Experimental Hematology

Clinical evidence demonstrates that ex vivo gene therapy and genome engineering of hematopoietic stem and progenitor cells (HSPCs) could represent one-time cures. However, while genome editing itself has become increasingly efficient and precise, the toxic conditioning required for hematopoietic stem cell transplantation remains a major barrier to broad clinical implementation of these otherwise curative therapies. In particular, the use of busulfan for myeloablative conditioning constitutes a major safety concern. While preclinical studies established CD117 as a promising target for antigen-specific therapy, clinical translation faced setbacks balancing efficacy and safety. To overcome current limitations, we generated a new CD117-blocking monoclonal antibody (CIM058) and demonstrate its potency to block wild-type HSPCs. To enable long-term blockade of host HSPCs even after transplantation, we used prime editing to engineer CIM058-resistant human CD34+ HSPCs. When combined, CIM058 and the epitope engineered CD34+ HSPCs ameliorated disease phenotype in a {beta}-thalassemia model. Our results suggest that this approach may overcome the reliance on busulfan or other myeloablative conditioning regimens with their associated morbidities, and by enabling toxin-free conditioning and in vivo selection of edited cells, may facilitate clinical implementation of these highly valuable genetic therapies.

Adult long-term hematopoietic stem cells (LT-HSCs) are classically defined by self-renewal, multilineage regenerative capacity, and relative quiescence, but how and when lifelong LT-HSCs are established during development remains unclear. Here, we demonstrate that Hoxb5 fetal liver HSCs exhibit bona fide LT-HSC activity, including long-term multilineage reconstitution and serial transplantation capacity, whereas Hoxb5- fetal liver HSCs display limited regenerative potential. Embryonic lineage tracing further demonstrates that E14.5 Hoxb5-expressing hematopoietic cells contribute broadly to adult hematopoiesis, including the adult HSC compartment, and give rise to functional adult LT-HSCs. Across developmental stages, single-cell transcriptional profiling revealed that fetal Hoxb5 HSCs remain highly proliferative while maintaining canonical LT-HSC transcriptional programs and superior repopulating activity relative to predominantly quiescent adult Hoxb5 HSCs. Fetal Hoxb5 HSCs also exhibited elevated ITGA4-mediated adhesion programs, and disruption of the ITGA4-VCAM1 axis impaired engraftment following transplantation. Together, these findings establish a developmental continuum linking fetal and adult LT-HSCs and identify enhanced ITGA4-mediated adhesion as a defining feature of fetal LT-HSCs.

Hematopoietic stem cells (HSCs) in the adult mouse can be prospectively isolated to near-purity through phenotypic markers, enabling detailed analysis of stem cell function. Homeobox B5 (Hoxb5) was previously identified as a definitive marker of long-term (LT) HSCs in adult bone marrow1. In contrast, fetal HSCs have not been purified to the same extent. Here, we show that Hoxb5 is expressed in fetal liver (FL) HSCs at embryonic day (E) 12.5-16.5 using a single-color tri-mCherry reporter driven by endogenous Hoxb5 regulation. Prospective purification by stringent multiparameter flow cytometry revealed Hoxb5 FL-HSCs to exhibit robust, multilineage reconstitution upon serial transplantation. Quantitative assays reveal that Hoxb5 enriches FL-HSCs to near-single-cell purity, analogous to its role in the adult bone marrow, underscoring its reliability in distinguishing LT-HSCs throughout hematopoietic ontogeny. Notably, Hoxb5 expression is not exclusive to FL-HSCs, as it is also detected across the fetal liver hematopoietic hierarchy and in fetal liver endothelial cells, suggesting developmental stage-specific regulation of its expression. In addition, single-cell RNA sequencing of FL-HSCs identified distinct transcriptional states defined by Hoxb5 expression. These findings establish Hoxb5 as a robust marker for enhancing the purification of fetal liver phenotypic HSCs (pHSC) and provide a framework for dissecting the molecular regulation of HSC ontogeny.

Myelodysplastic neoplasms (MDS) and related myeloid neoplasms such as chronic myelomonocytic leukaemia (CMML) are clonal haematopoietic stem cell disorders characterised by ineffective and dysplastic haematopoiesis. They are associated with peripheral cytopaenias, variable increases in immature blasts, and a risk of progression to acute myeloid leukaemia. Hypomethylating agents (HMA) can improve blood counts and reduce blasts, but responses are usually limited. Epigenetic rewiring of haematopoietic stem and progenitor cells (HSPC) by HMA enhances hematopoietic output but is influenced by clonal mosaicism, which requires tracking of response at the single cell level to achieve full understanding. We developed SCIMETAR-seq for single-cell interrogation of DNA methylation, target amplicons, and mRNA in FACS-indexed HSPC, then deployed SCIMETAR-seq on CD34+ HSPC from longitudinal HMA-treated patient BM in vitro and in vivo. HMA-induced LINE-1 (L1) demethylation was positively correlated with cell cycling; being lowest in quiescent HSC and highest in erythrocyte progenitors. Erythrocyte progenitor frequencies were particularly increased by HMA exposure. SRSF2 p.P95 genotype did not influence HMA-induced L1 demethylation but was enriched into cells with a CMP immunophenotype, which were transcriptionally biased away from MEP towards granulocytic progenitors. Despite a lack of L1 demethylation in quiescent HSC/MPP after 7 days of HMA treatment in vivo, their transcriptomes were enriched for TNF-, TGF{beta}- and WNT-signaling, suggesting that extrinsic factors secreted by other BM cells in response to HMA mediates reprogramming of quiescent HSC during HMA therapy in vivo.

Hematopoietic stem and progenitor cell (HSPC) niches support lifelong production of blood and immune cells. Recently, we identified a gene signature unique to HSPC niche endothelial cells that is highly conserved across species and developmental time, and includes the scavenger receptors, stab1/2 and mrc1a. Whether these receptors support HSPC development in the niche remains unclear. To investigate this, we used chemical inhibition and CRISPR mutagenesis in zebrafish and found that loss of stab2, and to a lesser degree stab1, reduced the number of embryonic HSPCs labeled by runx1:mCherry. Subsequent analyses of runx1:mCherry; cd41:GFP double transgenic embryos revealed an imbalance in the HSPC pool in stab2 mutants, with reductions in sub-populations containing stem cells and erythroid progenitors, the latter of which was most decreased. Our findings suggest stabilin scavenger receptors support HSPC development in the fetal niche, which could inform clinical strategies for culturing and expanding HSPCs.

The ability to generate functional B cells from human pluripotent stem cells (hPSCs) would open new opportunities to develop novel B cell-based therapies to treat a range of human diseases and disorders. Towards this goal, we established a protocol that promotes the efficient development of B lineage cells from definitive hematopoietic progenitors generated from different hPSC lines. Flow cytometric and multi-omic scRNA-seq analyses revealed that B cell development from hPSCs transitions through the well-established pro-B, pre-B and naive B cell stages, accurately recapitulating B lymphopoiesis in the human adult bone marrow. Importantly, the naive B cells generated with this approach could be induced to mature into plasma cells that secrete antibodies and undergo class switching. Analyses of signaling pathways that regulate B lymphopoiesis in these cultures uncovered a potent inhibitory effect of IL-7 on functional IgH rearrangement, resulting in the development of abnormal cells that failed to undergo pre-B cell maturation. Finally, analysis of the different hPSC-derived hematopoietic programs revealed that both definitive and yolk sac progenitors display B cell potential, indicating that there are distinct developmental sources of human B lineage cells. Taken together, these findings demonstrate the efficient generation of B cells from hPSCs and, in doing so, provide a system for further investigating the earliest stages of human B lymphopoiesis and a source of appropriately staged plasma cells for future therapeutic applications.

Haematopoietic stem cells (HSC), while usually quiescent, can rapidly divide following specific stimuli (mobilization). These HSC can seed additional niches, allowing for the swift generation of essential blood cells. However, studies in mice and humans have clearly demonstrated that cycling bone marrow (BM) HSC (cells in the G1/S/G2/M phases) engraft and reconstitute the haematopoietic system poorly compared with HSC in the G0 phase1. This raises the question why mobilized HSC, immediately following 3 or more cell divisions2, efficiently reconstitute the haematopoietic system. We studied this phenomenon in human HSC using scRNAseq analysis. We found that mobilized HSC rapidly start transcribing genes associated with quiescence, specific for the G0 phase of the cell cycle. We hypothesize that this rapid switch from actively dividing to quiescent cells combined with our extensive RNA expression data will allow us to better define pathways involved in this process.

Hematopoietic stem cells (HSCs) generated from induced pluripotent stem cells (iPSCs) offer a promising patient-specific alternative to allogeneic transplantation, yet current differentiation protocols fail to fully recapitulate in vivo HSC maturation. During mouse development, yolk sac (YS)-derived macrophages populate the aorta-gonad-mesonephros (AGM) region at the time of HSC emergence, but the mechanisms by which they support ex vivo hematopoietic stem and progenitor cell (HSPC) generation remain poorly defined. Bulk RNA sequencing revealed that mature AGM CD206 macrophages upregulate pro-inflammatory cytokines and the adhesion molecule F4/80. Using F4/80 knockout embryos, we identify a previously unreported, niche-specific role for F4/80 in restraining the frequency and colony-forming activity of HSPC subsets in the AGM, while supporting endothelial cell maintenance; this effect was absent in the YS. Lineage-tracing with a Cdh5-CreERT2;Rosa26LSL-tdTomato pulse-chase system confirmed that both CD206 and CD206- AGM cells originate from early YS-derived endothelial precursors, with no evidence of local macrophage generation within the AGM. Functional co-culture assays further demonstrated that the ability of CD206 macrophages to enhance the progenitor potential of hemogenic endothelium is AGM-specific and not an intrinsic, ontogeny-determined property, as YS macrophages failed to confer the same benefit even when paired with AGM endothelial cells, and AGM macrophages were ineffective with YS endothelium. Differential expression and NicheNet ligand-receptor interaction analyses identified a small set of AGM-restricted macrophage genes - including Mmp2, Nrep, Ccl2, and Cxcl16 - which are predicted to interact with both endothelial and cluster cells during endothelial-to-hematopoietic transition. Together, these findings establish that AGM macrophages acquire niche-specific transcriptional and functional properties upon entry into the aortic microenvironment, independent of their YS origin, and identify candidate macrophage-derived factors and a novel regulatory role for F4/80 in shaping HSPC output. These insights may guide the refinement of iPSC-based HSC differentiation protocols through the targeted, temporally controlled addition of macrophage-associated signals.

Hematopoietic stem cells (HSCs) play a pivotal role in the lifelong maintenance of hematopoiesis. However, heterogeneity and age-related alterations in HSC populations hinders accurate HSC analysis. Here, we show that bone marrow (BM) macrophage fragments that preferentially express F4/80 adhere to proliferative rather than dormant HSCs. The adhesion of macrophage fragments to proliferative HSCs occurred throughout the process of BM cell preparation in vitro. Consistently, proliferative HSCs express genes involved in the adhesion of macrophage fragments at higher levels than dormant HSCs. Notably, by using that as a benchmark, dormant HSCs can be easily identified as F4/80lowHSCs throughout their lifespan, thereby revealing that they retain considerable stemness and remain functional with aging. Collectively, we propose a novel and straightforward method for the rapid identification, isolation, and analysis of distinct HSC subpopulations, which will be helpful for a wide range of hematological studies and will provide insights into HSC biology.

Manufacturing red blood cells (RBCs) from human induced pluripotent stem cells (iPSCs) can improve our understanding of embryonic erythropoiesis, foster innovative treatments for RBC-related diseases, and ultimately address clinical blood supply shortages. However, existing systems face low efficiency, enucleation failure, and uncertainty about the develop-mental wave of cultured RBCs. We successfully used self-organized hemanoids to improve iPSC-derived RBC generation. Based on the hypothesis that cellular interactions and 3D organization promote hematopoietic cell fate, we aimed to thoroughly characterize hemanoids. We visualized the spatiotemporal emergence of hematopoiesis by generating a CD43-GFP reporter iPSC line. Imaging and spatial transcriptomics analysis provided de-tailed insight into the hemanoid architecture, identifying stromal cells and hepatoblasts as potential erythropoiesis-supportive elements. The developmental stage mirrors extraembryonic hematopoiesis. Given the difficulties of accessing these early stages in vivo, our system offers a platform not only for further clinical translation but also for exploring hu-man embryonic blood wave dynamics.

Myelodysplastic syndromes (MDS) are driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs), leading to clonal expansion and ineffective hematopoiesis. Hypomethylating agents (HMAs; azacitidine or decitabine) are the standard of care for higher-risk MDS. However, their effects on the bone marrow (BM) microenvironment, and the extent to which these changes correlate with clinical response, remain poorly understood. We performed longitudinal analyses of BM aspirates, trephine biopsies, and peripheral blood samples from MDS patients treated with azacitidine in a clinical trial (NCT03493646), integrating CyTOF, 5' single-cell RNA and TCR sequencing, plasma proteomics, and multiplex immunofluorescence microscopy to characterize changes associated with azacitidine response. Clinical responders showed expansion of GzmBCD56CD8 T cells together with increased type I and type II interferon signaling within the T-cell compartment. Responders also exhibited marked alterations in circulating platelet- and myeloid-derived factors with the potential to remodel the BM niche. Spatial analyses revealed expansion of neighborhoods enriched for CXCL12-abundant reticular cells and CD8 T cells in responders, whereas HSPC-enriched neighborhoods were largely unchanged. In contrast, several HSPC-enriched neighborhoods expanded in non-responders. These microenvironmental changes were accompanied by evidence of enhanced myelopoiesis in clinical responders. Our findings support a model in which azacitidine response extends beyond direct effects on malignant hematopoietic cells to involve coordinated remodeling of the BM microenvironment which may be reinforced by platelet- and myeloid-derived signals that establish a feed-forward circuit promoting productive hematopoiesis.

Aberrant activation of TAL1, a key oncogenic driver, defines a major subgroup comprising [~]30% of childhood T-lineage acute lymphoblastic leukemias (T-ALLs). We and others have shown that somatic non-coding mutations within upstream and intronic cis-regulatory regions of TAL1 contribute to transformation by creating binding sites for MYB and other transcription factors. Here we investigated cis-regulatory mechanisms mediated by somatic mutations occurring in an intergenic region located 29 kilobase pairs downstream of the canonical TAL1 transcription initiation site, implicated in 6% of TAL1-expressing T-ALLs. These somatic variants include i) complex indels resulting in de novo MYB transcription factor binding sites (TFBSs) and ii) internal tandem duplications (ITDs) encompassing canonical MYB TFBSs. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed binding of the TAL1 core regulatory circuit (CRC) transcription factors MYB, GATA3, and RUNX1, resulting in enhancer activity mediated by sequences with the mutant allele. Strikingly, ChIP-seq peaks for the repressive H3K27me3 mark and the active H3K27ac mark co-existed across TAL1 regulatory sequences but enriched for different haplotypes. TAL1 transcription from the mutant haplotype initiated from a promoter located within exon 4 of the canonical TAL1 transcript, resulting in a short isoform normally expressed by hematopoietic stem cells (HSC). Interestingly, neither the isoform expression nor the enhancer activity could be predicted by the sequence-to-function deep learning artificial intelligence (AI) model AlphaGenome, emphasizing the importance of experimental validation. Our findings indicate that selection for cis-regulatory, non-coding variants leads to reactivation of enhancers normally active in HSC but silenced in differentiated lineages during normal hematopoietic cell development.

Germline variants in DDX41 are the most frequent genetic predisposition to adult hematologic malignancies. The most common variants are truncating, implicating loss of function in the pathogenesis. However, non-truncating variants account for 30-40% of cases, and their impact on essential DDX41 functions remains unknown. We utilized a genetic complementation assay to assess the functionality of 10 recurrent germline non-truncating variants of DDX41. All variants restored viability to Ddx41-deficient hematopoietic progenitor cells at exogenous expression levels. In contrast, the hotspot mutant p.R525H, which is somatically acquired at disease onset in >50% of patients, failed to restore viability. CRISPR-based modeling in cell lines and mice revealed heterogeneity: some variants were non-functional at endogenous expression levels whereas others maintained complete functionality, supporting normal cell proliferation and even lifelong hematopoiesis in a homozygous setting. Notably, co-expression of p.R525H with some variants caused impaired hematopoietic progenitor cell viability, indicating a dominant-negative effect of p.R525H. In contrast, other variants, all classified as variants of unknown significance, were unaffected by the presence of p.R525H. A screen of 100 disease-associated variants confirmed that many non-truncating germline variants are susceptible to p.R525H-mediated dominant-negative effects, whereas wild-type DDX41 is not. These findings indicate that DDX41 variant curation is complicated by variable effects on functionality and variant-specific interactions with somatically-acquired DDX41 mutations. The dominant-negative effect of p.R525H provides a mechanistic basis for the conclusion of recent patient cohort analyses that co-occurrence with a somatic hotspot mutation is a reliable indicator of DDX41-driven disease in carriers of non-truncating variants.

The JAK2V617F (JAK2VF) driver mutation is found in 95% of patients with polycythemia vera (PV), a progressive myeloproliferative neoplasm. Current treatments suppress excessive hematopoiesis but lack specificity for targeting JAK2VF cells, are unable to deplete mutant stem/progenitor cells and ultimately result in drug resistance. We discovered that the FDA-approved antibiotic, linezolid (LZD), ameliorates the PV phenotype across multiple model systems. LZD suppressed cell proliferation and STAT5 signaling, altered the cell cycle, and increased apoptosis of JAK2VF-harboring human erythroleukemia cells, but not in wild-type acute leukemia cells. Computational modelling indicated that LZD interacts specifically with mutant JAK2VF but not with wild-type JAK2 protein. We further showed that, in JAK2VF mice that faithfully recapitulate human PV, LZD mitigates disease burden by selectively targeting JAK2VF stem cells thereby normalizing spleen size and blood counts. LZD also inhibited hematopoietic colony formation by patient-derived peripheral blood mononuclear cells, with the more primitive progenitors being preferred targets. Importantly, LZD selectively decreased JAK2VF+ colony numbers, without impacting wild-type JAK2 colonies. In all, the data provide a firm foundation for evaluating LZD-like molecules as an effective therapy for PV and other myeloproliferative neoplasms. Key pointsO_LILinezolid acts as a JAK2V617FIZselective inhibitor in PV mouse models and PV patient samples while sparing wildIZtype hematopoiesis. C_LIO_LILinezolid acts directly on JAK2V617F hematopoietic stem cells. C_LI

Clonal hematopoiesis (CH) is an age-associated condiion defined by over-representation of hematopoietic stem cells (HSCs) and their progeny carrying somatic mutations or variants that confer a selective advantage. CH is associated with increased risk of hematologic malignancies (1), cardiovascular disease and inflammatory bone loss (2, 3). Chronic inflammation is increasingly recognized as a central mediator of CH-mutant hematopoietic stem and progenitor cell (HSPC) expansion underlying CH (4). DNA methyltransferase 3a ( Dnmt3a )-mutant cells produce higher levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) (5), and blocking these pathways reduces the competitive advantage of Dnmt3a -mutant HSPCs (4, 6). The upstream mediators initiating inflammatory signaling in CH are unknown. Strong candidates are S100A8 and S100A9, members of the S100 calcium-binding protein family that regulate inflammatory signaling in the hematopoietic system. These proteins form a heterodimer complex and activate innate immune signaling through receptors including Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) (7). S100A8/A9 signaling promotes production of pro-inflammatory cytokines and inflammasome activation leading to poor prognosis in myelodysplastic syndrome and myeloproliferative neoplasms (8, 9). Across multiple myeloid malignancies, neutrophils are the primary bone marrow (BM) source of this alarmin (8, 10, 11) and pharmacologic inhibition of S100A9 with tasquinimod reduces disease severity without disrupting normal hematopoiesis (10, 12). Given the role of S100A8/A9 in establishing an inflammatory milieu, here we investigated the role of S100A8/A9 in Dnmt3a -mutant hematopoiesis. We identify neutrophils as a major source of elevated S100A8/A9 in the BM of Dnmt3a -mutant mice and this increase correlates with production of the inflammatory cytokines TNFα and IL-6. We show that tasquinimod reduces TNFα and IL-6 levels and selectively reduces the Dnmt3a -mutant HSPC compartment.

Concurrent mutations in DNMT3A, NPM1, and FLT3 define a high-risk subtype of acute myeloid leukemia (AML) associated with increased relapse risk and inferior survival following standard chemotherapy. However, the mechanisms by which DNMT3A mutations promote treatment resistance in NPM1c-FLT3ITD AML remain unclear. Using genetically engineered murine models of Npm1c-Flt3ITD AML with or without Dnmt3aR878H (homologous to human DNMT3AR882H), we demonstrate that Dnmt3aR878H promotes chemotherapy resistance through epigenetic regulation of leukemia stem cell (LSC) quiescence. Integrated transcriptomic and epigenetic profiling revealed coordinated remodeling of DNA methylation and chromatin accessibility in LSC-enriched populations, characterized by preferential hypomethylation and increased accessibility at loci associated with stemness and quiescence programs. These data were confirmed in human DNMT3AR882H-NPM1c-FLT3ITD AML datasets with enrichment of quiescence-associated and stem cell enriched transcriptional programs. Conversely, Dnmt3a-mutant LSCs retained sensitivity to the cell-cycle independent regimen venetoclax plus azacitidine, but residual LSCs exhibited transcriptional plasticity and reversion to a de-differentiated state. We have identified LSC heterogeneity spanning primitive hematopoietic stem cell (HSC)-and progenitor-like states and our data demonstrate preferential maintenance of a quiescent HSC-like LSC subpopulation in Dnmt3aR878H-mutant AML following chemotherapy treatment. Pharmacologic induction of cell-cycle entry using pegylated interferon (pegIFN) disrupted the quiescent LSC state and restored chemotherapy sensitivity, identifying quiescence as a reversible and therapeutically actionable mechanism of resistance. These findings identify DNMT3A-mediated epigenetic regulation of LSC quiescence as a conserved mechanism of standard chemotherapy resistance and position therapeutic reactivation of quiescent LSCs as a promising strategy to overcome chemotherapy resistance and improve outcomes in high-risk DNMT3A-mutant AML.

The specification of hematopoietic stem cells (HSCs) is tightly regulated by multiple transcription factors and signaling pathways. Inflammatory signaling is pivotal for embryonic HSC development, but the mechanisms that activate it in vivo remain poorly understood. Here, we show that Toll-like receptor 7 (TLR7) is essential for the emergence of embryonic HSC in both zebrafish and mouse embryos. TLR7 deficiency reduces HSC numbers but not primitive or definitive progenitors. Conversely, the TLR7 agonist R848 enhances embryonic HSC development. Mechanistically, TLR7 signaling acts through interferon regulatory factor 5 (IRF5) to induce the expression of inflammatory cytokines, which subsequently activate Notch signaling to promote HSC emergence through a non-cell-autonomous mechanism. Notably, we identify microRNA-146a (miR-146a) as a potential endogenous activator of TLR7, inducing inflammatory signaling and promoting HSC development. Pharmacological treatment with miR-146a significantly increases HSC numbers in zebrafish embryos. Together, our findings reveal a crucial role for miR-146a-TLR7-IRF5 signaling axis in HSC emergence, providing insights into the endogenous factors that drive tonic inflammatory signaling during normal hematopoiesis and suggesting the translational potential of TLR7 agonists and miR-146a for stem-cell-based therapeutics. Significance StatementThe embryonic origin of hematopoietic stem cells (HSCs) requires inflammatory signals, but the endogenous factor that triggers this process remains elusive. We identify microRNA-146a (miR-146a) as a natural activator of Toll-like Receptor 7 (TLR7) signaling, which is essential for HSC emergence. This miR-146a-TLR7 axis functions through IRF5 and inflammatory cytokines to activate the Notch signaling, specifically promoting embryonic HSC development. Our work addresses the critical question of endogenous ligands that mediate tonic inflammatory signaling in normal hematopoiesis, uncovers novel crosstalk between miRNAs and innate immunity in HSC specification, and identifies promising candidates for stem cell-based therapeutics.

Myelodysplastic syndromes (MDS) are clonal hematopoietic malignancies characterized by ineffective hematopoiesis, dysplastic morphology, and risk of progression to acute myeloid leukemia. While genomic alterations intrinsic to malignant MDS disease-initiating cells have been well-characterized, molecular assessment of the bone marrow in situ has been limited. Here we present single cell spatial assessment of gene expression, T cell receptors, as well as MDS-defining mutations and RNA isoforms within fixed, decalcified human bone marrow core biopsies (41 MDS, 15 controls) paired with single cell immunogenomic analysis of bone marrow aspirates (35 MDS, 6 controls). Bone marrow spatial analyses of >7.47x106 cells identified hematopoietic and non-hematopoietic populations not readily captured in dissociated tissue. We developed computational methods to compare ecological niche structures, revealing enriched hematopoietic niches and reorganization of T cell immunity in MDS patient bone marrow. In situ genotyping of mutant cells revealed increased TGF{beta} expression in malignant megakaryocytes suppressing local T cell cytotoxicity. By contrast, TGF{beta} signaling was disrupted in mutant cells due to aberrant splicing of multiple TGF{beta} signaling components. This study provides a spatially resolved landscape of human MDS bone marrow and uncovers mechanisms by which malignant cells simultaneously promote intrinsic clonal persistence while rewiring the microenvironment for immune escape.

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O2) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor ICs were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.

Sickle cell disease (SCD) is caused by a point mutation in the {beta}-globin gene that promotes hemoglobin polymerization, leading to chronic hemolytic anemia, vaso-occlusive episodes, and progressive organ damage. The most efficacious therapies focus on reactivating fetal hemoglobin (HbF) expression to mitigate the pathological effects of sickle hemoglobin (HbS) polymerization. However, the predominantly used HbF inducer, hydroxyurea (HU), exhibits substantial interpatient variability in efficacy, and curative approaches such as gene therapy remain inaccessible to the vast majority of patients. Although all SCD patients share the same causative HBB glu7val mutation, differences in genetic background significantly influence disease severity and therapeutic response. We describe a SCD-specific induced pluripotent stem cell (iPSC) platform as a renewable and scalable preclinical model to interrogate treatment responses across the genetically diverse SCD patient population. By generating patient-specific iPSC-derived erythroblasts (iEry) representing distinct SCD genetic backgrounds, we demonstrate that this system faithfully recapitulates the heterogeneous HbF induction observed clinically in response to HU. Moreover, this platform enables the identification and evaluation of alternative therapeutic agents for HU non-responders and provides sufficient resolution to dissect drug-specific effects on erythroid differentiation and cellular phenotypes. Together, these findings support the use of iPSC-derived erythroid models as a versatile tool to advance precision therapeutic strategies for SCD. KEY POINTS- SCD iPSC-derived erythroid cells (iEry) reflect the diversity in HU-mediated HbF induction seen in SCD patients - SCD iEry recapitulate patient-specific treatment responses and can be used to identify therapeutic alternatives for HU non-responders - iEry provide a versatile platform to study the impact of novel HbF inducers on erythroid cell characteristics and differentiation parameters