomic profiles of hematopoietic and nonhematopoietic cell types in 5 young adult and 9 middle-aged C57BL/6J female mice, providing a web-accessible transcriptomic resource for the field. Among all assessed cell types, hematopoietic stem cells (HSCs) exhibited the greatest phenotypic variation in expansion among individual middle-aged mice. We computationally pooled samples to define modules representing the molecular signatures of middle-aged HSCs and interrogated, which extrinsic regulatory cell types and factors would predict the variance in these signatures between individual middle-aged mice. Decline in signaling mediated by adiponectin, kit ligand (KITL) and insulin-like growth factor 1 (IGF1) from mesenchymal stromal cells (MSCs) was predicted to have the greatest transcriptional impact on middle-aged HSCs, as opposed to signaling mediated by endothelial cells or mature hematopoietic cell types. In individual middle-aged mice, lower expression of Kitl and Igf1 in MSCs was highly correlated with reduced lymphoid lineage commitment of HSCs and increased signatures of differentiation-inactive HSCs. These signatures were independent of expression of aging-associated proinflammatory cytokines including interleukin-1β (IL-1β), IL-6, tumor necrosis factor α and RANTES. In sum, we find that Kitl and Igf1 expression are coregulated and variable between individual mice at the middle age and expression of these factors is predictive of HSC activation and lymphoid commitment independently of inflammation.

A subset of megakaryocytes regulates development of hematopoietic stem cell precursors

Understanding the regulatory mechanisms facilitating hematopoietic stem cell (HSC) specification during embryogenesis is important for the generation of HSCs in vitro. Megakaryocyte emerged from the yolk sac and produce platelets, which are involved in multiple biological processes, such as preventing hemorrhage. However, whether megakaryocytes regulate HSC development in the embryonic aorta-gonad-mesonephros (AGM) region is unclear. Here, we use platelet factor 4 (PF4)-Cre;Rosa-tdTomato+ cells to report presence of megakaryocytes in the HSC developmental niche. Further, we use the PF4-Cre;Rosa-DTA (DTA) depletion model to reveal that megakaryocytes control HSC specification in the mouse embryos. Megakaryocyte deficiency blocks the generation and maturation of pre-HSCs and alters HSC activity at the AGM. Furthermore, megakaryocytes promote endothelial-to-hematopoietic transition in a OP9-DL1 coculture system. Single-cell RNA-sequencing identifies megakaryocytes positive for the cell surface marker CD226 as the subpopulation with highest potential in promoting the hemogenic fate of endothelial cells by secreting TNFSF14. In line, TNFSF14 treatment rescues hematopoietic cell function in megakaryocyte-depleted cocultures. Taken together, megakaryocytes promote production and maturation of pre-HSCs, acting as a critical microenvironmental control factor during embryonic hematopoiesis.

Inflammation as a driver of hematological malignancies.

Hematopoiesis is a tightly regulated process that produces all adult blood cells and immune cells from multipotent hematopoietic stem cells (HSCs). HSCs usually remain quiescent, and in the presence of external stimuli like infection or inflammation, they undergo division and differentiation as a compensatory mechanism. Normal hematopoiesis is impacted by systemic inflammation, which causes HSCs to transition from quiescence to emergency myelopoiesis. At the molecular level, inflammatory cytokine signaling molecules such as tumor necrosis factor (TNF), interferons, interleukins, and toll-like receptors can all cause HSCs to multiply directly. These cytokines actively encourage HSC activation, proliferation, and differentiation during inflammation, which results in the generation and activation of immune cells required to combat acute injury. The bone marrow niche provides numerous soluble and stromal cell signals, which are essential for maintaining normal homeostasis and output of the bone marrow cells. Inflammatory signals also impact this bone marrow microenvironment called the HSC niche to regulate the inflammatory-induced hematopoiesis. Continuous pro-inflammatory cytokine and chemokine activation can have detrimental effects on the hematopoietic system, which can lead to cancer development, HSC depletion, and bone marrow failure. Reactive oxygen species (ROS), which damage DNA and ultimately lead to the transformation of HSCs into cancerous cells, are produced due to chronic inflammation. The biological elements of the HSC niche produce pro-inflammatory cytokines that cause clonal growth and the development of leukemic stem cells (LSCs) in hematological malignancies. The processes underlying how inflammation affects hematological malignancies are still not fully understood. In this review, we emphasize the effects of inflammation on normal hematopoiesis, the part it plays in the development and progression of hematological malignancies, and potential therapeutic applications for targeting these pathways for therapy in hematological malignancies.

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype.

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Ex vivo expansion potential of murine hematopoietic stem cells is a rare property only partially predicted by phenotype

The scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCs ex vivo. After 3 weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential for ex vivo self-renewal, with alternative outcomes revealed by single-cell multimodal RNA and ATAC sequencing profiling. While most HSC progeny offered only transient in vivo reconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations for ex vivo HSC expansion, with major implications also for assessment of normal HSC activity.

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

KBTBD4-mediated reduction of MYC is critical for hematopoietic stem cell expansion upon UM171 treatment

AbstractEx vivo expansion of hematopoietic stem cells (HSCs) is gaining importance for cell and gene therapy, and requires a shift from dormancy state to activation and cycling. However, abnormal or excessive HSC activation results in reduced self-renewal ability and increased propensity for myeloid-biased differentiation. We now report that activation of the E3 ligase complex CRL3KBTBD4 by UM171 not only induces epigenetic changes through CoREST1 degradation but also controls chromatin-bound master regulator of cell cycle entry and proliferative metabolism (MYC) levels to prevent excessive activation and maintain lympho-myeloid potential of expanded populations. Furthermore, reconstitution activity and multipotency of UM171-treated HSCs are specifically compromised when MYC levels are experimentally increased despite degradation of CoREST1.

Deciphering the metabolic heterogeneity of hematopoietic stem cells with single-cell resolution

Metabolic status is crucial for stem cell functions; however, the metabolic heterogeneity of endogenous stem cells has never been directly assessed. Here, we develop a platform for high-throughput single-cell metabolomics (hi-scMet) of hematopoietic stem cells (HSCs). By combining flow cytometric isolation and nanoparticle-enhanced laser desorption/ionization mass spectrometry, we routinely detected >100 features from single cells. We mapped the single-cell metabolomes of all hematopoietic cell populations and HSC subpopulations with different division times, detecting 33 features whose levels exhibited trending changes during HSC proliferation. We found progressive activation of the oxidative pentose phosphate pathway (OxiPPP) from dormant to active HSCs. Genetic or pharmacological interference with OxiPPP increased reactive oxygen species level in HSCs, reducing HSC self-renewal upon oxidative stress. Together, our work uncovers the metabolic dynamics during HSC proliferation, reveals a role of OxiPPP for HSC activation, and illustrates the utility of hi-scMet in dissecting metabolic heterogeneity of immunophenotypically defined cell populations.

CUX1 Levels Control Hematopoietic Stem Cell Fate and Plasticity

Hematopoietic stem cells (HSCs) sustain lifelong, multipotent output, however, the molecular mechanisms governing HSC homeostasis remain unclear. Elucidating this process is key to understanding the imbalanced differentiation and self-renewal evident in myeloid malignancies. CUX1 is a transcription factor that is monoallelically mutated or deleted in high-risk myeloid malignancies. We previously reported that CUX1 has critical, dose-dependent roles in hematopoiesis and myeloid neoplasia. The haploinsufficient nature of CUX1 in myeloid neoplasia and the broad dose-dependent requirement of CUX1 in hematopoiesis led us investigate the role of CUX1 in earliest stages of hematopoiesis. We generated a CUX1 reporter mouse in which mCherry is fused in-frame with the C-terminus of CUX1 to study how the level of CUX1 in HSCs affects their behavior. Overall, we find that CUX1 protein levels within the HSC compartment delineates stem cell activity. Stringently defined immunophenotypic HSCs with low CUX1 levels (CUX1 Dim) are phenotypically dormant, as assessed by transcriptional profiling and proliferation assays, as opposed to CUX1 Bright HSCs which have greater cell cycle participation. In transplantation assays, CUX1 Dim HSCs are initially platelet-biased but give rise to long-term, multilineage reconstitution. CUX1 Bright HSCs are initially erythroid-biased but lack long-term HSC activity and the capacity for self-renewal. In other words, despite immunophenotypic HSC markers, CUX1 Bright HSCs are not, by definition, HSCs. By most metrics, CUX1 Intermediate HSCs exhibit a distinct, albeit intermediate, phenotype. Therefore, along a continuous gradient of CUX1 expression, HSCs gradually lose the ability to self-renew as CUX1 expression increases. Remarkably, knockdown of CUX1 in CUX1 Bright HSCs re-endows these cells with the capacity for long-term multilineage chimerism, suggesting low levels of CUX1 are required for stem cell activity in HSCs. Inversely, overexpression of CUX1 in HSCs promotes their expansion and ultimate exhaustion, further demonstrating that the level of CUX1 controls HSC fate. Altogether, we report that low CUX1 levels are required for HSC self-renewal and implicate CUX1 as a molecular rheostat for HSC behaviors such as cell division and differentiation. Further, we describe an unprecedented capacity for cells without demonstrated HSC activity to acquire HSC capacity and a novel role for CUX1 in preventing this plasticity.

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.

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Heme is required not only in red blood cells for oxygen transport but also in non-erythroid cells for the proper function of hemoproteins. Cytochrome c is one of the hemoproteins and inevitable for mitochondrial oxidative phosphorylation, suggesting that heme possesses important roles for ATP synthesis and mitochondria maintenance. Heme also can skew the differentiation of hematopoietic stem and progenitor cells (HSPCs) by altering the functions of transcription factors. Recent CRISPR Cas9 dropout screens repeatedly showed that some of the heme synthases, such as CPOX, might be inevitable for the survival and/or proliferation of HSPCs. However, still little is known about the roles for heme synthesis in the maintenance (survival, proliferation and differentiation) of HSPCs. To reveal the roles for heme synthesis in HSPCs in vivo, we developed polyI:C inducible hematopoietic cell specific Cpox knock-out mice ( Cpox f/f; Mx1-Cre). PolyI:C treated Cpox f/f; Mx1-Cre mice (KO mice) showed significant increase in the numbers of hematopoietic stem cells (HSCs) and multipotent stem and progenitor cells (MPP2s and MPP3s) but decrease in the numbers of common myeloid progenitors (CMPs) and some of their progenies (i.e., Gr1 positive cells and platelets). In contrast, its effects on erythroid and lymphoid lineages were limited. This result suggests that heme synthesis inhibition in HSPCs (HSCs, MPP2 and MPP3s) can cause their differentiation arrest. On the other hand, competitive transplantation analysis of LSKs (lineage -sca1 +c-kit +) or HSCs revealed that myelopoiesis were markedly decreased compared to control, suggesting that heme synthesis in HSPCs is important for the proper reconstitution of hematopoiesis. These results indicate that cell intrinsic heme synthesis is inevitable for the maintenance of HSPCs and their lineage specific output in vivo. In depth, we revealed that mitochondrial respiratory activity of Cpox KO HSCs were increased compared to control HSCs, suggesting that alterations of mitochondrial status and/or functions by the inhibition of heme synthesis might contribute to the aberrance in Cpox KO HSPCs. Comprehensive gene expression analysis is now ongoing to support this notion. To further reveal the roles for heme synthesis in human HSPCs, we treated human leukemia cell lines (MV4-11, KG1a and U937) with heme synthesis inhibitors (SA or NMPP). Concentration-dependent cell growth inhibitory effect and cell death were observed with increased apoptosis factors (cleaved caspase 3 and cleaved PARP), suggesting that heme synthesis inhibition triggered apoptosis. We also revealed that CPOX inhibition under doxycycline inducible CRISPR Cas9 system caused cell growth inhibitory effect and cell death in these cells. Second mitochondria-derived activator of caspases (Smac) and cytochrome c were released to cytoplasm, and the inhibitor of apoptosis protein (XIAP) was decreased by heme synthesis inhibitions. Therefore, caspase-3 activation by accumulation of cytoplasmic cytochrome c and Smac might be the inducer of this apoptosis pathway. Comprehensive transcriptomic analysis (RNA-seq) revealed that heme synthesis inhibition induced the release of cytochrome c and Smac from mitochondria into cytoplasm by the transcriptomic upregulation of Oxidative Stress Induced Growth Inhibitor 1 (Osgin1) that is the known target of NRF2. Collectively, this study demonstrated the significance of heme synthesis in the maintenance of HSPCs especially in vivo (mouse) and in vitro (human). Dysfunctions of heme synthesis in HSPCs can cause their aberrance by the alterations in their mitochondrial status which can cause apoptosis and/or differentiation arrest. Considering the metabolic differences (i.e., demands for mitochondrial oxidative phosphorylation and subsequent ATP synthesis) between normal HSPCs and leukemia or unperturbed hematopoiesis and perturbed hematopoiesis (i.e., infections and hematopoietic stem cell transplantations), further analysis to reveal the roles for heme synthesis in the maintenance of HSPCs will pave the way to establish optical approaches in these clinical settings.

Modeling Cell Population Dynamics in Early Hematopoiesis Reveals Mechanisms of Myeloid Bias Mediated By NFκB-Dysregulation

Alterations in hematopoiesis are associated with immune dysregulation and perturbations in NFκB signaling. Aging, for example, is correlated with increased myeloid and decreased lymphoid cell output, as well as elevated levels of NFκB activity in hematopoietic stem and progenitor cells (HSPC) and inflammatory cytokines in the bone marrow. Further, patients with myelodysplastic syndrome (MDS) show increased expression of components of the NFκB signaling pathway in HSPC and mesenchymal cells, in addition to increased production of inflammatory cytokines. Despite being implicated in these contexts, it remains unknown how elevated NFκB signaling might affect the production and abundance of certain HSPC subsets. Here we interrogate how increased NFκB activity may alter the developmental dynamics within the HSPC compartment to give rise to a biased progenitor population. To investigate how myeloid cell overproduction arises in the bone marrow, we developed mathematical models to recapitulate HSPC population dynamics. To address the role of NFκB in the process, we leveraged data from the IκB - mouse model ( Nfkbia +/- Nfkbib -/- Nfkbie -/-), which has constitutively elevated NFκB activity. Cell counts of HSPC subpopulations demonstrated a myeloid biased alteration in multipotent progenitor (MPP) abundances in the IκB -mouse, reminiscent of what occurs with aging. Fitting the cell population dynamics model to these data suggested that accelerated differentiation of Short-Term HSC (STHSC) with skewing in their fate commitment is insufficient to account for the increase in MPP2 and MPP3 cells. To explore additional mechanisms contributing to myeloid bias, we utilized single-cell RNA sequencing (scRNAseq) data from IκB - versuscontrol HSPC. We first used pseudotime reconstruction to position cells along a continuous differentiation trajectory from HSCs to erythrocyte-megakaryocyte- (MPP2), myeloid- (MPP3), and lymphoid- (MPP4) primed progenitors and then fit these data to a partial differential equation (PDE) model of population dynamics. This analysis suggested not only a loss of lymphoid fate specification amongst HSC but also an increase in the expansion of early myeloid primed cells to produce more MPP3 cells. Transplantation of wildtype HSPC into a lethally irradiated IκB - mouse additionally showed that an inflamed microenvironment is sufficient to drive myeloid bias. Utilizing the same mathematical models to describe these data revealed similar perturbations in population dynamics of the transplanted HSPC. Finally, the scRNAseq pseudotime analysis identified differentially expressed genes along the differentiating lineages that indicated decreased HSC commitment towards the lymphoid fate and increased proliferation of early myeloid primed cells, supporting the model predictions. We next leveraged scRNAseq datasets from HSPC isolated from MDS patients and healthy aged-matched controls to explore if these features of NFκB-mediated immune dysregulation are clinically relevant. These data demonstrated myeloid bias in progenitors at the transcriptional level and suggested elevated NFκB signaling amongst the HSPC from MDS patients. Finally, applying the same mathematical modeling and gene expression analyses developed for the murine datasets revealed common perturbations in developmental dynamics of MDS HPSC as the IκB - mouse models. These analyses motivate further study of the downstream consequences of immune dysregulation on hematopoiesis and the identification of perturbations in NFκB signaling in pathological contexts.

Metabolic Signatures and Pathway Rewiring in TET2-Mutated Myeloid Neoplasia

Background. TET2 mutations ( TET2 MT) are the most frequently observed somatic lesions in myeloid neoplasms (MN). TET2 belongs to an Fe2+ and α-ketoglutarate (αKG) dependent DNA-dioxygenase family that progressively oxidizes 5-methyldeoxycytidine (5mC), leading to promoter and enhancer mCpG demethylation essential for efficient transcription. TET2 accounts for the majority of DNA dioxygenase activity in hematopoietic stem and progenitor cells (HSPCs), and following its loss, the resulting accumulation of 5mC leads to a loss of lineage plasticity and transcriptional repression of tumor suppressor genes, thereby establishing a volatile pre-neoplastic state. TET2 is one of the major consumers of αKG, a major cofactor produced by the tricarboxylic acid (TCA) cycle. Therefore, loss of TET2 creates a metabolic perturbation in HSPCs. However, the impact of TET2 loss on the metabolism of leukemic cells and its subsequent effects on malignant evolution remains unclear. Methods. We generated isogenic TET2 -/- ( TET2 ko) leukemic cell models in THP1 and K562 using CRISPR-Cas9 knockout. We then utilized targeted and untargeted metabolic analyses by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry in these models. We aimed to investigate the intricate metabolic changes associated with TET2 and TET2 MT. To validate our findings, we extended our studies to primary cells derived from MN patients with and without TET2 lesions. We performed extensive cellular metabolism studies using 13C isotope labelled glucose and glutamine. Additionally, levels of 5mC and its oxidation products, namely 5-hydroxymethyldeoxycytidine, 5-formyldeoxycytidine, and 5-carboxydeoxycytidine were documented for correlating epigenetic modifications with TET2 activities. Results. Targeted metabolic analysis uncovered profound impacts of TET2 MT on key metabolic pathways including the TCA cycle, glycolysis, glutamine metabolism, and nucleoside biosynthesis (Figure A). Distinct changes were observed in cells from different lineage backgrounds, suggesting diverse metabolic pathway rewiring specific to cell origin. Despite the divergence among lineages, loss of TET2 created common and unique metabolic signatures, highlighting their crucial role in sustaining the survival and clonal advantages specific to TET2 MT. Representative signature compounds and their relative quantities in TET2 wildtype (WT) and TET2 MT are depicted in Figure A. The targeted metabolic analysis revealed a significantly lower level of glutamine in TET2 MTcells, suggesting a potential preference for exhausting glutamine as a source of energy production. For validation, we conducted 13C metabolic flux analysis (13C-MFA) using glucose (U-13C, 99%) and L-glutamine (U-13C, 99%). 13C-MFA revealed distinct enrichment patterns (Figure B). In the TCA cycle, the dominant isotopologues receiving [U-13C]-glucose were M+2 for citrate and isocitrate. However, for aKG, succinate (SA) and fumarate (FA), the M+2s were significantly lower in TET2 -/- compared to WT, suggesting that the major source for these metabolites were rewired from glycolysis to glutamine metabolism in TET2 -/-. This was confirmed by [U-13C]-glutamine labeling where dominant isotopologues, M+5 for aKG and M+4 for SA, FA and malate, were significantly higher at 24-hour time point in in TET2 -/- compared to WT. Untargeted metabolic analyses from isogenic cells were used to identify metabolic features associated with TET2 loss. These metabolic features were also assayed in primary AML cells with TET2 MT (n=12) and without: RUNX1 (n=20), SF3B1 (n=11), IDH1 (n=10), IDH2 (n=13), FH (n=3), as well as healthy donors as controls (n=6). Based on the molecular features identified in the TET2 ko isogenic cells, MN patients with TET2, IDH1/2, and germline FH mutations clustered more closely, suggesting shared metabolic signatures among these groups. In contrast, patients with RUNX1 and SF3B1 mutations formed separate clusters, demonstrating distinct metabolic profiles associated with these genetic alterations. Conclusion. Our findings provide comprehensive and novel insights into the metabolic consequences of TET2 inactivation in MN. The identified metabolic signatures and rewiring of key pathways may hold promise as potential therapeutic targets and offer opportunities for tailored treatment approaches in TET2MT MN.

15-Pgdh Inhibition Alternatively Activates Macrophages to Promote Hematopoietic Function during Aging

Aging results in the progressive decline of organ function and integrity across species. In the hematopoietic system aging is associated with diminished stem cell regenerative capacity and contributes to compromised tissue homeostasis and heightened vulnerability to age-related diseases including anemia, myelodysplastic syndromes, and hematological malignancies. Macrophages, integral components of the innate immune system, demonstrate remarkable functional plasticity and play diverse roles in tissue homeostasis, inflammation, and immunity. The balance between pro-inflammatory M1 and anti-inflammatory M2 macrophages significantly influences immune and inflammatory responses, and thus the progression of hematopoietic aging. We have previously demonstrated that the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) regulates hematopoietic stem cell (HSC) activity, that 15-PGDH inhibition (PGDHi) following transplantation results in a markedly accelerated recovery of neutrophils, platelets, and HSCs, and that 15-PGDH knockout animals display enhanced HSC function with age. Here we expand on our previous work and demonstrate in aged mice that (1) 15-PGDH expression and activity remains conserved (2) that PGDHi induces a gene expression signature consistent with alternatively activated M2 macrophages, (3) that prolonged PGDHi results in a significant increase in phenotypic HSCs in both the bone marrow and spleen and induces a gene signature consistent with enhanced HSC function, and (4) that PGDHi treated bone marrow (BM) displays a advantage during primary competitive repopulation studies and prevents age-associated myeloid bias during secondary transplants. Taken together we propose PGDHi represents an in vivo switch to alternatively activate macrophages, which has significant implications to not only prevent age-associated hematopoietic decline, but also as a novel strategy to treat age-associated hematologic disease.

Transcriptomic Signature and Functional Abnormalities of Bone Marrow Mesenchymal Stromal Cells Mediate Disease Progression of Myelodysplastic/Myeloproliferative Neoplasms

Background Mesenchymal stromal cells (MSC) are vital components of the bone marrow (BM) microenvironment niche and regulate hematopoietic stem and progenitor cell (HSPC) activity. While HSPC harbor primary disease driver mutations, MSC in myelodysplastic syndrome and myeloproliferative neoplasm (MDS/MPN) have been shown to influence the emergence and selection of leukemic clones and disease progression. However, it remains unclear if there are specific functional characteristics or genetic signals of MSC that interact with HSPC at different stages of disease progression. We examined BM samples from MDS/MPN patients with high rate of leukemic transformation in less than 2 years (HR) versus those with stable disease for 5 years (SD), to investigate the phenotypic differentiation, functional characteristics, and transcriptomic profiles of MSC and the HSPC counterparts. Methods We obtained BM samples at diagnosis and follow-up from MDS/MPN patients including chronic myelomonocytic leukemia (CMML) treated at Singapore General Hospital. We studied 5 HR and 13 SD patient samples to compare against 5 healthy donor samples. MSC were derived in MesenCult-ACF media (StemCell Technologies), and phenotypic characterization was done after 1-2 passages. HSPC were subjected to co-culture expansion with MSC in MyeloCult-H5100 for 7 days, and long-term culture-initiating cell assays (LTC-IC) using MSC as feeder cells for 6 weeks. Total RNA extracted from MSC and HSPC were subjected to bulk and single-cell RNA sequencing (scRNA-Seq) (10X Genomics platform). Principal component analysis (PCA) was used for the uniform manifold approximation and projection (UMAP). Gene set enrichment analysis (GSEA) was performed on differentially expressed genes to examine canonical signaling pathways. Cellchat algorithm was applied to predict intercellular communication between MSC and HSPC using scRNA-Seq data. Results Baseline clinical variables for the international prognostic scoring system (IPSS) including age at diagnosis, myeloblast percentage and cytogenetics were not significantly different between HR and SD patients. Decreased osteogenic and chondrogenic differentiation, but relatively preserved adipogenic differentiation capacity was observed in all MDS/MPN MSC samples. A significantly lower yield of CD34+ CD38- primitive HSPC with myeloid skewing was observed in the co-culture experiments with MDS/MPN MSC, compared to healthy MSC. Pre-treatment of SD-MSC feeder with the hypomethylating agent 5-azacytidine improved the clonogenic potential of the corresponding co-cultured HSPC, but did not restore the HSPC colony yield with HR-MSC feeder. Unbiased PCA of bulk RNA-Seq data demonstrated greater clustering of HR-MSC, while SD and healthy MSC overlapped significantly. Genes associated with mesenchymal multipotency and differentiation (PODXL20, FOXQ122, SERPINA923) were generally downregulated in MDS/MPN MSC, likely attributed to promoter hypermethylation. Significant differential upregulation of CD74 and IL-6 expression was observed in HR-MSC, which suggested a uniquely enriched inflammatory signature. GSEA results showed that pathways associated with G2M cell cycle checkpoint, DNA repair, glycolysis, and response to azacytidine were suppressed in HR-MSC. UMAP on scRNA-Seq data of longitudinal MSC samples identified distinct clustering of HR-MSC at disease progression. Cell-cell interaction analysis predicted a strong communication between collagen-encoding genes (COL1A1 and COL1A2) in HR-MSC at diagnosis and CD44, a negative regulator of HSPC proliferation, and the interaction was further enhanced at disease progression (Figure 1). Ongoing analysis of downstream gene expression changes and secretome profile of HSPC in MDS/MPN MSC-based in-vivo scaffold models will be presented. Conclusion Together, our data point towards a degree of co-development and communication between MSC and HSPC during disease progression of MDS/MPN. Further characterization of the unique functional and transcriptomic signatures in MSC may help to optimize disease prognostication and identify novel therapeutic targets in MDS/MPN patients.

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

Reducing DNA Damage Associated with Endogenous Genotoxic Stress in Hematopoietic Stem Cells

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. Endogenous formaldehyde is ubiquitous within cells as it is produced as a byproduct during normal cellular metabolism such as histone, RNA and DNA demethylation. Previous studies show that endogenous formaldehyde is a hematopoietic stem cell (HSC) genotoxin and metabolic carcinogen. We previously showed that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5), and Adh5-deficient mice therefore accumulate formaldehyde adducts in DNA that require the FA pathway to repair. Mice lacking Adh5 together with the FA pathway central component Fancd2 exhibit bone marrow failure and early lethality. Adh5 haploinsufficiency combined with Fancd2 deficiency, or Fancd2 -/- Adh5 +/- mice, demonstrates severely compromised HSC activity. However, the contributing factors that cause DNA damage and the protective mechanisms by which cells reduce DNA damage during endogenous genotoxic stress are not fully understood. Here, we show that the genetic inactivation of Lnk/Sh2b3 in Fancd2 -/- Adh5 +/- mice restores HSC function. Adh5 haploinsufficiency exacerbated Fancd2 deficiency, while Lnk deficiency rescued HSC homeostasis and transplantability of Fancd2 -/- Adh5 +/- mice. Moreover, Lnk deficiency increased HSC quiescence and reduced endogenous DNA damage in Fancd2 -/- Adh5 +/- mice. Importantly, Lnk deficiency does not participate in repairing formaldehyde-induced DNA damage, nor does it metabolize formaldehyde or reduce genomic formaldehyde-deoxyguanine adduct in vivo. Rather, Lnk deficiency reduced single-strand DNA (ssDNA) breaks and ssDNA binding protein RPA in HSCs during the cell cycle, thereby suppressing the ATR pathway activation upon endogenous replication stress. Moreover, loss of Lnk increased S-phase progression and fork recovery in Fancd2 -/- Adh5 +/- HSPCs at the stressed replication fork upon HU treatment, thereby preventing replication fork collapse and DNA damage. These findings highlight a new mechanism by which Lnk deficiency mitigates replication stress-induced DNA damage in response to endogenous genotoxic insult from cellular metabolism during HSC regeneration.

Human Hematopoietic Stem Cells Have Altered Mitochondrial Activity after Stem Cell Transplantation

Hematopoietic stem cells (HSCs) give rise to the entire blood system. With their high regenerative potential, they are sometimes the only curative option for many malignancies. The success of HSCT depends on both the quantity of HSCs and on their ability to produce life-long blood. However, their regenerative potential is not unlimited. Transplanted HSCs sustain injury that alters their subsequent ability to generate blood cells. This leads to ineffective hematopoiesis and can ultimately lead to graft failure. Injury to HSCs is also a risk factor for secondary neoplasms, including leukemia. Our goal is to elucidate the mechanisms behind post-transplant HSC decline. Mitochondria are critically important for HSC function (Filippi Exp Hematol 2021). During regeneration of the bone marrow, HSCs become activated and undergo mitochondrial reprogramming, including reorganization of the mitochondrial network and enhanced mitochondrial activity (Filippi et al Blood 2019, Ito et al Nat Rev Mol Cell Biol 2014). However, after transplantation, HSCs accumulate mitochondria that are abnormal in organization and activity. These abnormal mitochondria lead to decline of HSC function and ineffective hematopoiesis (Hinge et al Cell Stem Cell 2020). While these findings have been replicated in murine models, it has not been studied within the human system. Our hypothesis is that after transplantation, human HSCs have altered mitochondria leading to ineffective hematopoiesis. We examined changes in HSC function using post-transplant samples of patient's bone marrow and a xenotransplant system (human CD34+ peripheral blood stem cells transplanted into sublethally irradiated immunodeficient NOD/SCID/IL-2 receptor-γ null mice). We first measured mitochondrial membrane potential using tetramethyl rhodamine ethyl ester (TMRE) dye and analyzed via flow cytometry. Three to six months after transplant in the xenotransplant model, we found that the mitochondrial membrane potential of huCD34+CD38-CD90+ HSCs was similar to the non-transplanted HSCs. However, huCD34+CD38+ progenitors exhibited a drastic 3-fold increase in mitochondrial membrane potential in comparison to the non-transplanted, huCD34+CD38+ progenitor cells. The mitochondrial membrane potential of CD34+CD38+ progenitors from transplanted bone marrow patient samples was also higher than non-transplanted CD34+CD38+bone marrow control cells. To understand if alterations in mitochondrial activity affect HSC differentiation, we placed xenotransplanted HSCs into in vitro culture. Post-transplanted huCD34+ cells failed to expand within 6 days of in vitro culture (serum-free culture with self-renewing cytokines, SCF+TPO+FLT3) compared to huCD34+ control. In subsequent differentiation assays, post-transplanted huCD34+ cultures gave rise to myeloid cells only. This contrasts with control huCD34+ cultures that produced myeloid, erythroid, and megakaryocytic cells. Interestingly, if the post-transplanted HSCs were given serum and cytokines for differentiation after only 3 days in self-renewing conditions, the cultured cells expanded similarly to non-transplanted HSCs and generated myeloid, erythroid, and megakaryocytic cells. This suggests that transplanted HSCs have decreased proliferation and differentiation ability in comparison to non-transplanted HSCs and respond differently to cytokines in vitro. This could account for ineffective hematopoiesis observed in some post-transplant patients. In conclusion, this study indicates that both mitochondrial activity and in-vitro growth change in HSCs after transplantation, despite re-establishing quiescence. This indicates that human hematopoietic stem and progenitor cells incur injury after transplantation. These findings are clinically important as the changes in mitochondrial activity in HSCs may play a role in post-transplant hematopoietic stem cell decline. Future single cell RNA sequencing analyses will provide mechanistic information regarding post-transplant changes in metabolism and potential targets for improving HSC function in transplant patients.

Role of Lama4 Expression in Bone Marrow Niche for the Progression and Treatment Response of Chronic Myeloid Leukemia

Although tyrosine kinase inhibitors (TKIs) have dramatically improved the treatment outcomes of chronic myeloid leukemia (CML), TKIs cannot eliminate CML-initiating stem cells (LSCs), leading to CML persistence and relapse post TKI-discontinuation. Thus, it is imperative to develop more effective therapeutic options to overcome the treatment challenges. Targeting the bone marrow (BM) niche has become one of the research focuses in drug discovery since increasing evidence suggests that BM niches protect CML LSCs. Laminin alpha4 chain (Lama4), a functional chain for several laminin isoforms, is widely expressed in bone marrow (BM). We have recently shown the critical impact of LAMA4 expression in the microenvironment for hematopoiesis regeneration including megakaryocyte maturation and acute myeloid leukemia progression in mice (Cai et al., Blood, 2022). Furthermore, we have by RNA sequencing shown downregulation of LAMA4 mRNA in freshly isolated BM mesenchymal stem cells (MSCs) from newly diagnosed patients with CML (Dolinska, Cai, Mansson, et al., Blood, 2023). However, the functional impact of the LAMA4 expression on CML LSCs remains to be investigated. We have here explored the role of LAMA4 in CML development and drug response by using both mouse models and primary patient BM samples. To first assess the in vivo impact of LAMA4 expression in BM niches on CML development, we transplanted BM cells (CD45.2) from Scl- tTA× TRE-BCR-ABL1 (BCR-ABL1) mice prior or following tetracycline withdrawal into irradiated Lama4+/+ and Lama4-/- CD45.1mice and analyzed CML progression and BCR-ABL1 LSC activity by flow cytometry and droplet digital PCR (ddPCR). Following transplantation of unfractionated BM cells from the BCR-ABL1 mice with CML induced by tetracycline withdrawal, Lama4-/- recipients showed significantly increased platelet counts in blood compared to Lama4+/+ recipients at 2-8 weeks post-transplantation (P=0.005 at week 2, P= 0.008, n=7-8, at week 8), which is in contrast to the impaired platelet recovery in non-transplanted Lama4-/- mice following sublethal irradiation. This suggests that Lama4 deficient microenvironment is favorable for platelet production from BCR-ABL1 CML cells. Consistent with this, megakaryocyte-erythrocyte progenitors (MEPs) derived from BCR-ABL1+ cells were significantly increased in Lama4 -/- BM at the endpoint (8 weeks, p=0.02, n=3). Megakaryocyte progenitors (MkPs) seemed to be increased in Lama4-/- recipient BM (P=0.05, n=3). Intriguingly, when LIN -SCA1 +KIT + CD150 + hematopoietic stem cells from BCR-ABL1 mice without prior tetracycline withdrawal were transplanted, the donor-derived MEPs were significantly reduced in Lama4-/- BM (P=0.02, n=4-6), which is accompanied with a trend of reduction in MkP but no changes in platelets. ddPCR analysis indicated that the ratio of BCR-ABL1 + cell fraction to total CD45.2 + cells (detected by FACS) seemed to be lower in Lama4-/- recipients (33.6%) than that in Lama4+/+ recipients (41.5%), raising a possibility that Lama4-/- niches is less suitable for maintenance of BCR-ABL1 + cells. Together, these data point to a possible differential regulation of CML stem and progenitor cells by the Lama4 expression. More work will be done to dissect the regulatory mechanisms through in vitro co-cultures, transplantation and confocal imaging. In parallel, the functional impact of LAMA4 on human CML LSC survival was evaluated by a revised long-term culture-initiating cell assay using CML patient derived MSCs. The preliminary result indicated that recombinant LAMA4 peptides appeared to inhibit CML patient BM-derived CD34 +CD38 - cell growth. In conclusion, LAMA4 is downregulated in BM MSCs and endothelial cells of CML patients at diagnosis. Lama4-/- microenvironment might act differentially in regulating BCR-ABL1 + hematopoietic stem and progenitor cell activity post-transplantation. However, more work is required to validate the finding and the effect of LAMA4 peptides on the megakaryocyte differentiation and maturation.