Iron deficiency is a highly prevalent nutrient deficiency and the most common cause of anemia. Although iron deficiency exacerbates cardiovascular disease, the direct impact of iron deficiency on the vasculature remains unstudied. We assessed iron levels across the vascular endothelium in mouse and human endothelial cells and found resistance artery endothelial cells have the lowest iron stores, suggesting they may be especially impacted by iron deficiency. Anemia has previously been shown to increase arterial NO signaling in patients, and we have previously shown endothelial a-globin scavenges nitric oxide (NO) in the resistance artery endothelium. We hypothesized iron regulates vascular function through regulation of endothelial a-globin. To test this, we used a mouse model of iron deficiency anemia (IDA). In female mice, IDA increased NO signaling, which was rescued to control levels by repletion of vascular iron with iron dextran. Despite being similarly anemic and having a similar reduction in a-globin protein, there were no changes in NO signaling across groups in male mice. We further measured whether a-globin was in its heme-bound (holo a-globin) or heme-free (apo a-globin) state and found males did not fully lose its functional, heme-bound a-globin. Using endothelial specific a-globin knockout mice, we show loss of endothelial a-globin is necessary for increased NO signaling in IDA and for the rescue of NO signaling by iron dextran in female mice. Altogether, the data presented here demonstrate iron is a determinant of endothelial identity and modulates endothelial NO signaling through the regulation of a-globin.

Hemophilia A (HA) phenotype is predicted by factor VIII (FVIII) activity. Most HA adeno-associated virus (AAV) trials incorporate the B-domain-deleted FVIII-SQ variant and one-stage assay (OSA) FVIII activity exceeds chromogenic substrate assay (CSA) values by 1.5-2-fold. This contrasts with recombinant FVIII-SQ (rFVIII-SQ), suggests altered biochemical properties, and highlights the need to determine which assay reflects hemostatic function. In gene therapy treated mice and SPK-8011 trial participants, AAV-derived FVIII-SQ (AAV-FVIII-SQ) activation and function within the intrinsic tenase enzyme complex was compared to rFVIII-SQ. In both species, AAV-FVIII-SQ demonstrated normal cofactor function and A2-domain stability. In mice, the assay discrepancy persisted without von Willebrand factor (vWF), indicating it is not driven by altered vWF interactions. Both species demonstrated enhanced thrombin-mediated activation of AAV-FVIII-SQ, detectable by OSA but not CSA. Negative binomial regression analysis of 23 SPK-8011 participants, representing 99 cumulative patient-years, trended toward better prediction of annualized bleeding rate with OSA than CSA. In vivo evaluation of AAV-FVIII-SQ in mice demonstrated that OSA activity better correlated with hemostatic function and corresponded to rFVIII-SQ function. These findings support that OSA FVIII activity reflects AAV-FVIII-SQ function within the intrinsic tenase complex, captures enhanced activation not detected by CSA, and best predicts clinical outcome.

The t(4;14) chromosomal translocation drives overexpression of the histone methyltransferase NSD2 and defines a high-risk segment of multiple myeloma (MM) patients. Herein, we report the discovery of NSD2-LDD, a cereblon-recruiting and PWWP1-mediated ligand directed degrader (LDD) that selectively and potently eliminates full length and PWWP1 domain containing NSD2 protein isoforms. NSD2-LDD treatment induces global loss of H3K36me2 leading to promoter-proximal spreading of H3K27me3 and re-wiring of cis-regulatory interactions that reverse t(4;14) transcriptional programs. These effects suppress MM disease-associated phenotypes including stromal adhesion, three-dimensional colony growth and paracrine signaling. By integrating patient single cell profiles with model 3D epigenomic and spatial transcriptomics, we delineate t(4;14) disease state together with the tumor-intrinsic reprogramming and resultant remodeling of the bone marrow microenvironment upon NSD2 degradation. In cell line derived xenografts and genetically engineered mouse models of t(4;14), NSD2-LDD extends median survival accompanied by tumoral H3K36me2 loss and niche re-modelling. Although the NSD2-LDD response is restricted to PWWP1-containining models, collectively this work validates NSD2 as a tractable dependency and supports clinical development of NSD2 degradation as a novel, targeted therapeutic strategy in high-risk MM.

MLL-rearranged (MLLr) acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) involve reciprocal translocations of the KMT2A (MLL1) gene with various translocation partner genes, which yields oncogenic chimeric MLL1 fusion proteins (MLL-FPs). Menin, the protein product of the MEN1 gene, is essential for the leukemogenic activity of MLL-FPs. Revumenib is a small-molecule inhibitor that selectively disrupts the menin-MLL interaction, and it is now in clinical use for treatment of MLLr and NPM1-mutated (NPM1c) acute leukemia. Notably, menin also interacts with the JUND member of the AP-1 transcription factor family through a conserved protein sequence in the MLL1/2 binding pocket of menin. Despite this structural similarity, the impact of menin-MLL inhibitors on JUND function has remained unexplored. Here, we investigated the influence of menin-MLL inhibitors on JUND activity. Quantitative mass spectrometry analysis of MLLr leukemic cells demonstrated that menin-MLL inhibitors also disrupt menin-JUND interactions. Furthermore, CRISPR-mediated inactivation of JUND or pharmacological inhibition using JNK inhibitors synergistically enhanced the anti-leukemic effects of menin-MLL inhibitors leading to reduced cell proliferation, cell cycle arrest and apoptosis. RNA sequencing and chromatin binding assays revealed that menin-MLL inhibitor treatment increased JUND chromatin occupancy leading to upregulation of target genes and contributing to resistance against menin-MLL inhibitors. Immunocompromised mice engrafted with JUND-deficient leukemia cells exhibited reduced tumor burden compared to control mice engrafted with wild type leukemic cells. These findings reveal a role for JUND in MLLr AML and suggest that targeting JUND transcription factor activity enhances the efficacy of menin-MLL inhibitors towards MLLr leukemic cells.

Non‑hematopoietic stromal cells are essential regulators of hematopoiesis; however, their contribution to leukemogenesis and immune dysfunction remains poorly defined. Here, we identified fibroblast‑derived fibroblast growth factor 18 (FGF18) as a novel stromal cytokine that reprograms leukemia-immune interactions. Single-cell RNA sequencing of the bone marrow (BM) niche during acute myeloid leukemia (AML) revealed the upregulation of Fgf18 in stromal fibroblasts. Administration of recombinant FGF18 accelerated AML progression, whereas fibroblast-specific Fgf18 depletion markedly delayed disease development and improved the survival of mice. We performed a pooled CRISPR-Cas9 screen in AML cells and identified FGFR3 signaling as a critical mediator of leukemic fitness in the FGF18‑rich microenvironment. Genetic loss of Fgfr3 in AML cells recapitulated the effects of FGF18 deficiency and limited leukemic expansion in vivo. Mechanistically, FGF18 binds to its receptor, FGFR3, on AML cells, activating the AKT-mTOR signaling pathway and inducing interleukin (IL)-6 production. IL‑6 acts autocrinely to reinforce leukemic signaling and paracrinely to activate fibroblast JAK-STAT3 signaling, thereby amplifying stromal fibroblast FGF18 expression and forming a feed‑forward loop that suppresses CD8⁺ T‑cell effector function and weakens anti‑leukemic immunity. Clinically, elevated FGF18 expression correlates with poor prognosis in AML patients. To therapeutically target this malignant crosstalk, we generated an FGF18‑neutralizing antibody that disrupted the stromal-leukemia feedback loop, restored CD8⁺ T cell effector function, and synergized with anti-PD-1 therapy to elicit durable anti‑leukemic immunity in vivo. Collectively, these findings identify FGF18-dependent stromal-leukemia crosstalk that drives AML progression and immune dysfunction, highlighting FGF18 neutralization as a potential therapeutic strategy.

KMT2A-rearranged B-cell acute lymphoblastic leukemia (KMT2A-r B-ALL) is an aggressive subtype of leukemia, characterized by high relapse rates, therapy resistance, and poor prognosis. Although CD19-targeted immunotherapies have significantly benefited patients with relapsed/refractory (R/R) disease, relapses remain common and long-term survival is especially poor in KMT2A-r B-ALL patients. We recently identified the membrane-bound proteoglycan NG2 (CSPG4) as direct transcriptional target of KMT2A fusions, with its expression associated with poor prognosis, early relapse, and glucocorticoid resistance in KMT2A-r B-ALL. However, the molecular mechanism underlying the aggressiveness of KMT2A-r B-ALL remains poorly understood. Here, we identify the α4 integrin subunit (ITGA4) and NG2 as a key biological axis contributing to leukemic aggressiveness. NG2 expression promotes proliferation and migration of KMT2A-r B-ALL cells and it is associated with Rho GTPase activity in an ITGA4-dependent manner. In vivo studies using immunodeficient mice demonstrated that ITGA4 and NG2 cooperate to promote leukemia progression as combined genetic ablation of both genes significantly delayed disease onset and prolonged survival. Notably, Natalizumab (NTZ) - an FDA/EMA-approved monoclonal antibody targeting ITGA4 - delayed leukemia progression and potentiated the efficacy of standard-of-care chemotherapy in KMT2A-r B-ALL patient-derived xenograft (PDX) models. Collectively, our findings define a novel ITGA4-NG2 signaling axis that drives the aggressiveness of KMT2A-r B-ALL and support the repurpose of NTZ as an adjuvant therapeutic strategy for this high-risk leukemia subtype.

Dysregulation of galectins and global protein glycosylation have been reported in various cancers, but their role in myeloproliferative neoplasms (MPNs) have remained incompletely understood. We performed single-cell RNA sequencing (scRNA-seq) which revealed significant enrichment of galectin genes in MPN monocytes. Cell-cell communication analysis predicted monocytes as a pivotal mediator of cell interactions and galectin signaling as a robust input/output pathway for monocytes. We identified elevated expression of galectin-1 (Gal-1, LGALS1) in monocytes from both human MPN samples and mouse models. Mass cytometry (CyTOF) profiling of MPN blood samples demonstrated that recombinant galectin-1 (rGal-1) significantly increased levels of multiple inflammatory cytokines in monocytes without affecting other cell types. Incubation of CD14+ monocytes from MPN patients with rGal-1 led to markedly increased transcription and secretion of inflammatory cytokines. Mechanistically, we uncovered crosstalk between the TLR4 and Gal-1 signaling pathways, as evidenced by protein 3D modeling and co-immunoprecipitation. Notably, TLR4 inhibition abrogated Gal-1 mediated proinflammatory effects in monocytes. We further identified NF-κB-dependent signaling as a key downstream effector of Gal-1, as reporter assays demonstrated rGal-1 mediated activation of NF-κB signaling in a TLR4-dependent manner. We corroborated these findings in vivo in a murine model driven by MPLW515L in which genetic abrogation of Lgals1 ameliorated key MPN disease features, including leukocytosis and splenomegaly. Additionally, Gal-1 inhibition suppressed carrageenan-induced thrombosis and inflammation in vivo. In summary, we identify Gal-1 enrichment in MPN monocytes as a driver of monocyte-mediated inflammation through TLR4 and NF-κB activation and uncover a novel therapeutic avenue for MPNs.

The transition of hematopoietic stem cells (HSCs) from quiescence to lineage commitment requires precise post-transcriptional control, yet the contribution of mRNA isoform regulation remains poorly defined. Here, we identify a translationally controlled splicing program that contributes to HSC fate decisions. Using activity-based signatures of 305 splicing regulators, we uncover widespread post-transcriptional modulation of the spliceosome in stem and progenitor cells. The branch-point recognition factor Sf1 emerges as a key node, regulated by a conserved structured 5' UTR that cooperates with the RNA-binding protein Igf2bp2 to control its translation. Disrupting this cis-trans module reduces Sf1 protein synthesis and skews differentiation toward stem and erythroid programs. Mechanistically, Sf1-dependent alternative splicing remodels 5' UTRs of hematopoietic and DNA damage response genes, altering their translation and modulating DNA damage resolution. Together, these findings reveal an unrecognized translational layer controlling spliceosome activity and link RNA regulons, alternative splicing, and HSC fate determination.

We assessed the addition of the tyrosine kinase inhibitor Quizartinib, following intensive chemotherapy and as maintenance, in patients aged >60 years with AML or high-risk MDS, regardless of FLT3 mutation status. 463 patients (median age 68yrs) were randomised (1:1) to receive Quizartinib 40mg or not for 14 days immediately following chemotherapy courses 2 and 3, plus 28 additional days; those allocated Quizartinib were further randomised (1:1) to either 12 additional 28-day maintenance courses (long Quizartinib), or no further treatment (short Quizartinib). Median follow-up was 76 months. 314 patients were FLT3 wild type (WT); 116 had FLT3 mutations. The primary endpoint, overall survival (OS) unselected by FLT3 status, showed no significant difference (HR 0.99, 95% CI 0.79-1.24, p=0.937) and there was an increase in non-relapse mortality with Quizartinib (HR 1.64, 95% CI 1.04-2.59, p=0.032). In a pre-planned subgroup analysis, FLT3-mutated patients who received Quizartinib had significantly improved OS (HR 0.59, 95% CI 0.37-0.93, p=0.024) due to reduced relapse risk (HR 0.57, 95% CI 0.35-0.91, p=0.017) with greater benefit in the short Quizartinib group (HR 0.49, 95% CI 0.24-1.02, p=0.055). In FLT3-WT patients there was no survival benefit and no reduction in relapse risk. No significant differences were seen in time to hematologic count recovery or in the duration of hospitalisation. The most observed grade 3/4 adverse events were febrile neutropenia. In conclusion, the addition of Quizartinib to intensive chemotherapy, delayed until chemotherapy course 2, prolonged OS in older patients with FLT3-mutated AML but did not improve OS in non-FLT3 selected patients. ISRCTN-31682779, EudraCR-2013-002730-21.

Despite extensive investigation, the molecular control of developmental hemoglobin expression remains incompletely elucidated. Hemoglobin switching is controlled by transcription factors, miRNAs, and RNA-binding proteins (RBPs) that enforce gene regulatory changes through development. Here we examine the role of the heterochronically silenced N6 methyladenosine (m6A) RNA-binding protein IGF2BP1 that was previously described to regulate HBG1/2 indirectly by suppressing BCL11A expression through an unknown mechanism. We find that IGF2BP1 binds and activates HIC2, itself a BCL11A repressor. Furthermore, we identify that IGF2BP1 plays a BCL11A-independent role by direct binding to HBG1/2 to promote its translation. Stop codon-proximal m6A-modified coding sequences within HBG2 transcripts are necessary and sufficient for direct positive regulation mediated by IGF2BP1. This work deepens the mechanistic understanding of hemoglobin switching and suggests a physical relationship between heterochronic RBPs and globin transcripts.

The distinctive milieu of the bone marrow (BM), known as the BM niche, supports hematopoietic stem cells (HSCs) and serves as a foundation for hematopoietic regeneration. Myeloablative stress disrupts not only hematopoietic stem and progenitor cells but also essential BM niche components, including endothelial cells (ECs) and mesenchymal stromal cells (MSCs); disruption of the latter impairs efficient hematopoietic recovery. However, therapeutic strategies targeting niche restoration remain largely underdeveloped. Here, we demonstrate that the Hippo pathway effectors YAP/TAZ are critical for enabling ECs and MSCs to respond to BM injury, and that YAP/TAZ activation accelerates BM niche recovery, thereby promoting hematopoietic regeneration. We found that YAP/TAZ are rapidly activated in both MSCs and ECs following myeloablative stress, maintaining MSC multipotency and orchestrating vascular remodeling. Mechanistically, YAP/TAZ function as transcriptional hubs in MSCs, regulating key transcriptional factors such as Ebf1 and Ebf3. This regulation preserves MSC identity by preventing osteogenic and fibrogenic differentiation while promoting the expression of hematopoietic factors such as Cxcl12 and angiogenic factors. In addition, YAP/TAZ signaling in MSCs and ECs appeared to coordinately remodel sinusoidal vessels following BM injury. These YAP/TAZ-mediated niche responses are essential for HSC retention and hematopoietic regeneration following diverse myelosuppressive therapies. Notably, pharmacological activation of YAP/TAZ enhances BM niche reorganization and augments hematopoietic regeneration following myeloablative therapies. These findings establish YAP/TAZ as central regulators of BM niche resilience, providing a rationale for niche-targeted therapeutic strategies to enhance hematopoietic regeneration.

Steroid-resistant gastrointestinal graft-versus-host disease (SR-GI-GVHD) is a lethal complication of allogeneic hematopoietic stem cell transplantation with no clear therapeutic target. The role of neutrophil heterogeneity in its pathogenesis remains poorly defined. Through single-cell RNA sequencing of patient blood, we identified a specific neutrophil subset (CD16hiCD177⁺) that is markedly expanded in SR-GI-GVHD. This subset utilizes CD177 to bind endothelial CD31, facilitating transmigration into the intestine. Upon arrival, MyD88-mediated sensing of translocated gut bacteria triggers the release of neutrophil extracellular traps (NETs), directly causing epithelial damage. Mechanistically, we discovered that elevated levels of retinoic acid (RA) in patients drive the expansion and pathogenic programming of these neutrophils via the RARA-SPI1 transcriptional axis. In a murine model of GVHD, genetic deletion of Cd177 or Myd88 in donor cells attenuated disease. Crucially, pharmacological inhibition of the RA receptor with AGN193109 not only ameliorated GI-GVHD and improved survival but also restored sensitivity to corticosteroids. Together, these findings delineate a complete pathogenic circuit-from the metabolic driver retinoic acid and the RARA-SPI1 transcriptional axis to the CD177⁺ neutrophil cellular effector and its NETosis-mediated tissue damage. This establishes the RA-driven CD177⁺ neutrophil subset as a key mediator of SR-GI-GVHD and identifies RARA inhibition as a promising therapeutic strategy.

Myelodysplastic neoplasms (MDS) feature hematopoietic deficits driven in part by transcript splicing abnormalities. Thus far, such disease-driving transcripts have been identified in association with specific splicing factor mutations. However, conserved aberrant splicing-derived transcripts that drive MDS independently of mutational status remain poorly studied despite representing global therapeutic targets. Here, we characterize an MDS-associated MBD1 isoform (MBD1-L) as a novel member of this class of transcripts. Rather than originating from a mutant splicing factor, the abnormal production of MBD1-L is driven by reduced WTAP expression in MDS. Overexpression of MBD1-L in healthy human HSPCs recapitulates archetypal MDS defects, including reduced terminal GLYA+ erythroid differentiation, suppressed cell cycling and impaired in vivo reconstitution capacity during increased hematopoietic demand in xenotransplantation assays. An integrated multiomics approach assessing DNA binding of MBD1 isoforms, and resulting changes in chromatin accessibility, histone mark deposition and transcriptional changes, revealed that these defects arise from an isoform-specific switching of MBD1's binding behavior. The MBD1-L isoform refocuses MBD1-L's heterochromatin-promoting activity from methylated to unmethylated CpGs and thus enacting broad downregulation of CpG-rich promoters as well as secondary epigenetic effects mediated by its downstream target BCOR. Remarkably, we also find that directly reversing abnormal MBD1 splicing across a broad range of primary human MDS samples using nanoparticle-encapsulated ASOs enhances in vitro erythroid differentiation, supporting the utility of RNA therapies for MDS treatment. Thus, our findings demonstrate MBD1-L to be a global, disease-driving splice variant across MDS, and illustrate the potential for RNA-based therapies in the broad treatment of MDS.