Hematopoietic Stem Cell Quiescence Research Articles

Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3weeks to 18months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=.01) and hemoglobin levels (p<.05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p=.001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells

AbstractHematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin-dependent kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. Herein, we describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal, and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, supporting a concept, in which MAZ and NFY-A are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Assessment of Redox Patterns at the Transcriptional and Systemic Levels in Newly Diagnosed Acute Leukemia

Background: Acute leukemia is the result of clonal transformation and proliferation of a hematopoietic progenitor giving rise to poorly differentiated neoplastic cells. Reactive oxygen species play a role in maintaining the quiescence, self-renewal, and long-term survival of hematopoietic stem cells, but it is unclear how they would affect disease onset and progression. The aim is to evaluate, at the transcriptional and systemic level, the oxidative-inflammatory status in newly diagnosis acute leukemia patients. Methods: Seventy acute leukemia patients [26 acute lymphoblastic leukemia (ALL), 13 Acute Promyelocytic Leukemia (APL), and 31 Acute Myeloid Leukemia (AML)] and forty-one healthy controls were analyzed. Malondialdehyde and catalase activity were evaluated. Gene expression of NRF2, SOD, PRDX2, CAT, IL-6, and TNF-α was analyzed by real-time PCR. Results: Malondialdehyde concentration was similar in all groups studied. Catalase activity was significantly higher in AML and APL patients compared to controls, while ALL showed similar activity to the healthy group. NRF2, CAT, and PRDX2 expression levels were similar between groups, SOD expression was downregulated in all acute leukemia patients. TNF-α expression was lower in AML groups than in healthy individuals, and IL-6 mRNA expression was downregulated in ALL and APL. Conclusion: This is the first report that correlates transcriptional and systemic parameters associated with the oxidative inflammatory status in newly diagnosed acute leukemia. Some of the parameters evaluated could be used as biomarkers in the selection of an effective therapeutic strategy and will open new directions for the follow-up and evolution of this disease.

Yin Yang 1 regulates cohesin complex protein SMC3 in mouse hematopoietic stem cells

AbstractYin Yang 1 (YY1) and structural maintenance of chromosomes 3 (SMC3) are 2 critical chromatin structural factors that mediate long-distance enhancer-promoter interactions and promote developmentally regulated changes in chromatin architecture in hematopoietic stem/progenitor cells (HSPCs). Although YY1 has critical functions in promoting hematopoietic stem cell (HSC) self-renewal and maintaining HSC quiescence, SMC3 is required for proper myeloid lineage differentiation. However, many questions remain unanswered regarding how YY1 and SMC3 interact with each other and affect hematopoiesis. We found that YY1 physically interacts with SMC3 and cooccupies with SMC3 at a large cohort of promoters genome wide, and YY1 deficiency deregulates the genetic network governing cell metabolism. YY1 occupies the Smc3 promoter and represses SMC3 expression in HSPCs. Although deletion of 1 Smc3 allele partially restores HSC numbers and quiescence in YY1 knockout mice, Yy1−/−Smc3+/− HSCs fail to reconstitute blood after bone marrow transplant. YY1 regulates HSC metabolic pathways and maintains proper intracellular reactive oxygen species levels in HSCs, and this regulation is independent of the YY1–SMC3 axis. Our results establish a distinct YY1–SMC3 axis and its impact on HSC quiescence and metabolism.

Abstract 5539: PRDM16 is an intrinsic regulator of dormancy in bone disseminated prostate cancer cells

Abstract Metastatic prostate cancer (PCa) frequently occurs in the bone and can remain inactive for extended periods after primary tumor treatment. Understanding the intricate molecular mechanisms that govern this phenomenon could open the door to novel therapies, potentially reducing mortality rates. To address this, we developed a novel protocol to induce cellular dormancy in PCa cell lines (RM1, 22Rv1, LAPC4) in vitro. We noted that dormant cells exhibited distinctive clustering and could be reactivated when exposed to serum even after 21 days of stasis. The dormancy program was validated by multiple assays including cell cycle analysis, membrane dye retention, and Edu+ incorporation. RNASeq analysis showed significant transcriptional changes during dormancy induction, with 8 genes commonly upregulated across multiple human and mouse PCa cell lines, of which the transcription factor, PRDM16. We focused on PRDM16 since, 1) it is a known regulator of hematopoietic stem cell quiescence, 2) our emerging data points to bone morphogenetic protein-7 (BMP7), a well-known exogenous mediator of dormancy as an inducer of PRDM16 expression and 3) clinically, PRDM16 is suppressed in primary PCa and active metastases. First, we validated the upregulation of PRDM16 during dormancy in vitro. Silencing PRDM16 led to impaired dormancy phenotype and cell death, while its overexpression significantly mitigated proliferation rates and promoted survival. In vivo, we employed an intra-iliac immunocompetent model (RM1 &amp; C57BL6J) and demonstrated that overexpressing PRDM16 in active RM1 cells resulted in significant reduction of metastasis formation and Ki67 expression concurrent with extended survival when injected in vivo compared to control cells. Prostate cancer cells with PRDM16 over-expression were detected in bone sections as solitary or small clusters (&amp;lt;20 cells) demonstrating an inverse relationship between PRDM16 expression and metastatic outbreak. In defining the targets via which PRDM16 controls dormancy entry, we focused on RAR related orphan receptor C (RORC) based on bioinformatic analysis and publicly available PRDM16 ChIP-Seq. Our data demonstrate that pharmacological inhibition of RORC using GSK805 promoted the apoptosis of dormant PCa cells specifically when compared to their actively growing counterparts. In conclusion, our studies have revealed the critical role of PRDM16 in promoting PCa cell dormancy, in part via upregulating RORC. Translationally, clinically approved RORC inhibitors for non-cancerous conditions (psoriasis) have been developed. We expect, based on our results, that these inhibitors could be repurposed for the eradication of dormant PCa cells in patients with advanced disease. Citation Format: Mostafa M. Nasr, Tao Li, Ryan T. Bishop, Jeremy S. Frieling, Conor C. Lynch. PRDM16 is an intrinsic regulator of dormancy in bone disseminated prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5539.

TAL1-mediated regulation of hemocyte proliferation influences red blood phenotype in the blood clam Tegillarca granosa

Red blood phenotype, indicating the degree of red color in blood or hemolymph, is a significant economic trait in Tegillarca granosa, closely associated with its health condition and nutritive value. However, the molecular regulatory processes that account for the variation in the blood clams' red blood phenotype remain largely unexplored. In our previous studies, weighted gene co-expression network analysis (WGCNA) showed that TAL1 exhibits high intramodular connectivity in red blood-related module. Furthermore, TAL1 serves as a critical transcription factor in the survival and quiescence of hematopoietic stem cells, along with being essential in the terminal maturation processes in vertebrates. Consequently, we investigated the function of the TAL1 in blood clams by conducting a comparative analysis between high total hemocyte count (HTHC: THC ≥ 14 × 104 cells/μL) and low total hemocyte count (LTHC: THC ≤ 2.5 × 104 cells/μL) groups, as well as between negative control and Tg-TAL1 RNA interference groups of blood clams. The results revealed that, compared to LTHC group, there was a significant upregulation of TAL1 mRNA and protein expression levels in the HTHC group, which may be a crucial factor contributing to the alterations observed in THC. RNA interference of the TAL1 in blood clams resulted in a significant decline in THC, causing the blood color to become lighter. EdU staining experiments further confirmed a diminished cell proliferative vitality within T. granosa gills after TAL1 knockdown. Moreover, immunohistochemical analysis and immunofluorescence assays revealed a gradual escalation in TAL1 protein expression and cytoplasmic/nuclear ratio from gill filament chamber endothelial cells to gill filament chamber cells in gills, potentially representing different cell types in the maturation process of erythrocytes in blood clams. It suggesting that the gills might act as one of the hematopoietic tissues in blood clams, with hemocytes potentially originating from the gill filament chamber endothelium of the gills through division and differentiation. This research improves comprehension of the molecular mechanisms behind the variation in T. granosa's red blood phenotype, potentially assisting in the selective breeding of high THC T. granosa populations.

Exploring the intricate cross-talk between clonal expansion and the bone marrow niche

Haematopoietic stem cells (HSCs) reside within an intricate network of cells in the bone marrow (BM) niche. HSC crosstalk with niche compartments influences lineage determination and blood cell production, while independent niche interactions are essential for the maintenance of HSC quiescence. How different niche components influence the genetic diversity of HSCs represents an expanding field of investigation. As such, we will summarise the current knowledge of the contribution to the Darwinian evolution of mutant HSCs of both haematopoietic and non-haematopoietic cells residing in the BM. In this review, we will disentangle how somatic evolution associates with the niche at two stages: from (1) the stage of preleukaemic HSC expansion and clonal haematopoiesis (CH) to (2) leukaemia-initiating cells (LICs) and the development of myeloid malignancies with acute myeloid leukaemia (AML) being the most prevalent. We will finally describe current challenges such as limitations in models used in the field or the difficulty in studying specific genetic clones in isolation.

CD38 promotes hematopoietic stem cell dormancy.

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease.

NEAR-INFRARED DYE IR-780 ALLEVIATES HEMATOPOIETIC SYSTEM DAMAGE BY PROMOTING HEMATOPOIETIC STEM CELLS INTO QUIESCENCE.

Potential radiation exposure is a general concern, but there still lacks radioprotective countermeasures. Here, we found a small molecular near-infrared dye IR-780, which promoted hematopoietic stem cells (HSCs) into quiescence to resist stress. When mice were treated with IR-780 before stress, increased HSC quiescence and better hematopoietic recovery were observed in mice in stress conditions. However, when given after radiation, IR-780 did not show obvious benefit. Transplantation assay and colony-forming assay were carried out to determine self-renewal ability and repopulation capacity of HSCs. Furthermore, IR-780 pretreatment reduced the generation of reactive oxygen species (ROS) and DNA damage in HSCs after radiation. In homeostasis, the percentage of Lineage - , Sca-1 + , and c-Kit + cells and long-term HSCs (LT-HSCs) were improved, and more HSCs were in G0 state after administration of IR-780. Further investigations showed that IR-780 selectively accumulated in mitochondria membrane potential high LT-HSCs (MMP-high LT-HSCs). Finally, IR-780 promoted human CD34 + HSC reconstruction ability in NOD-Prkdc scid Il2rg null mice after transplantation and improved repopulation capacity in vitro culture. Our research showed that IR-780 selectively entered MMP-high LT-HSCs and promoted them into dormancy, thus reducing hematopoietic injury and improving regeneration capacity. This novel approach might hold promise as a potential countermeasure for radiation injury.

Fosl2 Deficiency Predisposes Mice to Osteopetrosis, Leading to Bone Marrow Failure.

Arthritis causes Fos-like 2 (Fosl2) inactivation, and various immune cells contribute to its pathogenesis. However, little is known about the role of Fosl2 in hematopoiesis and the possible pathological role of Fosl2 inactivation in the hematopoietic system in arthritis. In this study, we show that Fosl2 maintains hematopoietic stem cell (HSC) quiescence and differentiation while controlling the inflammatory response via macrophages. Fosl2-specific deletion in the hematopoietic system caused the expansion of HSCs and myeloid cell growth while affecting erythroid and B cell differentiation. Fosl2 inactivation enhanced macrophage M1 polarization and stimulated proinflammatory cytokines and myeloid growth factors, skewing HSCs toward myeloid cell differentiation, similar to hematopoietic alterations in arthritic mice. Loss of Fosl2 mediated by Vav-iCre also displays an unexpected deletion in embryonic erythro-myeloid progenitor-derived osteoclasts, leading to osteopetrosis and anemia. The reduced bone marrow cellularity in Vav-iCreFosl2f/f mice is a consequence of the reduced bone marrow space in osteopetrotic mice rather than a direct role of Fosl2 in hematopoiesis. Thus, Fosl2 is indispensable for erythro-myeloid progenitor-derived osteoclasts to maintain the medullary cavity to ensure normal hematopoiesis. These findings improve our understanding of the pathogenesis of bone-destructive diseases and provide important implications for developing therapeutic approaches for these diseases.

Phosphatase, Mg2+/Mn2+ dependent 1B regulates the hematopoietic stem cells homeostasis via the Wnt/β-catenin signaling.

Hematopoietic stem cells (HSCs) are primarily dormant in a cell-cycle quiescence state to preserve their self-renewal capacity and long-term maintenance. How HSCs maintain the balance between activation and quiescence remains largely unknown. Herein, we found that Phosphatase, Mg2+/Mn2+ Dependent 1B (Ppm1b) is required for the expansion of phenotypic HSCs in vitro. By using a conditional knockout mouse model in which Ppm1b was specifically depleted in hematopoietic cells, we demonstrated that loss of Ppm1b impaired the HSC homeostasis and hematopoietic reconstitution. Ppm1b deficiency mice also exhibited B-cell leukocytopenia, which is due to the compromised commitment and proliferation of B-biased lymphoid progenitor cells from CLPs. With the aid of a small molecular inhibitor, we confirmed the roles of Ppm1b in adult hematopoiesis that phenocopied the effects with loss of Ppm1b. Furthermore, transcriptome profiling of Ppm1b-deficient HSCs revealed the disruptive quiescence of HSC. Mechanistically, Ppm1b interacted with β-catenin and mediated its dephosphorylation. Loss of Ppm1b led to the decrease of the active β- catenin (non-phosphorylated) that interrupted the Wnt/β-catenin signaling in HSC, which consequently suppressed HSC expansion. Together, our study identified an indispensable role of Ppm1b in regulating HSC homeostasis via Wnt/β-catenin pathway.

Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient β-catenin/Hoxa9/Prmt1 axis

AbstractMaintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, β-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although β-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, β-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of β-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient β-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

Autophagy is essential for human myelopoiesis

Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.

Differential Effects of Endurance Exercise on Musculoskeletal and Hematopoietic Modulation in Old Mice.

One of the most important strategies for successful aging is exercise. However, the effect of exercise can differ among individuals, even with exercise of the same type and intensity. Therefore, this study aims to confirm whether endurance training (ETR) has the same health-promoting effects on the musculoskeletal and hematopoietic systems regardless of age. Ten weeks of ETR improved endurance exercise capacity, with increased skeletal muscle mitochondrial enzymes in both young and old mice. In addition, age-related deterioration of muscle fiber size and bone microstructure was improved. The expression levels of myostatin, muscle RING-finger protein-1, and muscle atrophy F-box in skeletal muscle and peroxisome proliferator-activated receptor-γ in the femur increased with age but decreased after ETR. ETR differentially modulated hematopoietic stem cells (HSCs) depending on age; ETR induced HSC quiescence in young mice but caused HSC senescence in old mice. ETR has differential effects on modulation of the musculoskeletal and hematopoietic systems in old mice. In other words, endurance exercise is a double-edged sword for successful aging, and great effort is required to establish exercise strategies for healthy aging.

Targeting the JAK2V617F Hematopoietic Stem Cell through Beta1 Integrin

The major barrier to progress in treatment of primary myelofibrosis (PMF) is the lack of effective therapies targeting the malignant JAK2 V617F hematopoietic stem cell (HSC), the source of disease progression and relapse. Expression of JAK2 V617F induces abnormal proliferation and differentiation of HSC, affecting all blood lineages but most prominently the erythro-megakaryocytic lineage, leading to clonal megakaryocyte proliferation, erythrocytosis and thrombocytosis. In our previously published work ( Matsuura et al, Blood 2020) we identified elevated expression of the α5 subunit of the α5β1 integrin receptor as a driver of JAK2 V617F megakaryocyte proliferation. Expression of β1 integrin subunit was similar in wild-type (WT) and JAK2 V617F megakaryocytes, but activation of β1 integrin, detected by the conformation-specific 9EG7 antibody, was elevated in JAK2 V617F megakaryocytes. Following up on those findings, we detected elevated β1 integrin activation in long-term hematopoietic stem cells (HSC LT, LSKCD48-CD150+) from JAK2 V617F transgenic mice ( Xing et al, Blood 2008) when compared to WT controls. To determine whether inhibition of β1 integrin in vivo reduces the number of HSC LT in JAK2 V617F transgenic mice, we examined HSC LT 24 hours after intravenous administration of 2mg/Kg of anti-β1 integrin inhibitory antibody HMβ1-1. This regimen led to a sharp reduction in number and percentage of HSC LT in JAK2 V617F transgenic mice, to 45% of control antibody-treated animals. The effect on WT HSC LT was negligible. HSC ST (LSKCD48-CD150-) and MPP2 (LSKCD48+CD150+) were also reduced, but the reduction was not statistically significant. MPP3/4 (LSKCD48+CD150-) and LSK were not affected. Megakaryocyte progenitors (LKCD41+CD150+) were significantly reduced. Inhibition of β1 integrin also sharply reduced peripheral blood platelet numbers in WT animals. The reduction in JAK2 V617F transgenic mice was not statistically significant. A longer observation time of 7 days demonstrated that while WT animals were able to mount a robust regenerative response with slight but significant increases in HSC LT and normalization of platelet numbers, in JAK2 V617F transgenic mice the number of HSC LT remained depressed, as did downstream stem/progenitor populations at various significance levels. To gain insight into the mechanisms that lead to reduction in HSC LT, we examined apoptosis, cell cycle and HSC mobilization, all processes known to be affected by β1 integrin. Although basal expression of the apoptotic marker annexin V was decreased in JAK2 V617F HSC LT compared to WT, no differences were detected upon administration of HMβ1-1 in both genotypes at 4 hours post-administration. Similarly, peripheral blood colony assays revealed a basally higher level of stem/progenitor mobilization in JAK2 V617F, which did not significantly increase upon HMβ1-1 administration. In contrast, the cell cycle profile, examined at 4 hours post-administration to account for the earliest effects, demonstrated a decrease in G0 phase cells and increase in S/G2/M cells indicating cell cycle induction and exit from quiescence in both WT and JAK2 V617F HSC LT. The results indicate that inhibition of β1 integrin disrupts the adhesion-mediated cell cycle suppression in both WT and JAK2 V617F HSC LT leading to exit from quiescence. However, the downstream consequences of cell division in HSC LT differ significantly, with an apparent loss of regenerative potential in JAK2 V617F HSC LT, at least at the observed timepoints. Longer observation times are required to determine whether continuous β1 integrin inhibition can lead to JAK2 V617F HSC LT exhaustion. More sensitive assays to monitor mobilization are warranted to quantify possible minute amounts of mobilized HSC LT. Our preclinical studies indicate that β1 integrin is an attractive target to specifically reduce the number of JAK2 V617F HSC LTin vivo. The role of β1 integrin in HSC LT has not been thoroughly studied due to lack of phenotype in β1 integrin-deficient HSC ( Brakebush et al, Immunity 2002). Our studies using the anti-β1 integrin antibody in WT HSC LT confirm previous findings. However, β1 integrin seems to be critical for maintenance of JAK2 V617F HSC LT, offering a possible therapeutic window. Understanding how β1 integrin supports quiescence in HSC LT will bring us a step closer to understanding the mechanisms that drive malignancy and fitness in JAK2 V617F HSC LT.

A c- MPL Splice Variant up-Regulated in Myeloproliferative Neoplasms Modulates Thrombopoietin Signaling and Induces Cycling of Hematopoietic Stem Cells and Inflammatory Progenitors

Introduction: Myeloproliferative neoplasms (MPN) are clonal disorders caused by mutations that activate JAK/STAT signaling through the thrombopoietin receptor, MPL, leading to overproduction of myeloid lineages. MPL is the only hematopoietic growth factor receptor expressed on the surface of hematopoietic stem cells (HSC) where it maintains their quiescence by tethering HSCs to osteoblasts in the endosteal niche. MPL is also expressed on megakaryocytes and platelets. Individuals born with loss of function mutations in the distal MPL cytokine receptor homology domain (CRHD) develop thrombocytopenia and progressive bone marrow failure. How MPL functions in MPN remains unclear. Here, we describe a novel MPL splice variant ( MPL-SV) that is present in CD34 + cells and platelets from healthy controls, but overexpressed in the MPN. We therefore sought to: 1) define MPL-SV function in hematopoiesis and MPN, and, 2) elucidate mechanisms underlying MPL-SV. Methods: After identifying a novel MPL-SV transcript from JAK2 V617F MPNplatelets, we compared its expression to MPL transcripts lacking the deletion in MPN patients and healthy controls. To assess its function, we generated transgenic (Tg) mice expressing varied levels of MPL-SV in HSC. Next, we examined responses of the Tg mice to thrombopoietin (THPO), hematopoietic stem progenitor cell (HSPC) frequency by flow cytometry, and HSPC function by colony-forming assays (CFA). To elucidate mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. Results: Using primers to amplify the MPL CRHD in platelets from patients with JAK2 V617F MPN, we discovered an MPL- SV lacking 7 amino acids in the MPL amino-terminal domain, a common hotspot for activating MPN mutations. MPL-SV is present in CD34 + cells and platelets of healthy controls, but overexpressed in MPN relative to MPL transcripts lacking the deletion. To examine its function, we generated transgenic (Tg) mice expressing MPL-SV in HSC. Tg miceexpressing a single copy of MPL-SV have unperturbed hematopoiesis, whereas Tg mice expressing 6 copies of the transgene ( MPL-SV-6) develop marked thrombocytopenia, mild splenomegaly, and a modest increase in red cells by 9-12 weeks of age. Baseline serum THPO levels are elevated in Tg MPL-SV-6 mice and they fail to increase platelet counts following exogenous THPO administration, suggesting defective function of MPL-SV in binding and/or internalizing THPO. To further assess MPL-SV function, we compared CFA from HSPC of Tg MPL-SV-6 and wildtype mice, which show a decrease in total colony forming units (CFU) from BM or spleen-derived MPL-SV-6 HSPC. Accordingly, there was a decrease in LSK cells, multi-potent progenitors (MPPs), and megakaryocytes (Mk) in BM by flow cytometry. By contrast, spleens show increases in LK cells and short-term HSC. Further analysis by scRNAseq of BM-derived LSK cells reveal a marked decrease in both quiescent HSC and HSC with a Mk-bias associated with a relative expansion in more proliferative HSC, including those with a bias towards erythroid and mature myeloid differentiation. Pathway analysis reveal that MPL-SV activates transcriptional networks involved in TNF-α signaling via NF-κB and oxidative phosphorylation. Analysis of cell-cell interactions by CellChat show decreased signaling through intercellular adhesion molecule 1 (ICAM-1) in MPL-SV-6 HSC compared to controls. Importantly, ICAM1 maintains quiescent HSC within their BM niche. Similarly, cell cycle analysis from scRNAseq indicate that quiescent HSC are stimulated to cycle and generate HSC primed to differentiate into erythroid and myeloid progeny. Conclusions: We discovered a novel MPL-SV that is up-regulated in MPN. In Tg mice, MPL-SV impairs responses to THPO and disrupts hematopoiesis by depleting quiescent HSC and BM megakaryocytes. Single cell transcriptomics suggest that MPL-SV drives expansion of cycling HSC and HSC primed to differentiate into progenitors involved in inflammation, including myeloid cells. Mechanistically, HSPC expressing MPL-SV-6 activate transcriptional networks involved in TNFα signaling via NF-κB pathways. Our results highlight the central role for MPL in hematopoiesis and suggest that aberrant MPL splicing contributes to MPN pathogenesis.

Mitigating Oxidative Stress Promotes Quiescence of Hematopoietic Stem Cells from Concurrent TLR4 Activation and IL-10R Blockade Mediated Inflammation

Inflammatory stress provides a clonogenic advantage towards CHIP mutant hematopoietic stem and progenitor cells (HSPCs) via different mechanisms including resistance to apoptosis, enhanced self-renewal and limited generation of reactive oxygen species. Reactive oxygen species (ROS) play a causative role in hematopoietic stem cell (HSC) dysfunction due to exit from quiescence and replication stress-induced stem cell exhaustion. Oxidative stress mediated decline in the fitness of WT hematopoietic stem cells (HSCs) may allow mutant HSCs to gain a selective advantage. Therefore, protection of WT HSCs from ROS may maintain their fitness, and prevent the selective expansion of mutant cells. To test whether the anti-oxidant N-Acetylcysteine (N-AC) protects HSCs from chronic inflammatory stress, we mice were challenged daily with lipopolysaccharide (LPS) (5μg, intraperitoneal) or saline control for 30 days while their drinking water was supplemented with N-AC at 2mg/ml or untreated regular water. At day 30, bone marrow was harvested for competitive repopulation assays using equal numbers of whole bone marrow cells from each competitor (Figure 1A). As expected, bone marrow from mice exposed to 30 days of chronic LPS on regular water showed inferior reconstitution compared to saline exposed mice. However, bone marrow from mice exposed to 30 days of LPS and maintained on N-AC supplemented water performed equivalently in competitive transplants as saline exposed mice. To demonstrate that this functional defect of HSCs stemming from chronic LPS treatment is dependent on increased HSC divisions, we challenged TetOP-H2B-GFP mice with LPS for 30 days and assessed H2B-GFP label retention in immunophenotypically defined long-term HSCs. Progenitor populations in the bone marrow demonstrated low levels of H2B-GFP expression while LT-HSCs showed high GFP signal indicating low turnover. Meanwhile, LPS treatment led to increased cell divisions in LT-HSCs compared to saline treated mice. We hypothesized that N-AC protects HSCs via reducing entry into cell cycle in response to inflammatory stimuli, which is ROS dependent. We assessed the impact of N-AC treatment on ROS in HSCs and its correlation with cell cycling. To induce acute inflammation, wildtype (WT) mice were injected with LPS (5μg) and IL-10R blocking antibody (0.1mg). IL-10R blocking antibody was used in conjunction with LPS because we have previously found that IL-10R blockade extends LPS induced HSC proliferation. Intracellular ROS was determined using the ROS sensitive dye, CM-H 2DCFDA (DCF). The DCF signal was increased in HSCs (Lin -, c-Kit +, Sca1 +, CD150 +, CD48 -) in response to TLR4 activation by LPS and the addition of αIL-10R further augmented this increase in ROS (Figure 1B). Pre-treatment with N-AC significantly reduced the increase in ROS mediated by LPS alone. Cell cycle status was determined by intracellular Ki67 staining of HSCs. LPS significantly increased HSC proliferation as determined by increased percentage of cells in the G1 phase of the cell cycle while IL-10R blockade further augmented HSC cycling. Mice pre-treated with N-AC did not show significant increase in HSC proliferation indicating protection from ROS mediated exit from quiescence. In conclusion, incorporation of N-AC into drinking water protected WT HSCs from inflammation induced exhaustion and promoted persistence of quiescence in HSCs. N-AC could potentially be utilized as a preventative method to preserve fitness of HSCs, particularly in those with chronic inflammatory stress. Moreover, preservation of HSC fitness should be evaluated for its ability to prevent emergence of clonal hematopoiesis.

High Mobility Group A1 Chromatin Regulators Drive Regenerative Capacity during Stress Hematopoiesis through Modulation of TNF-α and Aging Transcriptional Networks

Introduction: Chromatin structure and epigenetic modifications are key determinants of regenerative function and cell fate decisions, although mechanisms governing these properties in hematopoiesis are only beginning to emerge. The High Mobility Group A1 ( HMGA1) chromatin regulator gene is induced by growth factors and highly expressed during embryogenesis and in hematopoietic stem cells (HSCs), but silenced in differentiated tissues ( Cancer Res 2018,78:1890). In leukemia and solid tumors, HMGA1 is re-expressed where it activates enhancers that induce stem cell transcriptional networks, leading to aberrant proliferation and differentiation ( Blood 2022,139:2797; JCI 2023 133:e151601). HMGA1 also drives self-renewal by amplifying Wnt networks in intestinal stem cells ( Nature Comm 2017,8:15008). However, its role in normal hematopoiesis is unknown. We therefore sought to: 1) Define HMGA1 function in HSC, and, 2) Test the hypothesis that modulating HMGA1 epigenetic networks enhances regenerative function. Methods: First, we compared HMGA1 expression across hematopoietic stem and progenitor cells (HSPC) and differentiated progeny in humans and mice. Next, we generated mice with global Hmga1 knock-out ( Hmga1-KO) or Hmga1 deficiency confined to HSC (vav- Hmga1-KO) to assess HSPC function in clonogenic assays, competitive bone marrow transplantation (cBMT), and following irradiation. To elucidate underlying mechanisms, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells followed by pathway analysis. To assess HMGA1 function in human CD34 + cells, we used CRISPR to inactivate HMGA1. Results: HMGA1 is expressed in HSC, multipotent progenitors (MPP), and megakaryocyte-erythroid progenitors (MEP), with decreasing levels after differentiation, suggesting that HMGA1 functions in stem and progenitors. However, steady state hematopoiesis is unperturbed in both Hmga1-KO and vav- Hmga1-KO mice up to 18 months. By contrast, HSPC lacking Hmga1 show decreases in total colony forming units (CFU), but only after serial replating, suggesting that Hmga1 deficiency impairs long-term HSC (LT-HSC) regenerative function. Accordingly, in cBMT assays, Hmga1-KO HSC are outcompeted by HSC with both Hmga1 alleles, with most pronounced effects after serial transplantation. Serial cBMT from young donors (6 weeks) show decreases in LT-HSC, short-term HSC (ST-HSC), and progenitors from Hmga1 KO HSPC compared to control HSPC. Results were similar with older donors (4 months, 18 months). Following irradiation, vav- Hmga1-KO mice have impaired platelet recovery, although the frequency of HSPC in bone marrow were similar to controls. HSPC function in clonogenic assays is also impaired after irradiation, as evidenced by decreases in total CFU, GM-CFU and megakaryocyte (Mk)-CFU. To determine how HMGA1 modulates cell state, we compared scRNAseq in BM-derived LSK cells from adult mice (4 months old) with intact Hmga1 to those with Hmga1-KO. The transcriptomes in HSPC lacking Hmga1 were distinct from those with both Hmga1 alleles intact. The Hmga1 KO HSPC show a decrease in quiescent HSC (marked by high Procr expression), with a relative expansion in more proliferative HSC and early progenitors. In human CD34 + cells, CRISPR-mediated HMGA1 inactivation decreases total CFU, GM-CFU, and Mk-CFU. To elucidate underlying mechanisms, we applied gene set enrichment analyses to scRNAseq results which revealed that Hmga1 activates TNF-a signaling via NF-κB, a pathway that maintains HSC survival and regeneration following inflammatory stress. Hmga1 also represses aging-related transcriptional networks. Further, TNFa partially rescues HSC with Hmga1 deficiency in serial clonogenicity assays. Conclusions: HMGA1 deficiency impairs HSC regenerative capacity under conditions of stress hematopoiesis by depleting quiescent HSC. Mechanistically, HMGA1 induces transcriptional networks involved in survival signals, including TNFa -NF-κB networks, while repressing transcriptional networks activated with aging. Further, TNF-apartially rescues regenerative capacity in clonogenicity assays. Our findings highlight HMGA1 as a critical factor in stress hematopoiesis and suggest that modifying its function could enhance regenerative capacity and repress aging pathways following BMT or irradiation.

Single Cell Transcriptomics Reveal That HMGA1 Chromatin Regulators Drive Transcriptional Networks Involved in Megakaryocyte Expansion, Platelet Activation, and Interferon Signaling in JAK2 V617F Myeloproliferative Neoplasms

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling, thromboses, abnormal bleeding, and increased risk of transformation to myelofibrosis (MF) or acute myeloid leukemia (AML). Importantly, clinical outcomes are poor after transformation and actionable mechanisms that drive progression remain elusive. We discovered that High Mobility Group A1 ( HMGA1) chromatin regulators are required for transformation to AML in preclinical models of MPN by up-regulating transcriptional networks involved in GATA2 and proliferation (Li et al, Blood, 2022). Unexpectedly, loss of just a single Hmga1 allele within HSC prevents progression to MF in JAK2 V617F mice. Intriguingly, Hmga1 also drives fibrosis and tumor progression in pancreatic tumor models (Chia et al, JCI 2023). We therefore sought to elucidate mechanisms underlying HMGA1 early in progression that could be targeted in therapy to prevent MF. Methods : To elucidate Hmga1 function early in MPN, we performed single cell RNA sequencing (scRNAseq) of bone marrow (BM)-derived Lin -, Sca +, c-kit + (LSK) cells from JAK2 V617F mice at 16 weeks old before the development of MF with both Hmga1 alleles intact ( Hmga1+/+) compared to those with haploinsufficiency ( Hmga1+/-). We used gene set enrichment analysis (GSEA) to identify Hmga1-dependent transcriptional networks, CellChat to define cell interactions, and trajectory analysis to assess differentiation. To identify relevant networks in patients, we assessed HMGA1 pathways in transcriptomes from platelets, peripheral blood mononuclear cells, or CD34 + stem and progenitor cells from MPN patients and healthy, age-matched controls via bulk or scRNAseq. Results: We discovered that loss of just a single Hmga1 allele in HSPC is sufficient to dampen expansion of long-term, quiescent HSC (qHSC), megakaryocyte-biased HSC (Mk-HSC), and megakaryocyte-erythroid-biased HSC (MEP-HSC) while expanding HSC with a lymphoid bias (Ly-HSC) in JAK2 V617Fmice by scRNAseq. Pathway analysis in the most quiescent HSC clusters reveal that Hmga1 induces pathways involved in interferon α (IFNα) and interferon γ (IFNγ) signaling. Trajectory analysis show that JAK2 V617F qHSC with intact Hmga1 have diminished differentiation capacity, leading to decreased Ly-HSC. Because Hmga1 haploinsufficiency dramatically decreased megakaryocyte hyperplasia and thrombocytosis in JAK2 V617Fmice, we focused on HMGA1 transcriptional networks in Mk-HSC and MEP-HSC. Similar to qHSC, Hmga1 up-regulates networks involved in IFNα and IFNγ signaling in Mk- and MEP-biased HSC. In contrast to the qHSC, Hmga1 activates transcriptional networks involved in cell cycle progression in HSC poised to differentiate into Mk and MEP, including: 1) MYC Target pathways, 2) E2F Targets, and, 3) Mitotic Spindle genes. In addition, gene networks involved in oxidative phosphorylation are activated, which associate with proliferative states. Unexpectedly, we also discovered that Hmga1 up-regulates genes involved in coagulation and platelet activation, including genes encoding Platelet factor 4, von Willebrand factor, glycoprotein 2b, glycoprotein 3a, and P-selectin. Because megakaryocytes are associated with inflammatory signals in MPN, we queried inflammatory gene networks, which revealed that Hmga1 induces genes encoding IL6, IL1-β, and gene networks involved in signaling through TNFα and NF-κB. CellChat shows that Hmga1 increases MIF receptor-ligand signaling. Moreover, HMGA1 and similar networks are up-regulated in transcriptomes of platelets, PBMC, and CD34 + cells from patients with JAK2 V617F MPN. Conclusions: We uncovered a previously unknown epigenetic program in JAK2 V617FMPN whereby Hmga1 induces transcriptional networks that drive expansion in mutant HSC with greatest impact on HSC biased to differentiate into megakaryocytes (Mk-HSC, MEP-HSC). Mechanistically, Hmga1 up-regulates genes involved in cell cycle progression, platelet activation, and inflammatory signaling in Mk-biased HSC. HMGA1 also induces genes involved in IFNα and IFNγ signaling. Together, our studies reveal a new paradigm whereby HMGA1 drives progression early in MPN by activating transcriptional networks required for megakaryocyte expansion, inflammatory signaling, and IFN networks, the latter of which may sensitize JAK2 mutant HSC to IFN therapy.

FGF23 Inhibition Improves Hematopoietic Stem Cell Transplantation in β-Thalassemia

Clinical evidence has established a link between anemias and skeletal abnormalities, although the underlying molecular mechanisms remain elusive. β-thalassemia (βThal) represents a valuable model for studying congenital anemia and associated bone and bone marrow (BM) microenvironment defects. Osteoporosis has a high morbidity in βThal patients, and we demonstrated that impaired hematopoietic stem cell (HSC) function is due to the prolonged persistence in an altered βThal BM niche ( Aprile et al, 2020). The correction of the genetic cause of βThal, mutations in the β-globin gene, is achieved by transplantation of HSCs from healthy donors or autologous HSCs from patients upon gene therapy. In both scenarios, a comprehensive understanding of HSCs and the BM niche is crucial to obtain successful outcomes. Our research focused on the role of fibroblast growth factor 23 (FGF23), a phosphaturic hormone at the crossroads of bone and erythropoiesis. We demonstrated that the elevated erythropoietin characteristic of the disease, leads to increased FGF23 production in both the βThal mouse model ( th3) and βThal patients, negatively affecting bone homeostasis and the interaction between HSCs and the stromal niche. Notably, in vivo FGF23 inhibition by cFGF23 peptide in th3 mice fully rescued bone defects, BM niche and HSC function ( Aprile, Raggi et al, 2023). To mimic the allogenic transplantation, we transplanted wt and th3 cells in a competitive setting into untreated th3 or th3+cFGF23 recipients. We observed partially enhanced engraftment in the cFGF23-treated mice indicating that FGF23 inhibition improves the supportive capacity of the damaged niche. We hypothesized that prolonged cFGF23 administration to recipient mice after transplantation could further restore the BM niche and boost the graft, thus achieving therapeutic outcomes. To establish the proper dose regimen with withdrawal periods for prolonged cFGF23 treatment, we analyzed bone mineral density (BMD) and HSC cell cycle at 1 and 2 weeks after discontinuing cFGF23 administration. Our results showed that rescued BMD was sustained after the discontinuation of treatment for 1 and 2 weeks ( th3 vs. th3+cFGF23 _1wk vs. th3+cFGF23_2wks: 125.9±3.8 vs. 181±19.9 vs. 142±1.6 mg/cm 3, p&amp;lt;0.01). Ongoing histomorphometric analysis will provide a better characterization of bone quality. Additionally, we observed the restoration of defective quiescence in th3 HSCs after 1- and 2 weeks ( th3 vs. th3+cFGF23 _1wk vs. th3+cFGF23_2wks: 77.8±1,4 vs. 92.1±1.8 vs. 87.8±0.9 on Lin neg BM cells, p&amp;lt;0.001). To test autologous transplantation upon gene therapy, we transduced th3 Lin neg BM cells with a lentiviral vector carrying the β-globin gene (GLOBE LV) at MOI 5 resulted in a mean vector copy number of 1 and a transduction efficiency of 50%. We performed competitive transplantation using 1x10 5 (CD45.1) transduced donor th3 cells and 1x10 5 (CD45.2) competitor mock-transduced th3 cells into busulfan-conditioned (CD45.2) untreated th3 or th3+cFGF23 recipient mice. The treated recipient mice received two doses of cFGF23 every 2 weeks as a maintenance regimen. We observed higher engraftment of transduced cells in the peripheral blood of cFGF23-treated mice at 8 weeks post-transplant ( th3 vs. th3+cFGF23: 4.9±1.1 vs. 10.7±1.9 % of CD45.1 engrafted cells, p&amp;lt;0.05). Comparative evaluation of hematological parameters and molecular analysis of transduced cells are ongoing. Targeting FGF23 signaling provides a promising strategy to improve bone defects and HSC-niche alterations in βThal. Here, we show that the use of FGF23 inhibition in the transplantation setting in β-thalassemia results in superior engraftment of donor cells in treated recipients, indicating a potential application to ameliorate the clinical outcome.