Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using in vivo models, we uncover divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1, which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

Inflammation induces hematopoietic stem cell (HSC) expansion with compromised fitness, a culprit of hematopoietic aging. Elucidating mechanisms to maintain HSC fitness under inflammatory stress is crucial to preserve hematopoietic integrity.Using intravital imaging and HSC reporter mice (Mds1GFP/+; Flt3Cre), we revealed distinct stages of bone turnover across marrow cavities. HSCs within a subset of marrow cavities that lack bone resorption stayed nonmotile and solitary despite stress from cyclophosphamide/G-CSF (Nature, 2020), lipopolysaccharide (LPS, 35 μg/mouse)-induced acute inflammation, and aging. Harvesting cavity-resident HSCs under image guidance, we further revealed the superior colony-formation capacity of HSCs from nonresorptive cavities both at the steady state and under inflammation. These results suggest that local bone remodeling influences the functionality of compartmentalized HSCs.Notably, via transcriptomic analyses and in vivo tracking, marrow macrophages in resorptive (RE) cavities were found to be proinflammatory and frequently retrieve cellular cargos from HSCs, followed by incidences of HSC division. Further in vivo staining suggested surface calreticulin expression and elevation of MHC class I on HSCs, resembling the “grooming” behavior reported in zebrafish models from the Zon group (Science, 2022, 2024). Such interaction increases with LPS challenge and aging, and continues to occur predominantly in the RE cavities. Inhibiting bone resorption with zoledronic acid (1.2 μg/mouse) reduced grooming and restored HSC fitness under LPS challenge. Taken together, our findings present novel evidence of macrophage-HSC interactions in murine bone marrow that regulate HSC clonality and unveil previously unrecognized spatial heterogeneity of the HSC niche that may be targeted to intervene in hematopoietic decline under inflammatory stress.

Calcium signaling in blood vessels regulates their growth1,2, immune response3, and vascular tone4. Vascular endothelial cells are known to be mechanosensitive5-7, and it has been assumed that this mechanosensation mediates calcium responses to pulsatile blood flow8-10. Here we show that in larval zebrafish, the dominant trigger for vascular endothelial Ca2+ events comes from body motion, not heartbeat-driven blood flow. Through a series of pharmacological and mechanical perturbations, we showed that body motion is necessary and sufficient to induce endothelial Ca2+ events, while neither neural activity nor blood circulation is either necessary or sufficient. Knockout and temporally restricted knockdown of piezo1 eliminated the motion-induced Ca2+ events. Our results demonstrate that swimming-induced tissue motion is an important driver of endothelial Ca2+ dynamics in larval zebrafish.

Diamond-Blackfan Anemia (DBA) is a rare congenital bone marrow failure disorder with patients manifesting macrocytic anemia in infancy (Da Costa, et al. Blood 2020; Da Costa, et al. F1000Res. 2018). DBA is caused by germline heterozygous loss-of-function mutations in one of the twenty small- or large-subunit ribosomal protein (RP) genes. Current therapies for DBA include chronic red blood cell transfusions, glucocorticoid treatment, and allogeneic hematopoietic stem cell transplantation, all of which are associated with severe toxicities. The mutations in RP genes cause defects in ribosomes, leading to ribosomal stress and aberrant p53 activation. Over-activation of p53 is a crucial mediator of DBA-associated hematopoietic defects, including erythroid failure and anemia. Previous studies (Taylor, et al. Sci Transl Med. 2020; Taylor, et al. Exp Hematol. 2012) have demonstrated that calmodulin (CaM) inhibitors, including the FDA-approved anti-psychotic trifluoperazine (TFP), improve anemia in multiple models of DBA by reducing activation of p53 targets. However, TFP is associated with serious side effects, including neurotoxicity attributed to its high brain penetrance and interaction with dopamine D2 receptors (DRD2) in the central nervous system (CNS). We therefore embarked on a drug discovery program to identify novel CaM inhibitors with improved potency compared to TFP, but with activity against DRD2 eliminated and CNS penetrance attenuated. This effort resulted in the discovery of two novel small molecules, FTX-1 and FTX-2. Relative to TFP, FTX-1 and FTX-2 showed superior activity in reducing aberrant p53 activity in in vitro CD34+ human hematopoietic stem and progenitor cell (HSPC) models designed to mimic DBA via the introduction of ribosomal mutations in RPS19, RPL5, or RPL11 with CRISPR-Cas9 editing. Activation of p53 was assessed by monitoring the mRNA levels of CDKN1A/p21, a direct transcriptional target of p53. Compared to DMSO, 1 µM FTX-2 significantly attenuated p53 activity in the RPS19 model (p < 0.0001). This rescue was significantly greater than the positive control TFP, which rescued p53 activity at 3 µM (p < 0.01- P < 0.0001). FTX-2 was also significantly active in reducing p53 activity at 1 µM in the RPL5 and RPL11 models (p < 0.05 - < 0.0001, depending upon the donor and the experimental replicate), while 3 µM TFP was only active in the RPL5 model (p < 0.05-0.0001) but not in the RPL11 model. FTX-1 and FTX-2 were also effective in improving anemia in a rps29-/- zebrafish model of DBA. This model recapitulates the maturation arrest of red blood cells, growth retardation, decreased globin synthesis, and elevated p53 activity commonly observed in DBA patients. FTX-1 and FTX-2 significantly rescued hemoglobin levels at doses of 0.1 µM and 0.001 µM (p < 0.05 - <0.0001), respectively, compared to the positive control fluphenazine at 10 µM (p < 0.05 - <0.01). Lastly, 1 µM FTX-1 was effective in restoring erythroid development by increasing the BFU-E and CFU-E colonies in RPS19 DBA patient-derived HSPCs compared to vehicle (adjusted p-value < 0.05) as assessed using a colony formation assay. FTX-2 is currently being explored in toxicology and safety pharmacology studies in support of an Investigational New Drug (IND) application, with first-in-human studies anticipated in 2026.

The rational design of receptor agonists to control cell signaling is an emerging strategy for developing disease therapeutics. Creating a soluble cytokine-like agonist for the Notch receptor, which regulates cell fate in embryonic and adult development, is challenging, as receptor activation requires a mechanical force that is usually mediated by cell-associated transmembrane ligands. Here, we exploit computationally designed protein complexes with precise valencies and geometries to generate soluble cytokine-like Notch agonists. These molecules promote cell-cell bridging, cluster Notch receptors at cell synapses, and activate receptor signaling. We show that these agonists drive T cell differentiation from cord blood progenitors and human induced pluripotent stem cells (iPSCs) and in bioreactor production of T cells in liquid suspension. When delivered intravenously in mice, they stimulate cytokine production, expansion of antigen-specific CD4+ T cells, and antibody class switching. These de-novo-designed ligands can be broadly applied to optimize in vitro cell differentiation and advance immunotherapy development.

Stress erythropoiesis elevates the rate of red blood cell (RBC) production as a physiological response to stressors such as anemia or hypoxia. In acute anemia, RBC progenitors and precursors temporarily rewire their transcriptome, up- and downregulating hundreds of genes to accelerate the production of mature RBCs. Effective regeneration requires communication between critical cytokine signals (e.g., BMP4) and cis-regulatory elements on chromatin which coordinate transcriptional changes. To identify cis-regulatory changes that underlie anemia-specific gene expression and cellular responses, we analyzed chromatin accessibility in populations of cells enriched for red blood cell precursors isolated from mice at a range of time points after anemia induction. Early in the anemia response, chromatin is transiently open at AP-1-containing regions, correlated with increased Jun and Fos transcript/protein levels. Jun knockdown ex vivo decreases the percentage of KIT+ erythroid precursors after anemia induction. We observe a second rewiring event at time points consistent with anemia resolution, involving repression of GATA factor-accessible regions and activation of ETS factor-accessible regions. In both mouse in vivo models and human CD34+ cells stimulated with BMP4, accessibility changes at regions with prior associations to human blood phenotypes. Dozens of BMP4- and anemia-activated loci are sensitive to natural human variation. The representation of red blood cell trait-associated loci in ATAC-seq data remains durably elevated more than 1 month after anemia resolution. Together, these findings provide a framework to understand the early establishment and late resolution of a regeneration-dependent transcriptome in RBC precursors.

T lymphocytes, which are essential for cell-mediated immunity in vertebrates, rely on thymic seeding of lymphoid progenitors for commitment, differentiation and maturation. However, the epigenetic programming of lymphoid-primed progenitor migration and differentiation is incompletely understood. Here, we show that zebrafish embryos lacking the epigenetic modulator Atf7ip or Setdb1 methyltransferase exhibit decreased thymic homing and differentiation of lymphoid progenitor cells. We show that Atf7ip regulates T cell progenitor homing and differentiation via Setdb1-triggered H3K9 trimethylation. Atf7ip interacts with Setdb1 to catalyze H3K9me3 modification of the key immune regulator bach2b to derepress the expression of ccr9a and irf4a, thereby promoting lymphoid progenitor homing and intrathymic differentiation. In the absence of Atf7ip or Setdb1, replenishing irf4a or diminishing bach2b restores the thymic trafficking and differentiation of lymphoid progenitor cells. Notably, depletion of ATF7IP by two complementary cre-recombinase alleles in mice (CAG-CreERT2 and Mx1-iCre) impedes the migration of hematopoietic progenitors to the thymus, resulting in declined T lymphopoiesis. These findings establish the role of ATF7IP/SETDB1-mediated epigenetic programming in governing T lymphoid progenitor trafficking and differentiation, with implications for understanding the pathogenesis of human T lymphoid diseases.

Millions of platelet units are needed each year to manage thrombocytopenia and other conditions linked to excessive bleeding. These life-saving treatments still depend entirely on donated platelets, despite the numerous shortcomings associated with them, such as limited shelf life, supply shortages, unpredictable functionality, potential for infection, as well as immune-incompatibility issues. These challenges could be overcome with universal donor platelets generated from human induced pluripotent stem cell (hiPSC)-derived megakaryocytes (MKs). We recently developed expandable hiPSC-derived megakaryocytic cell lines (imMKCLs) as a potentially unlimited source for platelet production. imMKCL-derived platelets are functional and have already been tested in patients. In this study, we demonstrate through single-cell time-course imaging that imMKCL maturation is heterogeneous and asynchronous, with only a few imMKCLs generating platelets at any given time under static culture conditions. Using a chemical screen, we identify microtubule (MT) destabilizing agents, including vincristine (VCR), as promising hits, with a larger proportion of VCR-exposed imMKCLs developing proplatelet extensions and more platelets being produced per imMKCL. VCR use reduces the MT content of imMKCLs and results in the production of platelets with a diminished peripheral MT ring structure. Nevertheless, these platelets are functional, as evidenced by their normal response to agonists, their ability to attach to and spread on fibrinogen-coated surfaces, and their capacity to restore hemostasis in vivo. Interestingly, we also observed a negative correlation between the MT content of imMKCLs and platelet yields when we compared imMKCLs differentiated under static conditions (MThigh, low yield) to our turbulence-optimized VerMES™ bioreactor (MTlow, high yield). Taken together, our findings highlight the importance of MT dynamics in megakaryocyte biology, provide a possible explanation for the still poorly understood link between vinca alkaloid in vivo use and thrombocytosis, and bring us closer to realizing the clinical potential of affordable, off-the-shelf hiPSC-derived platelets.

The acquisition of somatic mutations that increase the competitive potential of hematopoietic stem and progenitor cell (HSPC) clones causes Clonal Hematopoiesis (CH). CH, where one HSPC clone becomes dominant, occurs in >10% of individuals over 65 and predisposes these individuals to hematological malignancy and cardiovascular disease. No approved therapies currently exist. We posited that metabolically profiling dominant, mutant HSPCs could reveal targetable metabolic vulnerabilities. We combined mosaic mutagenesis of CH-driving genes and HSPC color barcoding to isolate dominant HSPC clones in zebrafish. Untargeted metabolomics was performed on dominant asxl1- or ezh2-mutant or wildtype (WT) HSPCs. Only 7 of 90 detected metabolites differed significantly. Two choline-related metabolites were decreased in dominant HSPC clones: betaine (FC -0.68, p<0.01) and acetylcholine (FC -0.42, p<0.01). This metabolic signature was confirmed in an independent asxl1-mutant CH cohort using a standard containing choline metabolite profiling assay. We hypothesized that decreased betaine and acetylcholine levels reflected increased partitioning of choline into phospholipid biosynthesis (e.g., phosphatidylcholines). Lipid profiling of asxl1-mutant and WT zebrafish HSPCs revealed a global decrease in phosphatidylcholine levels; species 16:1/18:1 (FC 1.33, p<0.05), 16:0/16:0 (FC 1.44, p<0.05), and 16:0/18:2 (FC 1.50, p<0.05) were the most affected. Primary human CD34+ HSPCs from two donors were edited via CRISPR/Cas9 in exon 12 of ASXL1 and showed increased levels of the same phosphatidylcholine species observed in zebrafish after 7 days of culture (16:1/18:1, FC 2.73; 16:0/16:0, FC 2.15; 16:0/18:2, FC 2.76). To determine if phosphatidylcholine biosynthesis was required to maintain clonal dominance zebrafish with large asxl1-mutant clones (>10% VAF) were treated with RSM-932A, an inhibitor of the first enzyme in phosphatidylcholine synthesis, for 5 days followed by monthly blood collection. After 2 months of treatment, RSM-932A decreased asxl1 VAF by 49.95% compared to a 42.61% increase in controls (p<0.01). RSM-932A did not affect lineage or clonal output in WT zebrafish. We next used a genetic barcoding system (GESTALT) in zebrafish to track dominant clones during monthly RSM-932A dosing. In clones contributing <60% of blood (3/5), RSM-932A reduced the clone below detection after 6 months. To validate these findings in a human model, primary human CD34+ HSPCs edited in ASXL1 or AAVS1 (control) were treated with RSM-932A. ASXL1-mutant HSPCs increased in number after 14 days and in EdU positivity after 4 days compared to AAVS1 control but these effects were completely abrogated by RSM-932A (total HSPC number, p<0.0001; Edu positivity, p<0.01). To test whether this treatment may be generalizable to other CH mutations, HSPCs (Lin-Sca+cKit+CD34-CD150+) were isolated from mice with inducible, mutant DNMT3aR878H and ROSA26-tdTomato alleles. RSM-932A reduced the number of DNMT3aR878H-tdTomato+ cells by 49.12% (p<0.01) after 21 days of culture. To understand what underlies the need for more phosphatidylcholine synthesis transcriptomic analysis of dominant asxl1-mutant and WT zebrafish HSPCs was performed and lpcat2 (p<0.05), known to catalyze the last step of phosphatidylcholine into platelet activating factor (PAF), was upregulated. When ASXL1-mutant human HSPCs were cultured with RSM-932A and PAF, RSM-932A's suppressive effect on HSPC expansion (p<0.01, day 14) and EdU incorporation (p<0.05, day 4) was overcome. To address the potential of RSM-932A as a therapeutic approach primary, CD34+ HSPCs from a patient with myelodysplastic syndrome (ASXL1 VAF 17%) were cultured for 14 days with RSM-932A with and without PAF. RSM-932A reduced ASXL1 VAF by 37.27% (p<0.05), but RSM-932A with PAF was not different from control. In sum, inhibiting phosphatidylcholine biosynthesis in ASXL1 or DNMT3a mutant HSPCs suppresses their clonal expansion in a PAF dependent manner. This is the first identification of choline metabolism as a targetable metabolic liability in CH-causing HSPCs.

T cell-mediated tumor killing underlies immunotherapy success. Here, we used long-term in vivo imaging and high-resolution spatial transcriptomics of zebrafish endogenous melanoma, as well as multiplex imaging of human melanoma, to identify domains facilitating the immune response during immunotherapy. We identified cancer regions of antigen presentation and T cell engagement and retention (CRATERs) as pockets at the stroma-melanocyte boundaries of zebrafish and human melanoma. CRATERs are rich in antigen-recognition molecules, harboring the highest density of CD8+ T cells in tumors. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within CRATERs, characteristic of antigen recognition. Following immunostimulatory treatment, CRATERs expanded, becoming the major sites of activated CD8+ T cell accumulation and tumor killing. In humans, elevation in CRATER density in biopsies following immune checkpoint blockade (ICB) therapy correlated with a clinical response to therapy. CRATERs are structures that show active tumor killing and may be useful as a diagnostic indicator for immunotherapy success.

Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery.

Macrophages maintain hematopoietic stem cell (HSC) quality by assessing cell surface Calreticulin (Calr), an "eat-me" signal induced by reactive oxygen species (ROS). Using zebrafish genetics, we identified Beta-2-microglobulin (B2m) as a crucial "don't eat-me" signal on blood stem cells. A chemical screen revealed inducers of surface Calr that promoted HSC proliferation without triggering ROS or macrophage clearance. Whole genome CRISPR-Cas9 screening showed that Tlr3 signaling regulated b2m expression. Targeting b2m or Tlr3 reduced the HSC clonality. Elevated B2m levels correlated with high expression of repetitive elements (RE) transcripts. Overall, our data suggest that RE-associated dsRNA could interact with TLR3 to stimulate surface expression of B2m on HSPCs. These findings suggest that the balance of Calr and B2m regulates macrophage-HSC interactions and defines hematopoietic clonality.

Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome commonly associated with mutations or deletions of ribosomal genes, leading to protein haploinsufficiency and resulting in nucleolar stress and p53 activation. Clinically, DBA usually manifests after birth and is characterized by macrocytosis, reticulocytopenia and a paucity of erythroid precursors in the bone marrow. Patients can also present with additional defects in other hematopoietic lineages, suggesting a defect in hematopoietic stem and/or progenitor cells. Notably though, it is still unclear whether these defects are common to all ribosomal protein (RP) mutations or specific to large vs.small ribosomal subunits. Furthermore, the role of RPs in fetal hematopoiesis is poorly understood due to the lack of clinically relevant mouse models for ribosomopathies. To address these questions, we generated Rps19fl/fl and Rpl5fl/fl conditional mouse models - representing the most mutated genotypes in DBA - using CRISPR/Cas9 technology and crossed them to a Vav-iCre mouse. Vav-iCre;Rps19fl/+ mice recapitulated clinical features of DBA, including macrocytic anemia and reticulocytopenia, and died on post-natal day 10 (P10) due to bone marrow failure without stress erythropoiesis in the spleen. These mice exhibited a progressive exhaustion of the hematopoietic stem and progenitor cell (HSPC) compartment by E17.5, resulting in a 40% reduction in the number of erythroid cells at the BFU-E stage. Flow cytometry analyses of terminal erythroid differentiation further demonstrated a 30% reduction in the number of basophilic erythroblasts at E17.5. In contrast with Vav-iCre;Rps19fl/+ mice, Vav-iCre;Rpl5fl/+ mice died perinatally from severe anemia. They also differed markedly from Rps19 haplo-insufficient mice as they exhibited a progressive expansion of the HSPC compartment. Rather, the defect in Rpl5 haplo-insufficient mice was restricted to the erythropoietic compartment, with an accumulation of BFU-E and CFU-E erythroid progenitors and a 70% decrease in the number of basophilic erythroblasts by E17.5. To elucidate the mechanisms underlying the specific HSPC phenotypes in Rps19 and Rpl5 haplo-insufficient mice, we performed scRNAseq analyses of fetal liver cells at E13.5. While Vav-iCre;Rps19fl/+ mice presented global alterations of the transcriptome at each stage of differentiation, Vav-iCre;Rpl5fl/+ mice only presented defects in erythroid lineage cells, beginning at the proerythroblast stage. Comparative analyses revealed Uba52, encoding the core RPL40 ribosomal protein, as a differentially regulated gene and western blot analyses confirmed decreased expression of RPL40 in both models, albeit to different extents. RPL40 is associated with translation elongation, and accordingly, we observed acceleration of translation by polysome profiling in the Rps19 model, along with decreases in the phosphorylation of the elongation factor eEF2 in the ckit+ population, corroborating the polysome profiling results. By E17.5, the phosphorylation of both the translation initiation factor eIF2a and elongation factor eEF2 were reduced, indicative of increased translation in the mutant mice. Accordingly, we observed reduced mTOR signaling in both these models. Furthermore, Vav-iCre;Rps19fl/+ fetal livers demonstrated activation of the p53 pathway, consistent with clinical findings. However, to our surprise, expression of Runx1, a transcription factor that mediates ribosome biogenesis, was significantly increased. Importantly, activation of both p53 and Runx1 pathways played critical roles in the pathological hematopoiesis occurring in Rps19 haplo-insufficient mice as erythroid defects were rescued by conditional deletion of either p53 or Runx1. Deletion of one or both Runx1 alleles significantly rescued fetal HSPC defects but bi-allelic p53 loss was needed to fully rescue fetal HSC exhaustion and prevent neonatal lethality in Vav-iCre;Rps19fl/+ mice. Taken together, our results unravel distinct requirements for Rps19 and Rpl5 during fetal hematopoiesis and provide novel insights into the mechanism(s) behind ribosomal protein haploinsufficiency leading to DBA.

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (TIE:EGFP). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP+ melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP+ regions and preferentially phagocytose TIE:EGFP+ melanoma cells compared to TIE:EGFP- melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized niche microenvironments in the marrow made of sinusoidal vascular endothelial cells (ECs) and other perivascular supportive cells. Pathological conditions such as primary myelofibrosis (PMF) in the marrow cause HSPC niche defects and pan-cytopenia. Extramedullary HSPC niche can form in the liver or the spleen sinusoids during PMF, but the process is inefficient. Here, we aim to identify the transcription factor (TF) code that specifies the sinusoidal vascular EC fate in the HSPC niche. We performed differential gene expression analysis on ECs from adult zebrafish kidney marrow and liver and identified TF candidates that were uniquely upregulated in the marrow sinusoidal ECs, namely tfec, mafbb, foxp4, irf8, and hoxb8a. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs were reprogrammed to upregulate key genes known for HSPC niche supportive functions, including mrc1a (log2FC=4.9, p<0.005), lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Transplant assay of liver cells into irradiated adult hosts showed that primary HSPCs occupy the newly reprogrammed liver vascular niche (7/26 in the reprogram group vs. 0/20 in the control group) (p=0.0296). Therefore, TFs tfec and mafbb were sufficient in reprogramming adult liver sinusoidal ECs to become HSPC niche in vivo. Furthermore, we aim to translate our findings to program human iPSC-derived ECs to support primary HSPCs in vitro. We engineered human iPSC lines with inducible overexpression of human ETV2, TFEC and MAFB. Upon ETV2-directed differentiation hiPSCs into ECs, the overexpression of TFEC and MAFB significantly upregulated the expression of sinusoidal endothelial and HSPC niche supportive genes, such as MRC1 (log2FC=3.6, p<0.005), STAB2 (log2FC=10.3, p<0.005), JAG1 (log2FC=1.7 p<0.005), and CXCL12 (log2FC=5.1 p< 0.005). Methylcellulose assays showed that CD34+CD45+ HSPCs co-cultured with hiPSC-derived ECs induced with ETV2 plus TFEC and MAFB contained significantly more colony-forming units (CFUs) compared to those co-cultured with ECs induced with ETV2 alone or HSPCs cultured without ECs: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Transplant of HSPCs into immunodeficient mouse hosts showed that HSPCs co-cultured with TFEC and MAFB induced ECs have significantly better engraftment potential than control HSPCs (p=0.0164). In summary, TFs TFEC and MAFB could program human iPSC-derived ECs to adopt HSPC niche fate and support primary human cord blood-derived CD34+ HSPCs in vitro. Our findings provide a method to engineer human HSPC niche-like sinusoidal ECs to enhance engraftment of HPSC, which could help transplantation therapies.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

Immunotherapy using T cells modified with chimeric antigen receptors (CAR) has shown significant efficacy against lymphoid malignancies. However, the process of preparing autologous CAR T cells is labor-intensive. Utilizing human induced Pluripotent Stem Cells (iPSCs) to generate CAR T cells offers the potential for off-the-shelf allogeneic cancer immunotherapies. Yet, achieving fully mature iPSC-T cells with robust functionality remains a challenge. Previously, we established a stroma-free culture method for differentiating iPSCs into T cells. Expanding on this approach, we conducted screenings to identify epigenetic regulators affecting lymphoid development, pinpointing the H3K9 histone lysine methyltransferase G9a/GLP as a suppressor of T cell fate. Through ATAC-seq and RNA-seq analyses, we discovered that G9a/GLP regulates chromatin accessibility and gene expression patterns related to lymphoid differentiation and controls the lineage choice between myeloid and lymphoid cells in HSPCs. Inhibiting G9a/GLP promotes lymphopoiesis in zebrafish, indicating its conserved role in T cell development across species. Notably, chemically-induced epigenetic reprogramming via G9a/GLP inhibition facilitates the generation of highly functional iPSC-T cells closely resembling mature alpha-beta T cells from peripheral blood. These reprogrammed iPSC-T cells expressing an anti-CD19 CAR demonstrated improved effector responses and tumor cell-killing ability in vitro. Moreover, CAR iPSC-T cells derived with G9a/GLP repression exhibited robust antitumor activity in a xenograft lymphoma mouse model, leading to enhanced survival. Remarkably, rechallenging mice treated with epigenetically modulated iPSC-CAR-T cells showed resistance to tumor cell reoccurrence. These findings offer a pathway for efficiently producing clinically relevant iPSC-derived T cells for adoptive cell therapies.

Gene expression is a process through which genetic information is decoded and manufactured into a functional gene product. Different cell types within an organism exhibit distinct biological functions, despite containing the same genetic material, or DNA sequence. This in part can be attributed to the various ways gene expression in a cell is regulated. At a microscopic level, a DNA template is "transcribed" into RNA and then "translated" into a protein, the functional gene product. This process involves the coordination between multiple dynamic events, which are subject to regulation at each step. These regulatory events happen in multiple levels, namely the transcriptional, the posttranscriptional, the translational, and finally, the posttranslational level. Processes integral to multicellular organismal development occur through a series of gene regulatory events, cascading in a gene product that determines cell fate. Proper regulation of gene expression is crucial to the development of healthy living organisms. Since transcription is the very first step of gene expression, predicting transcriptional control that occurs during human development and disease is critical. Combination of multiple genome-wide next-generation sequencing approaches ("multi-omics") within a particular biological system could be a means to achieve that. This may eventually help in developing therapeutics for human disorders related to the particular tissue type. Here, we have used multiomics by combining ChIP-seq, RNA-seq, and ATAC-seq in human hematopoietic stem and progenitor cells. This approach enabled us highlight certain hitherto unknown transcriptional control mechanisms during red blood cell development from hematopoietic stem cells. The performed analyses were able to predict gene sets, their enhancers, and the hematopoietic master and signaling transcription factors that control them during human red cell differentiation.

Stress granules (SGs) are crucial in RNA regulation, affecting cell fate and function. SGs contain RNAs, some of which can be methylated. We studied m6A RNA modifications during the human CD34+ HSPCs (hCD34+) differentiating into erythroid cells and found that mRNAs encoding many erythroid-specific proteins had decreased methylation during differentiation. Increased levels of ALKBH5 demethylase during erythropoiesis control the levels of the 3'UTR methylation of these mRNAs. hCD34+ carrying ALKBH5 mutations demonstrated a block in erythropoiesis, and mass-spectrometry studies of the mutant cells showed decreased levels of SG proteins, including the core granule protein ATXN2. ALKBH5 directly regulates the methylation of the mRNA of ATXN2. ATXN2 overexpression accelerated the erythroid differentiation of HSPCs and rescued the erythroid differentiation of ALKBH5 mutant cells. Very few SGs are found in normal human erythroid progenitors. SGs accumulated substantially in ALKBH5 mutant cells, and surprisingly overexpression of ATNX2 reduced SG numbers to normal. Polysome analysis demonstrated m6A-modified RNAs to be enriched in the pre-polysome fractions that were less translated. This work establishes a mechanism by which during stress, ATXN2 facilitates the release of SG-stored m6A-modified RNAs including erythroid-specific and SG-enriched RNAs that are loaded onto functional ribosomes, allowing better translation and accelerated erythroid differentiation during stress. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Stress granules confer cancer cells the ability to withstand harsh biological conditions and impart tumorigenic translational states through mRNA sequestration. Despite advances in in vitro knowledge, mechanistic understanding of the endogenous influence of stress granules during tumorigenesis in vivo remains understudied. To address this, we use a zebrafish melanoma model where oncogenic human BRAF V600E is driven by the melanocyte master regulator mitfa in a p53 -/- background. Through vector-based genetic engineering, endogenous melanoma induction in these zebrafish can be developmentally staged from single cell to tumor. BRAF V600E ::p53 -/- melanocytes form a cancerized field (CF), from which a subset upregulate the melanocyte master regulator mitfa, creating a cancer precursor zone (CPZ). Transformation of CPZ cells to an embryonic, neural crest-like state facilitates their formation of an early tumor patch that inevitable develops into an overt tumor. Single cell RNAseq analysis of cells representing each melanoma stage showed that CPZ cells upregulate core stress granule markers, including g3bp1, tia1, tia1l, and nufip2. Immunostaining of g3bp1 in CF, CPZ, patch, and tumor cells revealed a significant induction of stress granules from the CF to CPZ transition (2 vs 10 stress granules/cell; N=3; p=0.034) that persists during the patch and tumor stage, indicating cell stress accompanies melanoma initiation and progression. Genetic disruption of g3bp1 through cell autonomous, melanocyte-specific CRISPR editing in zebrafish (N=10-13) causes delayed CPZ onset (P=0.012) and fewer CPZs to form (P=0.021). In turn, g3bp1-edited zebrafish have delayed tumor formation (P=0.026) and develop fewer tumors (P=0.006). To decipher the RNAs sequestered by stress granules during melanomagenesis, we performed RIPseq for G3BP1 in human A375 melanoma cells stressed with sodium arsenite. RNAs significantly bound to G3BP1 upon stress induction include major tumor suppressors, such as CDKN2A, RBM5, BIK, and RHOB. Together, these results suggest that endogenous g3bp1-mediated stress granule formation in melanoma initiating cells is tumorigenic in vivo and operates through the sequestration of tumor suppressive RNAs. Citation Format: Kyle D Drake, Emily Formato, Leonard Zon. g3bp1-mediated stress granule formation drives melanoma initiation in zebrafish [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A012.