Hematopoietic Stem Cell Function Research Articles

Epigenetics of hematopoietic stem cell aging.

The development of new antiaging medicines is of great interest to the current elderly and aging population. Aging of the hematopoietic system is attributed to the aging of hematopoietic stem cells (HSCs), and epigenetic alterations are the key effectors driving HSC aging. Understanding the epigenetics of HSC aging holds promise of providing new insights for combating HSC aging and age-related hematological malignancies. Aging is characterized by the progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. During aging, the HSCs undergo both quantitative and qualitative changes. These functional changes in HSCs cause dysregulated hematopoiesis, resulting in anemia, immune dysfunction, and an increased risk of hematological malignancies. Various cell-intrinsic and cell-extrinsic effectors influencing HSC aging have also been identified. Epigenetic alterations are one such mechanism. Cumulative epigenetic alterations in aged HSCs affect their fate, leading to aberrant self-renewal, differentiation, and function of aged HSCs. In turn, these factors provide an opportunity for aged HSCs to expand by modulating their self-renewal and differentiation balance, thereby contributing to the development of hematological malignancies.

Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of the cell cycle

AbstractLoss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment and limit the global variability in gene expression and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation.

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47phox-deficient chronic granulomatous disease

The p47phox-deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 cDNA knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67phox, an NADPH oxidase subunit that closely interacts with p47phox and is predominantly expressed in myeloid cells. This approach restored p47phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47phox-deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47phox and p67phox, ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

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 myc-associated zinc finger protein (MAZ) and nuclear transcription factor Y subunit alpha (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.

Abstract PO2-06-12: Patients with metastatic triple-negative breast cancer who receive trilaciclib prior to cytotoxic chemotherapy exhibit improved survival after receiving subsequent anticancer therapy

Abstract Background In an open-label, randomized, phase 2 trial in patients (pts) with metastatic triple-negative breast cancer (mTNBC; NCT02978716), administering trilaciclib prior to gemcitabine plus carboplatin (GCb) significantly prolonged overall survival (OS; a key secondary endpoint) versus administering GCb alone (median 19.8 vs 12.6 months; hazard ratio 0.37; P < 0.0001), with a nonsignificant trend toward improved progression-free survival (median 9.0 vs 5.7 months; hazard ratio 0.62; P = 0.13; Tan et al. Clin Cancer Res. 2022). Given murine data suggesting trilaciclib may preserve the long-term function of hematopoietic stem and progenitor cells (He et al. Sci Transl Med. 2017) and improve T-cell memory (Goel et al. Nature. 2017; Lelliott et al. Cancer Discov. 2021; Heckler et al. Cancer Discov. 2021), we examined survival of pts with mTNBC who received subsequent lines of therapy after receiving GCb with or without trilaciclib. Methods In the phase 2 trial, pts with ≤ 2 prior chemotherapy regimens for locally recurrent or metastatic TNBC were randomized 1:1:1 to receive GCb on days 1 and 8, trilaciclib prior to GCb on days 1 and 8, or trilaciclib alone on days 1 and 8 and prior to GCb on days 2 and 9. The 2 trilaciclib arms were combined for this analysis. To determine if trilaciclib improved outcomes in pts who received additional therapy after GCb, a post hoc survival analysis was performed using data from pts who received any subsequent anticancer therapy (SACT). Results In the subanalysis of data from pts with mTNBC who received any SACT after the phase 2 trial, clinical characteristics such as demographics, disease variables, duration from study treatment to first SACT, and type of SACT were balanced between the trilaciclib (n = 43) and placebo (n = 20) arms. The most common SACTs were gemcitabine, capecitabine, eribulin, taxanes, checkpoint inhibitors, carboplatin, and anthracyclines. Median OS in pts who had previously received trilaciclib was 32.7 months versus 12.8 months in those who had received placebo, with increasing separation of survival curves over time. Median OS from start of first SACT was 17.4 months in the trilaciclib arms vs 5.8 months in the placebo arm. Improved survival and sustained separation of curves was also observed in pts unable to receive SACT (trilaciclib, n = 25; placebo, n = 14), although the magnitude of benefit was smaller (median 9.4 vs 5.4 months). Mechanistic in vivo murine and in vitro human studies are underway to examine the effect of trilaciclib on the generation of memory T cells. These studies will assess the preferential differentiation of specific memory T-cell subsets along with in vivo assessment of memory T-cell responses following a rechallenge. Further data will be presented to evaluate whether the enhanced generation and preservation of a memory T-cell pool is supportive of improved outcomes in pts receiving SACT. Conclusions Data from the phase 2 study suggest that pts with mTNBC who received trilaciclib prior to cytotoxic chemotherapy have prolonged survival, which is notably improved for pts who are able to receive SACT following discontinuation of trilaciclib. The survival benefit from trilaciclib extends to pts who subsequently received immune checkpoint inhibitors and/or chemotherapy, and also to those who did not receive any SACT. Improved OS in pts receiving trilaciclib may be associated with protection of hematopoietic stem and progenitor cells and formation of a memory T-cell pool, which is critical for long-term immune surveillance and in eliciting rapid recall responses. OS will be evaluated in pts with mTNBC in the ongoing phase 3 trial of trilaciclib prior to GCb (PRESERVE 2; NCT04799249) and phase 2 trial of trilaciclib prior to sacituzumab govitecan (NCT05113966). Citation Format: Shom Goel, Joyce O'Shaughnessy, Kun-Hui Lu, Andrew Beelen, Symantha Melemed, John Yi, Michael Danso, Antoinette Tan. Patients with metastatic triple-negative breast cancer who receive trilaciclib prior to cytotoxic chemotherapy exhibit improved survival after receiving subsequent anticancer therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-12.

Advances in hematopoietic stem cells ex vivo expansion associated with bone marrow niche.

Hematopoietic stem cells (HSCs) are an ideal source for the treatment of many hematological diseases and malignancies, as well as diseases of other systems, because of their two important features, self-renewal and multipotential differentiation, which have the ability to rebuild the blood system and immune system of the body. However, so far, the insufficient number of available HSCs, whether from bone marrow (BM), mobilized peripheral blood or umbilical cord blood, is still the main restricting factor for the clinical application. Therefore, strategies to expand HSCs numbers and maintain HSCs functions through ex vivo culture are urgently required. In this review, we outline the basic biology characteristics of HSCs, and focus on the regulatory factors in BM niche affecting the functions of HSCs. Then, we introduce several representative strategies used for HSCs from these three sources ex vivo expansion associated with BM niche. These findings have deepened our understanding of the mechanisms by which HSCs balance self-renewal and differentiation and provided a theoretical basis for the efficient clinical HSCs expansion.

Unveiling the role of hypoxia-inducible factor 2alpha in osteoporosis: Implications for bone health.

Osteoporosis (OP) has become a major public health problem worldwide. Most OP treatments are based on the inhibition of bone resorption, and it is necessary to identify additional treatments aimed at enhancing osteogenesis. In the bone marrow (BM) niche, bone mesenchymal stem cells (BMSCs) are exposed to a hypoxic environment. Recently, a few studies have demonstrated that hypoxia-inducible factor 2alpha (HIF-2α) is involved in BMSC osteogenic differentiation, but the molecular mechanism involved has not been determined. To investigate the effect of HIF-2α on the osteogenic and adipogenic differentiation of BMSCs and the hematopoietic function of hematopoietic stem cells (HSCs) in the BM niche on the progression of OP. Mice with BMSC-specific HIF-2α knockout (Prx1-Cre;Hif-2αfl/fl mice) were used for in vivo experiments. Bone quantification was performed on mice of two genotypes with three interventions: Bilateral ovariectomy, semilethal irradiation, and dexamethasone treatment. Moreover, the hematopoietic function of HSCs in the BM niche was compared between the two mouse genotypes. In vitro, the HIF-2α agonist roxadustat and the HIF-2α inhibitor PT2399 were used to investigate the function of HIF-2α in BMSC osteogenic and adipogenic differentiation. Finally, we investigated the effect of HIF-2α on BMSCs via treatment with the mechanistic target of rapamycin (mTOR) agonist MHY1485 and the mTOR inhibitor rapamycin. The quantitative index determined by microcomputed tomography indicated that the femoral bone density of Prx1-Cre;Hif-2αfl/fl mice was lower than that of Hif-2αfl/fl mice under the three intervention conditions. In vitro, Hif-2αfl/fl mouse BMSCs were cultured and treated with the HIF-2α agonist roxadustat, and after 7 d of BMSC adipogenic differentiation, the oil red O staining intensity and mRNA expression levels of adipogenesis-related genes in BMSCs treated with roxadustat were decreased; in addition, after 14 d of osteogenic differentiation, BMSCs treated with roxadustat exhibited increased expression of osteogenesis-related genes. The opposite effects were shown for mouse BMSCs treated with the HIF-2α inhibitor PT2399. The mTOR inhibitor rapamycin was used to confirm that HIF-2α regulated BMSC osteogenic and adipogenic differentiation by inhibiting the mTOR pathway. Consequently, there was no significant difference in the hematopoietic function of HSCs between Prx1-Cre;Hif-2αfl/fl and Hif-2αfl/fl mice. Our study showed that inhibition of HIF-2α decreases bone mass by inhibiting the osteogenic differentiation and increasing the adipogenic differentiation of BMSCs through inhibition of mTOR signaling in the BM niche.

Crosstalk between DNA Damage Repair and Metabolic Regulation in Hematopoietic Stem Cells.

Self-renewal and differentiation are two characteristics of hematopoietic stem cells (HSCs). Under steady physiological conditions, most primitive HSCs remain quiescent in the bone marrow (BM). They respond to different stimuli to refresh the blood system. The transition from quiescence to activation is accompanied by major changes in metabolism, a fundamental cellular process in living organisms that produces or consumes energy. Cellular metabolism is now considered to be a key regulator of HSC maintenance. Interestingly, HSCs possess a distinct metabolic profile with a preference for glycolysis rather than oxidative phosphorylation (OXPHOS) for energy production. Byproducts from the cellular metabolism can also damage DNA. To counteract such insults, mammalian cells have evolved a complex and efficient DNA damage repair (DDR) system to eliminate various DNA lesions and guard genomic stability. Given the enormous regenerative potential coupled with the lifetime persistence of HSCs, tight control of HSC genome stability is essential. The intersection of DDR and the HSC metabolism has recently emerged as an area of intense research interest, unraveling the profound connections between genomic stability and cellular energetics. In this brief review, we delve into the interplay between DDR deficiency and the metabolic reprogramming of HSCs, shedding light on the dynamic relationship that governs the fate and functionality of these remarkable stem cells. Understanding the crosstalk between DDR and the cellular metabolism will open a new avenue of research designed to target these interacting pathways for improving HSC function and treating hematologic disorders.

Bone Marrow Adipose Tissue.

Bone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, skeletal remodeling, and immune system control. Bone marrow adipose tissue (BMAT) was long simply considered a filler of space, but now it is known that it instead constitutes an essential element of the BM microenvironment that participates in homeostasis, influences bone health and bone remodeling, alters hematopoietic stem cell functions, contributes to the commitment of mesenchymal stem cells, provides effects to immune homeostasis and defense against infections, and participates in energy metabolism and inflammation. BMAT has emerged as a significant contributor to the development and progression of various diseases, shedding light on its complex relationship with health. Notably, BMAT has been implicated in metabolic disorders, hematological malignancies, and skeletal conditions. BMAT has been shown to support the proliferation of tumor cells in acute myeloid leukemia and niche adipocytes have been found to protect cancer cells against chemotherapy, contributing to treatment resistance. Moreover, BMAT's impact on bone density and remodeling can lead to conditions like osteoporosis, where high levels of BMAT are inversely correlated with bone mineral density, increasing the risk of fractures. BMAT has also been associated with diabetes, obesity, and anorexia nervosa, with varying effects on individuals depending on their weight and health status. Understanding the interaction between adipocytes and different diseases may lead to new therapeutic strategies.

Advance of research on the role of BCL11A in the occurrence and treatment of β-Thalassemia

β-Thalassemia is a single-gene disease caused by mutations in β-globin and has a distinct geographical characteristics. Current treatment for patients with moderate to severe thalassemia has mainly relied on long-term blood transfusion and/or hematopoietic stem cell transplantation. B cell lymphoma/leukemia 11A (BCL11A) as a transcriptional repressor plays a vital role in monitoring γ/β hemoglobin switching, maintaining the normal function of hematopoietic stem cells, and regulating erythrocyte differentiation and lymphocyte development. With the rapid progress in gene editing technology, the BCL11A as a therapeutic target for β-thalassemia has shown promising results. This article has systematically summarized the regulatory mechanism and therapeutic potential of the BCL11A, with an aim to provide new ideas for the treatment of β-thalassemia.

Aging brain: exploring the interplay between bone marrow aging, immunosenescence, and neuroinflammation.

Aging is a complex process characterized by a myriad of physiological changes, including alterations in the immune system termed immunosenescence. It exerts profound effects on both the bone marrow and the central nervous system, with significant implications for immunosenescence in neurological contexts. Our mini-review explores the complex relationship between bone marrow aging and its impact on immunosenescence, specifically within the context of neurological diseases. The bone marrow serves as a crucial hub for hematopoiesis and immune cell production, yet with age, it undergoes significant alterations, including alterations in hematopoietic stem cell function, niche composition, and inflammatory signaling. These age-related shifts in the bone marrow microenvironment contribute to dysregulation of immune cell homeostasis and function, impacting neuroinflammatory processes and neuronal health. In our review, we aim to explore the complex cellular and molecular mechanisms that link bone marrow aging to immunosenescence, inflammaging, and neuroinflammation, with a specific focus on their relevance to the pathophysiology of age-related neurological disorders. By exploring this interplay, we strive to provide a comprehensive understanding of how bone marrow aging impacts immune function and contributes to the progression of neurological diseases in aging individuals. Ultimately, this knowledge can hold substantial promise for the development of innovative therapeutic interventions aimed at preserving immune function and mitigating the progression of neurological disorders in the elderly population.

Aging-induced MCPH1 translocation activates necroptosis and impairs hematopoietic stem cell function.

DNA damage contributes to the aging of hematopoietic stem cells (HSCs), yet the underlying molecular mechanisms are not fully understood. In this study, we identified a heterogeneous functional role of microcephalin (MCPH1) in the nucleus and cytoplasm of mouse HSCs. In the nucleus, MCPH1 maintains genomic stability, whereas in the cytoplasm, it prevents necroptosis by binding with p-RIPK3. Aging triggers MCPH1 translocation from cytosol to nucleus, reducing its cytoplasmic retention and leading to the activation of necroptosis and deterioration of HSC function. Mechanistically, we found that KAT7-mediated lysine acetylation within the NLS motif of MCPH1 in response to DNA damage facilitates its nuclear translocation. Targeted mutation of these lysines inhibits MCPH1 translocation and, consequently, compromises necroptosis. The dysfunction of necroptosis signaling, in turn, improves the function of aged HSCs. In summary, our findings demonstrate that DNA damage-induced redistribution of MCPH1 promotes HSC aging and could have broader implications for aging and aging-related diseases.

Asymmetric cell division of hematopoietic stem cells: recent advances, emerging concepts, and future perspectives

Hematopoietic stem cells (HSCs) can self-renew and differentiate for the entire life of an organism to produce new blood cells when needed. This process is regulated by asymmetric cell division (ACD), an evolutionarily conserved mechanism whereby cell fate determinants are unequally segregated into the daughter cells during division to instruct different cell fates. After many years of controversy, recent technical advances in microscopy, imaging, and bioinformatics make it now possible to visualize and quantify how factors segregate asymmetrically in dividing HSCs and lead to predictable changes in daughter cell fates many days later. While the molecular processes behind ACD in HSCs are still poorly understood, accumulating evidence suggests that lysosomes and other organelles, including mitochondria, autophagosomes, mitophagosomes, and recycling endosomes can segregate asymmetrically and act as cell fate determinants during divisions. Asymmetric segregation of lysosomes and mitochondria has been shown to predict mitochondrial activity, translation, and differentiation of HSC daughter cells and their offspring. This discovery and recent seminal findings show that lysosomes, once considered to be merely the trash bin of the cell, regulate many aspects of HSC biology and are crucial for the maintenance of quiescence and stem cell function. Here we provide a historical perspective and discuss the recent advances in our understanding of ACD and the role of lysosomes in HSC function. We discuss the limitations of past studies, talk about emerging concepts, and suggest critical next steps required to move the field forward.

Abstract 7077: The oncogenic transcription factor ERG has distinct roles and signaling in different prostate cancer cell types

Abstract ERG is the most common oncogene in prostate cancer, as it is aberrantly expressed in about 50% of cases due to the TMPRSS2/ERG gene rearrangement. ERG is not normally expressed in the adult prostate. The normal function of ERG is confined to hematopoietic stem cells and blood vessels. Aberrant expression of ERG in prostate epithelial cells is oncogenic when combined with mutations that activate the PI3K/AKT signaling pathway. The RAS/MAPK pathway can also regulate ERG function through direct phosphorylation of ERG by ERK. The function of these regulatory events and how they can be exploited to develop therapy for ERG-positive prostate cancer remains unclear. Here we find that ERG expression in prostate epithelial cells with low PI3K/AKT pathway activity drives epithelial to mesenchymal transition and stemness properties. This function requires ERG phosphorylation via a positive feedback loop with the TLR4 signaling pathway, and can be repressed by a TLR4 inhibitor. However, activation of the PI3K/AKT pathway rearranges the ERG cistrome and alters ERG function, where ERG now promotes luminal epithelial differentiation. In the presence of activated AKT, new ERG targets include genes involved in vascular development and ERG phosphorylation is regulated through a positive feedback loop with the VEGF signaling pathway. Despite these differences in cellular differentiation, we find that ERG can promote stemness and tumorigenesis in both AKT-high and AKT-low cellular backgrounds. ERG functions in prostate epithelial cells appear to mirror ERG functions in hematopoietic stem cells and blood vessels. These findings suggest strategies of combinatorial inhibition of ERG function through signaling pathway inhibition. Citation Format: Saranya Rajendran, Peter C. Hollenhorst. The oncogenic transcription factor ERG has distinct roles and signaling in different prostate cancer cell types [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 7077.

Abstract 4398: TET2 accelerates clonal hematopoiesis in an age-dependent manner

Abstract Aging is a process of systemic deterioration and the most significant risk factor for cancers. Clonal hematopoiesis (CH) commonly occurs with aging and links to higher mortality, leukemia risk, and cardiovascular diseases. Age-related CH involves the abnormal clonal expansion of hematopoietic stem cells (HSCs) bearing somatic mutations in genes frequently mutated in leukemia, including genes encoding epigenetic regulators, such as the DNA demethylase TET2. While such mutations are known to alter the HSC epigenome, the mechanisms through which these mutations drive HSC self-renewal, myeloid transformation, and inflammatory response remain elusive. In this study, we hypothesize that aging and CH mutations cooperatively reshape the HSC transcriptomic landscape and enhancing HSC competitive advantage that facilitates clonal expansion. Recently, TET2-mutant CH in humans has been strongly associated with epigenetic age acceleration. Using single-cell multi-omic analyses and flow-cytometric phenotyping, we demonstrated, for the first time, that HSC aging processes at transcriptomic, epigenomic, and cellular levels are accelerated by Tet2 deficiency in mice, in an age-dependent manner. Tet2-deficient HSCs increase the HSC aging scores at an older age of greater than or equal to 16 months, but not at a young or mid-age, based on gene expression and chromatin accessibility at a single-cell resolution. This age acceleration was further validated by epigenetic clock analysis of DNA methylation mapping. Moreover, we found that Tet2 deficiency drives a significant expansion of HSCs, a cellular hallmark of HSC aging, in an age-dependent manner. Specifically, we observed increased expression of S100a6, Cdk6, Egr1 in Tet2-deficient HSCs from older mice, suggesting that Tet2 deficiency hijacks the HSC expansion strategy during HSC aging by enhanced self-renewal. We also observed that the differentially expressed genes between Tet2-deficient older HSCs and control older HSCs are associated with open chromatin accessibility. Many of these genes are enriched in the targets of specific transcription factors (TFs), such as Egr1, Creb1, Stat family and Hoxb8. These target genes play critical roles in inflammatory pathways and HSC functions. In conclusion, our findings reveal that Tet2 deficiency significantly contributes to the acceleration of HSC aging, and hijacks the HSC expansion strategy (e.g., self-renewal) during HSC aging, which exacerbates age-related clonal hematopoiesis. By elucidating the transcriptomic and epigenomic alterations in Tet2-deficient HSCs, our study provides novel insights into how age and somatic mutations collaborate to promote the pathogenesis of age-related hematological diseases. These discoveries not only enhance our understanding of HSC aging mechanisms but also offer potential biomarkers for early detection and targets for therapeutic intervention in age-associated clonal disorders. Citation Format: Shilpita Karmakar, Yang Liu, Efthymios Motakis, Kaustav Sengupta, Travis Roeder, Kristina Mujica, Jennifer Trowbridge, Hideyuki Oguro, Sheng Li. TET2 accelerates clonal hematopoiesis in an age-dependent manner [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 4398.

The histone acetyltransferase KAT6B is required for hematopoietic stem cell development and function

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.