The myeloid transcription factors PU.1 and C/EBPα are essential for monocyte/macrophage development, and their dysregulation has been linked to myeloid malignancies and immune disorders. While their binding to enhancers for myeloid coding genes is established, their control of noncoding regulatory RNAs remains poorly understood. Using a comprehensive collection of putative and verified enhancers, we profiled the PU.1 cistrome and transcriptome, identifying a subset of noncoding genes that are both associated with PU.1-bound enhancers and regulated by PU.1. Notably, PU.1 induces expression of LOUP, an enhancer RNA transcribed from a locus containing a conserved PU.1 cis-regulatory element cluster characterized by features of myeloid-specific enhancers. Disruption of a PU.1-binding motif in the LOUP promoter and the enhancer reduced LOUP promoter activity, while mutation of another PU.1-binding site within the LOUP gene body and the enhancer- which modulates enhancer-promoter interaction- diminished both Pu.1 and Loup levels in mice. The myeloid transcription factor C/EBPα, which binds to the enhancer, is necessary for PU.1 and LOUP expression as inducible deletion of Cebpa in mice led to their downregulation. LOUP depletion impaired monocyte/macrophage marker and inflammatory cytokine expression as well as phagocytic function. Collectively, our findings reveal that PU.1 and the enhancer RNA LOUP form a previously unrecognized feed-forward loop, induced by C/EBPα, that drives their mutual expression and establishes a regulatory circuit. This circuit programs monocyte to macrophage differentiation as well as innate immune function, providing important implications for inflammatory diseases and myeloid malignancies.

The bone marrow microenvironment (BMME) is essential for hematopoiesis and immunity, yet spatiotemporal single-cell analysis during leukemogenesis remains challenging. We characterized the BMME in femurs from wild-type and chronic myeloid leukemia (CML) mice at 7, 14 and 21 days post-induction by highly multiplexed and 3D microscopy. Using a 54-marker CODEX panel, we profiled 2,033,725 cells in 55 regions of interest and identified 41 cell-types. During CML progression, we observed myeloid and progenitor cell expansion, increased PD-L1+ leukemic cells, PD-1 upregulation on CD4+ and CD8+ T-cells, and a profound loss of B-cells, plasma cells and bone cells. Advanced CML exhibited a striking expansion of immature, pericyte-deficient vasculature that disrupted vascular niches and impaired hematopoietic stem/progenitor cell positioning. Spatial mapping revealed leukemia-specific cellular neighborhoods enriched in PD-1+CD8+ T-cells, suggesting localized immune exhaustion. Early CML showed increased contacts between plasmacytoid dendritic cells and megakaryocytes, whereas advanced CML featured heightened megakaryocyte emperipolesis of non-leukemic granulocytes. Megakaryocytes were morphologically irregular in CML mice and patient BM biopsies. In contrast, in mice with acute myeloid leukemia, vasculature and megakaryocytes were reduced, while remaining megakaryocytes retained normal morphology. Laser-capture microdissected megakaryocytes from newly diagnosed CML patients had reduced cytoskeleton gene expression, which was reversed in advanced cases treated with tyrosine kinase inhibitors. 3D imaging revealed vascular disorganization and depleted megakaryocytes in the diaphysis, underscoring region-specific pathology. Together, this study provides a spatiotemporal single-cell atlas of the BMME during leukemic progression, showing how leukemic cells reprogram it to support their expansion and immune evasion.

Intractable diarrhea is a recently described complication following B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma (MM) with reported mortality rates of 36-50%. The optimal clinical management is unknown. Here, we report a series of five patients who presented with severe diarrhea after BCMA CAR T-cell treatment. We hypothesized that the Janus kinase (JAK) inhibitor, ruxolitinib, might be an effective therapy based on its success in graft-versus-host-disease (GVHD) after allogeneic bone marrow transplant and other immune-driven diarrhea syndromes. Three patients received ruxolitinib, all of whom experienced rapid clinical improvement. Among the two with matched pre- and post-treatment biopsies, both showed signs of histopathologic response, including one with CAR T cell-associated indolent T-cell lymphoproliferative disease of the gastrointestinal tract (ITLPD-GT).

The administration of intravenous (IV) iron to treat anemia during acute infection remains controversial due to concerns of exacerbating the infection. We conducted a retrospective cohort study using the TriNetX Research Network (2000 to June 2025) to evaluate the safety and efficacy of IV iron administration in adults with iron-deficiency anemia and infection (methicillin-resistant Staphylococcus aureus [MRSA] bacteremia, pneumonia, urinary tract infection [UTI], colitis, or cellulitis). Patients must have received antibiotics within 2 days of infection for inclusion and were stratified by IV iron exposure. Propensity matching (1:1) was performed within each cohort. Survival was significantly higher (p<0.001 for each infection type) at both 14 and 90 days in patients who received IV iron (MRSA bacteremia 97.6% vs 95.0% and 88.6% vs 83.8%; pneumonia 95.7% vs 91.5% and 84.7% vs 78.1%; UTI 97.6% vs 95.7% and 89.1% vs 85.6%; colitis 97.6% vs 95.5% and 89.7% vs 83.8%; and cellulitis 98.5% vs 97.4% and 92.2% vs 89.2%). Hemoglobin recovery 60-90 days after infection was significantly greater (all p<0.001) when IV iron was administered across all subgroups (MRSA bacteremia +1.3 vs +1.0 g/dL; pneumonia +1.3 vs +1.0 g/dL; UTI (+1.4 vs +1.0 g/dL; colitis +1.5 vs +0.7 g/dL; and cellulitis +1.4 vs +0.9 g/dL). The findings observed for each infection type studied suggest that IV iron administration during acute infection does not exacerbate infection and is associated with improved survival and enhanced recovery from anemia in hospitalized patients. Prospective studies are needed to confirm these findings and expand their applicability.

T-cell acute lymphoblastic leukemia (T-ALL) results from the malignant transformation of thymocytes blocked in their differentiation. Surface expression of the γδ T-cell receptor (γδTCR) is surprisingly frequent in T-ALLs, questioning the susceptibility of the γδ-lineage to leukemogenesis. Among 1233 T-ALL phenotyped in our center, 33% (n=403) expressed a TCR, of which 47% (n=191 (113 adults, 78 children)) were positive for γδTCR (γδTCR+). By integrating oncogenetic, immunogenetic, phenotypic and bulk transcriptomic data, we were able to delineate two distinct γδTCR+ T-ALL subtypes, with likely distinct physiological counterparts. The first (75% of cases) was characterized by ectopic expression of homeodomain-containing oncogenes (HD+), Vβ-Jβ rearrangements, phenotypic (including surface pre-TCRα chain) and transcriptional profiles reminiscent of cortical thymocytes, and was therefore termed cortical-like γδ T-ALLs. Transduction of murine T-cell progenitors and human CD34+ cells with HOXA9 or TLX3 (HD+ oncogenes) led to a differentiation bias toward γδTCR expressing thymocytes. The second (26%) was negative for these features, exhibited phenotypic and transcriptional profiles reminiscent of γδ thymocytes and was therefore termed bona fide γδ T-ALLs. These findings were validated in the COGAALL0434 cohort. Although bona fide γδ T-ALLs were enriched for early T-cell progenitor (ETP)-like and KMT2A-rearranged cases, they mostly eluded the phenotypic definition of ETP-ALLs. Similar to the ETP-like subtype, bona fide γδ T-ALLs were associated with a poor initial response to chemotherapy, but were sensitive to the BCL2-inhibitor venetoclax. Our results reveal developmental heterogeneity behind γδTCR expression in T-ALLs, and suggest that overrepresentation of this subtype reflects αβ-lineage commitment repression by HD+ oncogenes.

Quadruplet (QUAD) induction and autologous stem cell transplantation (ASCT) leads to high rates of measurable residual disease (MRD)-negativity with improved outcomes in multiple myeloma (MM). The t(11;14) confers unique biology and different kinetics of treatment response. We analyzed MRD trajectories of patients treated with QUAD/ ASCT and MRD-adapted post ASCT management. Of the 302 patients assessed, 47 (16%) had t(11;14)+. Median follow up was 45.8 months. MRD negativity (10-5) for t(11;14)+ vs. t(11;14)- was 9% vs. 31% (P<0.001), 36% vs. 59% (P=0.004), and 53% vs. 75% (P=0.004) at post-induction, post-ASCT and any time on treatment, respectively. The rates of sustained MRD negativity (S-MRD)<10-5 were 38% vs. 46% (P=0.43). Median time to MRD<10-5 was 13.6 vs 7.7 months (P=0.002) for t(11;14)+ vs. t(11;14)-, respectively. PFS was superior for t(11;14)+ patients (P=0.012), with 4-year PFS rates of 90% vs. 72%. In multivariable analysis, S-MRD<10-5 (HR 0.41, P=0.002) and t(11;14)+ (HR 0.36, P=0. 048) were associated with reduced risk of progression or death, with no progression seen in t(11;14)+ patients who achieved S-MRD<10-5. In the setting of QUAD/ASCT therapy and MRD-adapted post ASCT management, t(11;14)+ NDMM in associated with improved prognosis despite slow conversion to MRD negativity.

Management of recurrent gastrointestinal (GI) bleeding is a clinical unmet need for patients with Von Willebrand disease (VWD) and is linked to the presence of gut vascular malformations (angiodysplasia). We previously demonstrated that von Willebrand factor (VWF) regulates angiogenesis and vascular integrity. VWF controls the storage of the angiogenesis regulator Angiopoietin-2 (Angpt-2) in endothelial cells (EC), suggesting a candidate for the genesis of angiodysplasia; however, no direct evidence of the role of Angpt-2 in VWF-dependent angiogenesis is available. Using VWF-deficient Human Umbilical Vein Endothelial Cells (HUVEC) and endothelial colony forming cells (ECFCs) from severe VWD patients, we find that loss of VWF results in increased Angpt-2 expression through the positive feedback loop Angpt-2-TIE2-AKT-FOXO1-Angpt-2. In the gut of VWF-deficient mice, Angpt-2 expression is increased whilst Angpt-1 expression is decreased, suggesting that VWF regulates the Angpt/Tie2 balance in the gut. Moreover, the intestinal vasculature in the jejunum of VWF-deficient mice appears abnormal, with hyper-sprouting and lumen formation defects. The findings reveal VWF-deficient mice as a model to study gut angiodysplasia. We investigate sprouting angiogenesis in vitro using a fibrin bead assay and find increased sprouting in VWF-deficient EC. We develop a 3D-microfluidic model of angiogenesis and find that ECFCs from severe VWD patients exhibit defective remodeling and abnormal lumen formation, reminiscent of the defects in the gut of VWF KO mice. Importantly, inhibition of Angpt-2 reduces sprouting in VWF-deficient HUVEC and normalises vascular remodeling in VWD ECFCs, suggesting that Angpt-2 inhibitors may be effective in VWD patients with GI bleeding and angiodysplasia.

Oncogenic mutations of SF3B1 are common in myeloid cancers, chronic lymphocytic leukemia (CLL) and select solid tumors. Their mechanistic basis for promoting oncogenesis has been investigated in detail, with the stereotyped missplicing of mRNA protein coding sequences most intensively studied. These changes, in genes such as MAP3K7, BRD9, and ABCB7, typically lead to loss-of-function, thus contributing to cancer pathogenesis.Here we systematically analyzed the impact of mutant SF3B1 on non-coding regions of mRNA transcripts across disease types, in both cell lines and primary patient specimens. This identified numerous novel and highly reproducible splicing alterations in such regions. Studies of one target gene, DCAF16, revealed multiple complex mutation-induced alterations in its 5' and 3' untranslated regions (5', 3' UTRs). Remarkably, these were mechanistically associated with increased DCAF16 protein levels in SF3B1 mutant cells, representing the first time that oncogenic SF3B1 has been shown to increase levels of a target protein in a gain-of-function manner. DCAF16 is a substrate recognition adapter for the DDB1/CUL4 E3 ubiquitin ligase complex. Novel protein degrader small molecules which co-opt DCAF16 to degrade BRD4 as a neosubstrate demonstrated preferential selectivity for SF3B1 mutant cancers and CLL primary patient specimens due to increased DCAF16 protein levels. In turn, this reveals the therapeutic relevance of mutant SF3B1 dysregulation of transcript untranslated regions and uncovers a novel strategy for the treatment of these important neoplasms.

Plasminogen activator inhibitor 1 (PAI-1) is an inhibitor of fibrinolysis, thereby promoting blood clot stabilization. PAI-1 contributes to thrombosis, diet-induced obesity, and age-associated diseases such as diabetes, cancer, and Alzheimer's disease. Circulating PAI-1 increases with age, contributing to the increased thrombotic risk in age-related diseases. In contrast, partial PAI-1 deficiency protects patients from cardiovascular morbidity and extends lifespan. Decreasing circulating PAI-1 levels has both experimental and therapeutic value. RNA gene therapy can regulate the levels of target proteins, including those not amenable to traditional small-molecule or antibody-based therapies. Here, we developed a therapeutic approach to induce long-lasting PAI-1 knockdown in vivo with siRNA-lipid nanoparticles (siPAI-1). One dose of siPAI-1 resulted in 90% knockdown of plasma PAI-1 and lasted 10 days post-administration with no overt toxicity. siPAI-1 decreased thrombus weight following complete ligation of the inferior vena cava (IVC) in young and aged mice, and increased survival in aged mice four days post-IVC ligation. Hepatic PAI-1 mRNA expression in diet-induced obese mice was >10-times higher than in healthy mice and was exponentially correlated with body weight. One dose of siPAI-1 in obese mice resulted in 70% knockdown of circulating PAI-1. Furthermore, siPAI-1 normalized the supraphysiologic concentration of PAI-1 in aged mice, and prolonged lifespan in a fast-aging mouse model. Thus, siRNA-mediated PAI-1 knockdown represents a long-term anti-thrombotic approach and effective strategy to limit pathologic impact of PAI-1 inaging and in age-related diseases.

Sufficient levels of fetal hemoglobin (HbF) can ameliorate the pathophysiologic basis of sickle cell disease and β-thalassemia postnatally. DNA methylation has long been posited to mediate silencing of HbF expression, but this has been controversial. Recent publications provide definitive evidence for the critical role of HBG gene promoter methylation in silencing and insight into the mechanisms involved. The data support a model in which methylation of CpG sites in the HBG promoters and repressive transcription factors recruit the MBD2-NuRD chromatin remodeling complex, which enforces silencing. These findings have implications for the treatment of β-globin disorders.

Graft-versus-host disease (GvHD) remains one of the major complications following allogeneic haematopoietic cell transplantation. Currently, immunosuppressants are used for GvHD prophylaxis and treatment in the majority of transplant recipients. Due to their systemic, non-specific mode of action, this treatment regimen is frequently associated with severe toxic side effects, opportunistic infections as well as cancer relapse when treating haematologic malignancies. By using short-term ex vivo modulation of haematopoietic cell transplants with the anti-human CD4 antibody MAX.16H5, we have developed a novel immune tolerance-inducing strategy enabling potent GvHD prevention. Functional in vitro assays and transcriptome profiling data suggest impaired TCR signalling and a shift towards an IL-10-dependent regulatory phenotype as the primary mechanism of action of anti-human CD4 antibody treatment, leading to a significantly reduced activation and proliferation of CD4+ and CD8+ T cells. A one-time incubation of haematopoietic transplants with MAX.16H5 prolongs survival of NSG mice and reduces signs of GvHD manifestation as effectively as repeated application with clinically applied immunosuppressants - making it a safe and effective immunotherapy for GvHD prevention.

Mutations in GATA1 that cause skipping of exon 2, which encodes the N-terminus, are associated with the myeloid leukemia of Down syndrome and Diamond-Blackfan anemia (DBA). To elucidate the molecular function of this N-terminal region, we employed single-cell RNA sequencing (scRNA-seq) on fetal liver cells from Gata1 mutant embryos that express only the short isoform of GATA1 (GATA1s) lacking the N-terminus of full-length GATA1 (GATA1FL). scRNA-seq revealed defects in erythropoiesis and aberrant upregulation of glycolytic genes, including PKM, which encodes pyruvate kinase to catalyze the final and irreversible step of glycolysis. Using precision nuclear run-on sequencing (PRO-seq) and cleavage under targets and release using nuclease (CUT&RUN) following acute GATA1 deletion in erythroid cells, we identified PKM as a direct target of GATA1. Substitution of GATA1FL with GATA1s induced histone lactylation at the PKM promoter, increased PKM expression and activity, and enhanced glycolytic flux in erythroid progenitors, without affecting mitochondrial respiration. Importantly, PKM expression is also significantly elevated in DBA patients with RPS19 mutations, which is associated with reduced levels of GATA1, further supporting a link between GATA1s-driven defective erythropoiesis and dysregulated glycolysis. Together, these findings reveal that GATA1 controls not only heme metabolism, but also glycolytic reprogramming.

By integrating short-read whole-genome sequencing and RNA sequencing (RNA-seq) data with long-read RNA-seq, we dissect the complex genomic architecture of PAX5 intragenic tandem multiplication, revealing that these complex rearrangements result in in-frame transcripts that likely encode proteins with altered domains.

Malaria, caused by Plasmodium parasites, accounts for ~250 million cases and over 600,000 deaths annually. One leading cause of mortality and morbidity is cerebral malaria (CM). Platelets mediate CM pathogenesis, although the exact mechanisms remain largely unknown. We examined if the mechanistic target of rapamycin (mTOR) pathway in platelets contributes to malaria pathogenesis. Our results demonstrate activation of the mTOR pathway in platelets ex vivo after co-incubation with Plasmodium falciparum-infected red blood cells and in vivo in Plasmodium berghei ANKA (PbA)-infected mice. When mTOR was specifically deleted in platelets (mTORplt-/-), mice with experimental cerebral malaria (ECM) had significantly increased survival. Survival differences were independent of parasitemia and thrombocytopenia. PbA-infected mTORplt-/- mice demonstrated significantly reduced platelet deposition in the brain resulting in improved cerebral blood flow and reduced brain vascular permeability. Plasma heme levels, generated during malaria, correlated significantly with intracerebral platelet accumulation in the PbA-infected mTORplt+/+ mice, but not in PbA-infected mTORplt-/- mice. In vitro experiments demonstrated that heme activates platelet mTOR downstream of Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling, predominantly through C-type lectin-like receptor 2. Blockage of heme-induced platelet activation with cobalt protoporphyrin significantly reduced platelet mTOR activation and decreased ECM-associated mortality. In conclusion, our findings demonstrate that platelet mTOR amplifies platelet activation responses induced by heme and deletion of platelet mTOR reduces platelet deposition in the brain, which we propose impedes symptomatic disease progression and malaria-associated mortality.

Relapse remains a major barrier to survival in B-cell acute lymphoblastic leukemia (B-ALL). Both activation of B-cell signaling pathways and increased glucose consumption have been linked to chemo-resistance and relapse risk. Here, we connect these observations, showing that B-ALL cells with active signaling, marked by high phosphorylated ribosomal protein S6 (pS6+), are glucose dependent. Isotope tracing confirms that pS6+ cells are highly glycolytic and rely on glucose for de novo nucleotide synthesis. Uridine, but not other purines or pyrimidines, rescues pS6+ cells from glucose deprivation, highlighting uridine as essential for survival. Active mTOR signaling in pS6+ cells drives de novo pyrimidine synthesis by activating CAD (Carbamoyl phosphate synthetase 2, Aspartate transcarbamylase, and Dihydroorotase), which catalyzes the first steps of de novo pyrimidine synthesis. Inhibiting signaling abolishes glucose dependency and CAD phosphorylation. Primary pS6+ cells express high levels of pyrimidine synthesis proteins, including dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in pyrimidine synthesis. Increased DHODH expression correlates with relapse and poor event-free survival. Most B-ALL molecular subtypes exhibit DHODH activity. BAY-2402234, a DHODH inhibitor, effectively kills pS6+ cells in vitro, with IC50 values correlating with pS6 signaling strength across 14 B-ALL patient-derived xenografts (PDX). In vivo, DHODH inhibition prolongs survival and reduces leukemia burden in pS6+ B-ALL models. These findings link active signaling to pyrimidine dependency and relapse risk, highlighting DHODH inhibition as a promising therapeutic strategy for chemo-resistant B-ALL.