BCMA- or GPRC5D-directed T-cell immunotherapies have substantially improved the survival of patients with relapsed/refractory multiple myeloma (MM). Despite these advances, a subset of patients does not respond, and most patients will eventually relapse. Tumor heterogeneity, resulting in rapid selection of both antigen-negative and antigen-low cells, is a critical issue affecting response to T-cell immunotherapies targeting single tumor-associated antigens. In addition, antigen escape (due to deletions, mutations, or epigenetic alterations) is frequently observed in patients who experience disease progression after CAR T-cell infusion or during BsAb treatment. Simultaneous targeting of two tumor-associated antigens may improve efficacy by addressing heterogeneous target expression and preventing antigen escape. Various dual-targeting strategies are currently evaluated in MM, including the combination of two single-antigen targeting BsAbs. Notably, the efficacy of the combination of teclistamab and talquetamab appears to have enhanced anti-MM activity, compared to the corresponding conventional BsAbs alone in similar patient populations. Furthermore, dual-antigen targeting with T-cell redirecting trispecific antibodies (e.g., ramantamig [BCMAxGPRC5D] and ISB2001 [BCMAxCD38]) has already demonstrated promising results in heavily pretreated MM with several studies ongoing in earlier stages of the disease. Studies with limited numbers of patients have demonstrated that CAR T-cell products with specificity for more than one antigen are also effective in advanced MM, but at this time none of the dual-targeting CAR T-cell products is clearly superior to targeting BCMA alone with ciltacabtagene autoleucel (cilta-cel). Dual-targeting should eventually be compared in large phase 3 trials with the classical approach of serial treatment with mono-targeting agents with target switch.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of circPCMTD1 in BCR/ABL1-positive myeloid leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR/ABL1 translocations. RNA sequencing and mass cytometry experiments identified aberrant activation of the DNA damage response (DDR) pathway as a downstream effect of circPCMTD1 depletion. DNA fiber assays, Comet assays and profiling of DDR markers (phospho-H2AX, phospho-CHK1, etc.) further underscored the pronounced effect of circPCMTD1 depletion in increasing genotoxic stress and inhibiting leukemic cell growth. circPCMTD1 targeting also led to aberrant DDR activation in leukemia patient blasts with BCR/ABL1 translocations. In in vivo experiments, circPCMTD1 knock-down prolonged the survival of mice engrafted with BCR/ABL1-positive leukemia cells. Mechanistically, we found that circPCMTD1 is enriched in the cytoplasm and associates with the ribosomes of leukemic blasts. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 generates a 127 amino-acid peptide product (cPCMTD1-127aa). Using a custom-produced antibody, we found that the cPCMTD1-127aa interacts with the BCR/ABL1 oncoprotein, as well as with the BLM, TOP3A and RMI1 proteins, which form the BTR complex and regulate DNA repair and genome stability. cPCMTD1-127aa enhanced BTR complex formation, thereby increasing cellular tolerance to genotoxic stress. In summary, we identify and characterize circPCMTD1 as a molecular vulnerability and potential therapeutic target in BCR/ABL1-positive leukemias.

Acute myeloid leukemia (AML) is a complex hematological malignancy with multiple disease sub-groups defined by somatic mutations and heterogeneous outcomes. Although genome-wide association studies (GWAS) have identified a small number of common genetic variants influencing AML risk, the heritable component of this disease outside of familial susceptibility remains largely undefined. Here we perform a meta-analysis of four published GWAS plus two new GWAS, totalling 4710 AML cases and 12938 controls. We identify a new genome-wide significant risk locus for pan-AML at 2p23.3 (rs4665765; P=1.35x10-8; EFR3B, POMC, DNMT3A, DNAJC27) which also significantly associates with patient survival (P=6.09x10-3). Our analysis also identifies three new genome-wide significant risk loci for disease sub-groups, including AML with deletions of chromosome 5 and/or 7 at 1q23.3 (rs12078864; P=7.0x10-10; DUSP23) and cytogenetically complex AML at 2q33.3 (rs12988876; P=3.28x10-8; PARD3B) and 2p21 (rs79918355; P=1.60x10-9; EPCAM). We also investigated loci previously associated with risk of clonal hematopoiesis (CH) or clonal hematopoiesis of indeterminate potential (CHIP) and identified several variants associated with risk of AML. Our results further inform on AML etiology and demonstrate the existence of disease sub-group specific risk loci.

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.