Aberrant activation of BCL11B ("BCL11B-a") defines a subtype of lineage ambiguous leukemias with T-lymphoid and myeloid features, co-occurring activating FLT3 mutations, and a stem/progenitor immunophenotype and gene expression profile. As with other lineage ambiguous leukemias, optimal treatment is unclear and there are limited targeted therapeutic options. Here, we investigated the efficacy of BCL-2 and FLT3 inhibition with venetoclax and gilteritinib, respectively, in preclinical models of BCL11B-a leukemia. Despite variation in response to single agent therapies, the combination of venetoclax plus gilteritinib (VenGilt) was highly effective in all models evaluated. BH3 profiling suggested that resistance to venetoclax monotherapy was due to the tumor-intrinsic dependence on additional BCL-2 family proteins prior to drug treatment. Longitudinal single cell RNA-seq analysis identified mitochondrial pathways and a pro-lymphoid gene expression signature as potential drivers of rare cell survival on VenGilt therapy. These data support clinical evaluation of venetoclax in combination with gilteritinib in BCL11B-a lineage ambiguous leukemias.

The regulated secretion of von Willebrand factor (VWF) from Weibel-Palade bodies (WPB) in endothelial cells is fundamental to hemostasis. This process relies on recruiting Rab GTPases and their effectors to the WPB membrane, with the guanine nucleotide exchange factor (GEF) MAP-kinase activating death domain (MADD) playing a central role. Biallelic variants in MADD lead to a pleiotropic neurological and developmental disorder that can include bleeding abnormalities. This study investigates the impact of pathogenic MADD variants on VWF secretion using patient-derived endothelial cells. We isolated endothelial colony forming cells (ECFCs) from three pediatric patients with biallelic MADD variants and unaffected heterozygous family members. All patients exhibited low VWF plasma levels (22-30 IU/dL). Proteomic analysis of patient-derived ECFCs revealed an absence of MADD peptides, reduced VWF, and downregulation of proteins involved in the exocytotic machinery, including Rab3D and the Rab3/27 effector Slp4-a. Functional assays demonstrated diminished Rab27A and Rab3D activity and their failure to localize to WPBs in patient cells. Biochemical and live-imaging studies showed that histamine-induced VWF and VWFpp secretion were significantly reduced in patient cells due to delayed and reduced degranulation of WPBs. Our findings demonstrate the critical role of MADD in maintaining the secretion competence of WPBs and the magnitude of VWF secretion by regulating the recruitment of the endothelial exocytotic machinery. This study highlights the in vivo significance of WPB exocytosis in maintaining plasma VWF levels and establishes MADD as the first causal gene for quantitative von Willebrand Disease (VWD) in patients without pathogenic VWF variants.

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic system throughout life, and their functional decline contributes to hematological disorders and organismal aging. Understanding the molecular mechanisms that govern HSC function is critical for developing interventions for treating and preventing aging-related diseases. Here, we show that DCAF8, a substrate recognition component of Cullin-RING E3 ubiquitin ligases, is highly expressed in HSCs and undergoes a progressive decline with age. Loss of DCAF8 in mice results in impaired function in HSCs, characterized by increased number yet decreased self-renewal capacity, which associates with cellular senescence and elevated DNA damage. Mechanistically, DCAF8 mediates the degradation of DOCK11, a guanine nucleotide exchange factor for CDC42. In the absence of DCAF8, DOCK11 accumulates, leading to elevated CDC42 activity and consequential loss of polarity of HSCs. Knocking out Dock11 mitigates the senescence, DNA damage, and self-renewal defects of Dcaf8-/- HSCs. This study highlights a critical role of DCAF8 in preventing HSC senescence via the DOCK11-CDC42 axis and suggests potential therapeutic targets for preventing functional decline in HSCs.

Chimeric antigen receptor (CAR) T cells exhibit high response rates in B cell malignancies, but most patients eventually relapse. A key mechanism of treatment failure is the loss or downregulation of tumor antigen expression, yet strategies to modulate cell surface levels of CAR T cell targets remain largely unexplored. Here we identify B cell maturation antigen (BCMA), a central CAR T cell target in multiple myeloma (MM), as a highly short-lived protein that undergoes K48-linked polyubiquitylation at the plasma membrane, leading to its p97-dependent degradation via the ubiquitin-proteasome system (UPS). This previously unprecedented mechanism of plasma membrane protein regulation enables significant enhancement of BCMA expression via proteasome inhibitors (PI). The clinically approved PI carfilzomib (CFZ) significantly enhances the efficacy of BCMA-directed CAR T cells against both PI-sensitive and refractory MM cells in vitro and in vivo. Notably, treatment of ten patients with CFZ under the compassionate use CarCAR protocol - after relapse following BCMA CAR T cell therapy - resulted in increased BCMA expression in all patients. However, clinical responses were observed only in those with residual and/or expanding CAR T cells, suggesting restored CAR T cell function. These findings provide a rationale for the use of CFZ treatment in relapsed or refractory MM following BCMA CAR T therapy, advocate for future trials combining CFZ with BCMA CAR T cells and provide a framework for exploring UPS-dependent degradation of other immunotherapy antigens.

TP53-Y220C is a recurrent hotspot mutation in cancers and leukemias. It is observed predominantly in acute myeloid leukemia (AML)/myelodysplastic syndromes among hematological malignancies and is associated with poor outcome. The mutation creates a structural pocket in the p53 protein. PC14586 (rezatapopt) is a small molecule designed to bind to this pocket and thus restore a p53-wild type (p53-WT) conformation. We demonstrate that PC14586 converts p53-Y220C into a p53-WT conformation and activates p53 transcriptional targets, but surprisingly induces limited/no apoptosis in TP53-Y220C AML. Mechanistically, MDM2 induced by PC14586-activated conformational p53-WT and the nuclear exporter XPO1 reduce the transcriptional activities of p53, which are fully restored by inhibition of MDM2 and/or XPO1. Importantly, p53-WT protein can bind to BCL-2, competing with BAX in the BH3 binding pocket of BCL-2 and also binds to BCL-xL and MCL-1. However, such binding by PC14586-activated conformational p53-WT is not detected. Pharmacological inhibition of the BCL-2/BAX interaction with venetoclax fully compensates for this deficiency, induces massive cell death in AML cells and stem/progenitor cells in vitro and prolongs survival of TP53-Y220C AML xenografts in vivo. Collectively, we identified transcription-dependent and -independent mechanisms that limit the apoptogenic activities of reactivated conformational p53-WT and suggest approaches to optimize apoptosis induction in TP53-mutant leukemia. A clinical trial of PC14586 in TP53-Y220C AML/myelodysplastic syndromes has recently been initiated (NCT06616636).

Marstacimab is a monoclonal antibody that targets the tissue factor pathway inhibitor to rebalance hemostasis. Previous phase 1 and 2 trials established marstacimab safety and efficacy in adults with severe hemophilia A or B. BASIS is an open-label, phase 3 trial of marstacimab in males aged 12-74 years with severe hemophilia A (factor VIII <1%) or moderately severe to severe hemophilia B (factor IX ≤2%). Participants without inhibitors received on-demand (OD) or routine prophylaxis (RP) factor replacement during a 6-month observational phase (OP) before receiving once-weekly subcutaneous 150 mg marstacimab prophylaxis during a 12-month active treatment phase (ATP). Primary endpoints were annualized bleeding rate (ABR) for treated bleeds vs prior OD or RP during the OP and safety. Of 128 participants enrolled in the OP, 116 received marstacimab in the ATP. In the OD group (n=33), mean ABR (95% CI) decreased from 39.86 (33.05-48.07) in the OP to 3.20 (2.10-4.88) in the ATP, demonstrating superiority of marstacimab (estimated ABR ratio, 0.080 [0.057-0.113]; P < .0001). In the RP group (n=83), mean ABR decreased from 7.90 (5.14-10.66) in the OP to 5.09 (3.40-6.78) in the ATP, demonstrating noninferiority and superiority of marstacimab (estimated ABR difference, -2.81 [-5.42 to -0.20]; P = .0349). There were no deaths or thromboembolic events. Weekly subcutaneous marstacimab reduced ABR compared with OD or RP therapy in the OP in individuals with severe hemophilia A or moderately severe to severe hemophilia B without inhibitors. Marstacimab was safe and well tolerated with no unanticipated side effects. This trial was registered at www.clinicaltrials.gov as # NCT03938792.

Loss-of-function (LoF) mutations frequently found in human cancers are generally intractable by classical small molecule inhibitor approaches. Among them are mutations affecting polycomb-group (PcG) epigenetic regulators, EZH2 and ASXL1 frequently found in haematological malignancies of myeloid or lymphoid lineage, and their concurrent mutations associates with particularly poor prognosis. While there is clear need to develop novel and effective treatments for these patients, the lack of appropriate disease models and mechanistic insights have significantly hindered the progresses. Here we show that genetic inactivation of Asxl1 and Ezh2 in murine haematopoietic stem/progenitor cells results in highly penetrant haematological malignancies as observed in corresponding human diseases. These PcG proteins regulate both coding and non-coding genomes, leading to marked reactivation of transposable elements (TEs) and DNA damage responses in PcG LoF mutated cells, which create a novel vulnerability for PARP inhibitors (PARPi)-induced synthetic lethality. Using both mouse models and primary patient samples, we demonstrate that Asxl1/Ezh2 mutated cells are highly sensitive to PARPi that induce excessive DNA damage and significantly extend disease latency. Intriguingly the observed PARPi-sensitivity can be specifically overridden by reverse transcriptase inhibitors that interrupt target-site primed reverse transcription (TPRT) and life cycle of TEs. This mechanism is contrastingly different from the current concept of BRCAness associated PARPi-induced synthetic lethality, which largely rely on deficient homologous recombination and is independent on reverse transcriptase inhibitors. Together, this study reveals a novel application and mechanism of PARPi-induced synthetic lethal targeting of blood cancers with reactivated TEs such as those carrying PcG epigenetic mutations.

In inflammation, circulating neutrophils indirectly damage the skeleton by inducing formation of bone-resorbing osteoclasts. However, neutrophil progenitors in marrow have no known physiological function. A bone-protective role for the neutrophil lineage was recently suggested when a profound defect in bone structure was observed in mice with neutropenia due to Granulocyte Colony Stimulating Factor (G-CSF) deletion coupled with STAT3 hyperactivation in bone cells. Here, we tested the existence of this protective effect by manipulating neutrophil progenitors in bone marrow by Ly6G antibody (aLy6G) treatment. Two protocols revealed an inverse relationship between marrow neutrophil progenitors and osteoclasts. Two weeks of aLy6G treatment increased marrow immature neutrophils by 25% and halved osteoclast mRNA markers in cortical bone. In contrast, coupling six weeks of aLy6G with anti-rat IgG2a to maintain antigenicity reduced marrow pre-neutrophils by 50%. This doubled trabecular osteoclast surface, halved trabecular bone mass, and significantly reduced high density bone mass both in control mice, and in mice with bone-specific STAT3 hyperactivation. In culture, isolated pre-neutrophils dose-dependently inhibited osteoclastogenesis independent of direct contact. We conclude that neutrophil progenitors directly inhibit osteoclast formation by releasing soluble factors. This identifies a novel action of hematopoietic cells in marrow to protect bone structure.

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A conditional knockout mice have reduced laser-injury induced cremaster arteriolar thrombus formation and prolonged FeCl3 induced carotid arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced cytosolic ATP release to amplify agonist-stimulated calcium-PI3K-AKT signaling. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which positively regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

The human genome contains regulatory DNA elements, known as enhancers, that can activate gene transcription over long chromosomal distances. Here, we showed that enhancer distance can be critical for gene silencing. We demonstrated that linear recruitment of the normally distal strong HBB enhancer to developmentally silenced embryonic HBE or fetal HBG promoters through deletion or inversion of intervening DNA sequences led to strongly reactivated expression in adult erythroid cells and ex vivo differentiated hematopoietic stem and progenitor cells. A similar observation was made for the HBA locus in which deletion-to-recruit of the distal enhancer strongly reactivated embryonic HBZ expression. Overall, our work assigned function to seemingly nonregulatory genomic segments; by providing linear separation, they may support genes to autonomously control their transcriptional response to distal enhancers.

Suppression of plasminogen activation and/or plasmin activity (PA) reduces blood loss and decreases hemorrhage-related death. However, whether the endogenous PA system is a biological mechanism to prevent intravascular thrombus formation is debated, and the potential that reduced PA may increase venous thrombosis/thromboembolism (VTE) risk cautions against the use of antifibrinolytic agents. We aimed to determine the contribution of PA to VTE. Type 1 plasminogen-deficient humans enrolled in the HISTORY registry (https://clinical trials.gov; NCT03797495) reported pathologic pseudomembrane formation, but not unprovoked VTE. When subjected to an experimental model of venous thrombosis, compared to Plg+/+ mice, neither partial (Plg+/-) nor complete (Plg-/-) deletion of plasminogen altered thrombus mass or thrombus nucleated cell, platelet, or fibrin(ogen) content at 24 or 6 hours after thrombus induction. Administration of tranexamic acid (TXA) to mouse plasma in vitro or healthy mice in vivo dose-dependently delayed and suppressed plasma plasmin generation for up to 3 hours. However, mice administered TXA did not have significantly altered thrombus mass or thrombus composition at 24 or 6 hours after thrombus induction, despite unexpectedly persistent TXA in plasma. In a genome-wide association study, variants in gene regions encoding PA pathway proteins were not significantly associated with VTE risk. In the UK Biobank repository, plasminogen protein levels were not significantly associated with VTE risk. These data from genetic, pharmacologic, and proteomic analyses of mice and humans indicate that perturbations in PA do not increase VTE risk. Collectively, these results suggest PA is not a molecular regulatory mechanism to protect against VTE.

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-triggered mortality. Recently, we demonstrated the critical role of Fc-dependent complement activation in anti-CD36-mediated murine TRALI. In this study, we found that C5-/- mice were protected, and administration of anti-C5 rescued wild-type mice from anti-CD36-mediated TRALI. However, C5aR1-/- mice were not protected against anti-CD36-mediated TRALI, implying a possible role of C5b-9 (membrane attack complex [MAC]). Accordingly, elevated levels of MAC were detected in bronchoalveolar lavage fluid and lung tissue of mice with anti-CD36-induced TRALI. Inhibition of MAC formation by administration of anti-C7-blocking monoclonal antibody alleviated TRALI in mice, suggesting the critical role of the MAC in the pathology of anti-CD36-mediated TRALI. Furthermore, anti-C7 treatment also led to favorable outcome in murine TRALI induced by anti-major histocompatibility complex class 1, indicating the potential broader applicability of MAC inhibitors in the treatment of antibody-mediated TRALI. Therefore, this approach may be promising to further explore the treatment of patients with TRALI.

Macrophage activation syndrome (MAS) is believed to be caused by inappropriate proliferation and activation of the mononuclear phagocytic system. Adult-onset Still disease (AOSD) is characterized by neutrophil activation and a cytokine storm, which can lead to the severe and potentially life-threatening complication of MAS. RNA sequencing revealed that neutrophils may play a distinct role and enhance the innate immunity of patients with AOSD with MAS (AOSD-MAS). In the CpG-induced secondary hemophagocytic lymphohistiocytosis (HLH) model, the depletion of neutrophils significantly reduced cytokine levels with effects comparable with monocyte depletion. Significant enrichment was observed in the type I/II interferon and JAK-STAT pathways in neutrophils from patients with AOSD-MAS. Treatment of 10 patients with refractory AOSD-MAS with ruxolitinib led to the resolution of inflammatory parameters and clinical symptoms. RNA sequencing and ex vivo assays confirmed that ruxolitinib suppressed aberrant NETosis and STAT3/STAT5 signaling. In vivo, PAD4 knockout further confirmed the pathogenic role of NETosis in a secondary HLH model. Moreover, the selective inhibition of STAT3 or STAT5 alleviated systemic inflammation. Ten functional variants were identified in genes related to the JAK-STAT pathway, however, their clinical relevance requires further validation. These findings suggest that ruxolitinib has the potential to facilitate disease remission in patients with refractory AOSD-MAS by broadly inhibiting JAK-STAT signaling and modulating neutrophil activation and NETosis.

Immunotherapy has become standard of care in the treatment of diffuse large B-cell lymphoma (DLBCL). Changes in immunophenotypes observed at first diagnosis predict therapy outcome but little is known about the resolution of these alterations in remission. Comprehensive characterization of immune changes from fresh, peripheral whole blood revealed a functionally relevant increase of myeloid-derived suppressor cells, reduced naïve T-cells, and an increase of activated and terminally differentiated T-cells before treatment which aggravated after therapy. Suggesting causal relation, injection of lymphoma in mice induced similar changes in the murine T cells. Distinct immune imprints were found in breast cancer and AML survivors. Identified alterations persisted beyond five years of ongoing complete remission and in DLBCL correlated with increased pro-inflammatory markers such as IL-6, B2M, or sCD14. The chronic inflammation was associated with functionally blunted T-cell immunity against SARS-CoV-2-specific peptides and reduced responses correlated with reduced Tn-cells. Persisting inflammation was confirmed by deep sequencing and by cytokine profiles, together pointing towards a compensatory activation of innate immunity. The persisting, lymphoma-induced immune alterations in remission may explain long-term complications, have implications for vaccine strategies, and are likely relevant for immunotherapies.

Insufficient eradication of cancer cells and survival of drug tolerant clones are major relapse driving forces. Underlying molecular mechanisms comprise activated prosurvival and antiapoptotic signaling, leading to insufficient apoptosis and drug resistance. The identification of programmed cell death pathways alternative to apoptosis opens up possibilities to antagonize apoptosis escape routes. We have earlier shown that acute lymphoblastic leukemia (ALL) harbors a distinct propensity to undergo cell death by receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis, activated by small-molecule second mitochondria-derived activators of caspase (SMAC) mimetics. Despite demonstrated safety and tolerability of SMAC mimetics in clinical trials, their efficacy as single agent seems still limited, highlighting the need for combinatorial treatments. Here, we investigate so far unexplored regulatory mechanisms of necroptosis and identify targets for interference to augment the necroptotic antileukemia response. Ex vivo drug response profiling in a model of the bone marrow microenvironment reveals powerful synergy of necroptosis induction with histone deacetylase (HDAC) inhibition. Subsequent transcriptome analysis and functional in vivo CRISPR screening identify gene regulatory circuitries through the master transcription regulators specificity protein 1 (SP1), p300, and HDAC2 to drive necroptosis. Although deletion of SP1 or p300 confers resistance to necroptosis, loss of HDAC2 sensitizes cells to RIPK1-dependent cell death by SMAC mimetics. Consequently, our data inform strong in vivo antileukemic activity of combinatorial necroptosis induction and HDAC inhibition in patient-derived human leukemia models. Thus, transcriptional dependency of necroptosis activation is a key regulatory mechanism that identifies novel targets for interference, pointing out a strategy to exploit alternative nonapoptotic cell death pathways to eradicate resistant disease.

The prognosis for relapsed or refractory (R/R) nucleophosmin 1-mutated (NPM1m) acute myeloid leukemia (AML) is poor and represents an urgent unmet medical need. Revumenib, a potent, selective menin inhibitor, was recently approved for the treatment of R/R acute leukemia with a KMT2A translocation in patients aged ≥1 year based on results from the phase 1/2 AUGMENT-101 study. Here we present results from patients with R/R NPM1m AML enrolled in the phase 2 portion of AUGMENT-101. Enrolled patients received revumenib with or without a strong CYP3A4 inhibitor every 12 hours in 28-day cycles. Primary endpoints were rate of complete remission (CR) or CR with partial hematologic recovery (CRh; CR+CRh), and safety and tolerability. Secondary endpoints included overall response rate (ORR) and duration of response. As of September 18, 2024, 84 patients received ≥1 dose of revumenib. Median age was 63 years; 1 patient was aged <18 years. The protocol-defined efficacy-evaluable population for the primary analysis included 64 adult patients (≥3 prior lines of therapy, 35.9%; prior venetoclax, 75.0%). The CR+CRh rate was 23.4% (1-sided P=.0014); the ORR was 46.9%. Median duration of CR+CRh was 4.7 months. Five of 30 responders (16.7%) proceeded to hematopoietic stem cell transplant (HSCT); 3 resumed revumenib after HSCT. Treatment-related adverse events led to treatment discontinuation in 4 patients (4.8%). Revumenib demonstrated clinically meaningful responses in this heavily pretreated, older population with NPM1m AML, including remissions that enabled HSCT. The safety profile of revumenib was consistent with previously reported results. This trial was registered at www.clinicaltrials.gov as NCT04065399.

Coagulation factor Va (FVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, FVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the FVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of FVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here, we report the cryo-EM structure of the FVa-APC complex at 3.15 Å resolution in which the protease domain of APC engages R506 in the A2 domain of FVa through electrostatic interactions between positively charged residues in the 30-loop and 70-loop of APC and an electronegative surface of FVa. The auxiliary γ-carboxyglutamic acid and epidermal growth factor domains of APC are highly dynamic and point to solvent, without making contacts with FVa. Binding of APC displaces a large portion of the A2 domain of FVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch," or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC, which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the FVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.