The B-cell maturation antigen (BCMA)-targeting bispecific T-cell engager (BiTE®) pavurutamab (AMG 701) directs the cytotoxic T-cell response toward multiple myeloma (MM) cells. This phase 1/1b, open-label, dose-exploration and dose-expansion study evaluated the safety, tolerability, and efficacy of pavurutamab monotherapy in patients with triple-class relapsed/refractory MM (RRMM). Pavurutamab (5-18,000 µg) was administered intravenously weekly with step-up dosing in week 1. Overall, 172 patients received at least one dose of pavurutamab; 73 received the recommended phase 2 dose (RP2D; 18,000 µg), with two different step-up dosing regimens in phase 1b. Twelve patients had dose-limiting toxicities, which included cytokine release syndrome (CRS) and increased transaminases. None occurred at the RP2D. The most frequently reported treatment-emergent adverse events included CRS (74.4%), anemia (61.0%), neutropenia (47.1%), and hypophosphatemia (45.3%). Grade 3 or higher infections were noted in 60 patients (34.9%). The overall response rate was 46.5% among all patients and 65.8% (≥very good partial response [VGPR], 60.3%) among 73 patients treated at the RP2D. At median follow-up of 17.2 months, median duration of response was 36.6 months (95% confidence interval [CI]: 22.3-not estimable). Median progression-free survival for all patients was 5.5 months (95% CI: 2.8-10.1) and 16.8 months (95% CI: 5.0-not estimable) with the RP2D. Pavurutamab exposure generally increased in a dose-proportional manner, with high soluble BCMA levels correlating with lower exposure level. The acceptable safety profile, pharmacokinetics, and preliminary efficacy of pavurutamab supports anti-BCMA T-cell engager therapy in heavily pretreated patients with RRMM. The trial was registered at www.ClinicalTrials.gov as # NCT03287908.

Intercellular communications between multiple myeloma (MM) cells and osteoclast precursor cells (pre-OCs) contribute extensively to the occurrence and development of myeloma-related bone destruction. However, key interacting modes and the substances exchanged involved in this communication remain unclear. In this study, we discover that tunnelling nanotubes (TNT) directly connect MM and pre-OCs. Using stable isotope labeling with amino acids in cell culture (SILAC) assay and a positive-negative double selection strategy, we identify Filamin-A (FLNA) as a major protein transported from MM to pre-OCs. FLNA acts as a molecular clutch linking ECM-bound MAC1 to the cytoskeleton, activating Rho and MAPK signaling pathways and promoting F-actin polymerization, which subsequently enhances osteoclast differentiation by modulating cellular stiffness, traction force, and deformability. Additionally, FLNA directly binds vinculin and promotes its recruitment to podosome, thereby enhancing functions of podosome and bone resorption capacity of osteoclasts. Conditional depletion of Flna in mice suppresses podosome activity and reduces stiffness, traction force, and deformability in pre-OCs, leading to significantly impaired osteoclast differentiation and increased bone mass. In the Vk*MYC mouse model of myeloma, administration of the TNT inhibitor Latrunculin B disrupts FLNA transport to osteoclasts and alleviates osteolytic bone disease. These findings highlight the critical role of MM-transferred FLNA in osteoclastogenesis and suggest that targeting TNTs may represent a therapeutic strategy to limit pathological bone resorption associated with multiple myeloma.

Acute myeloid leukemia (AML) is a multi-clonal disease, existing as a milieu of clones with unique but related genotypes as initiating clones acquire subsequent mutations. However, bulk sequencing cannot fully capture AML clonal architecture or the clonal evolution that occurs as patients undergo therapy. To interrogate clonal evolution, we performed simultaneous single-cell molecular profiling and immunophenotyping on 43 samples from 32 NPM1-mutated AML patients at different timepoints in disease progression. Here we show that diagnosis and relapse AML samples display similar clonal architecture patterns, but signaling mutations drive increased clonal complexity, specifically at relapse that correlates with overall survival. We uncovered unique genotype-immunophenotype relationships regardless of disease state, suggesting leukemic lineage trajectories can be hard-wired by the mutations present. Analysis of longitudinal samples from patients on front-line AML therapy identified dynamic clonal and immunophenotypic changes consistent with the genotype-immunophenotype relationships we identified.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy arising from the neoplastic transformation of immature T cells during their development in the thymus. Deciphering the developmental programs whose dysregulation drives T-ALL pathogenesis is critical for the development of novel targeted therapies, which remain an urgent unmet need for the treatment of this disease. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of numerous physiological processes including cancer. However, the specific role of miRNAs in human T-cell development and T-ALL pathogenesis remains largely unexplored. In this study, we comprehensively evaluated miRNA expression profiles across human T-cell development using microarray analysis and identified a dynamic expression pattern of miR-16-2, which is upregulated during early pre-T cell proliferative stages up to the resting stage of immature thymocytes immediately preceding TCRab expression, and is subsequently downregulated. We also confirmed the coordinated regulation of miR-15b expression, consistent with the reported clustered genomic location of both miRNAs. Notably, functional studies identified the miR-15b/16-2 cluster as a negative regulator of early thymocyte proliferation and demonstrated that overexpression of miR-15b/16-2 in T-ALL cells impaired leukemic growth in vitro and tumor progression in patient-derived xenotransplantation assays. Mechanistically, miR-15b/16-2 represses the expression of the genes encoding BCL-2 and CYCLIN D3, thereby promoting apoptosis and cell cycle dysregulation in T-ALL cells, characterized by an accumulation of G0-phase cells and a defective transition to the G2/M phase. Overall, these findings support a novel tumor-suppressive function for miR-15b/16-2 in T-ALL and highlight its potential as a promising therapeutic target.

Children with Down syndrome (DS) have an elevated risk of developing myeloid leukemia in DS (ML-DS). In addition to mutations in GATA1, which generate the truncated isoform GATA1-short (GATA1s), ML-DS requires additional somatic gene mutations, most frequently in cohesion and polycomb repressive complex 2 (PRC2) genes. Here, we show that PRC2 insufficiency underlies ML-DS pathogenesis. Transplantation of Gata1s fetal liver cells followed by deletion of the cohesion subunit Stag2 and/or the PRC2 component Ezh2 induced megakaryocyte-biased differentiation and expansion of megakaryocytic progenitors, culminating in lethal myelofibrosis. Mechanistically, loss of Stag2 or Ezh2 reinforced Gata1s-driven reduced chromatin accessibility at erythroid transcription factor target loci in pre-megakaryocyte/erythroid progenitors (pre-MegEs), thereby promoting megakaryocytic skewing. Ezh2 loss attenuated the Gata1s-mediated global elevation of H3K27me3 in pre-MegEs, resulting in derepression of a broad set of PRC2 target genes and establishing a functionally PRC2-insufficient state. Similarly, Stag2 loss induced a moderate but significant degree of PRC2-insufficient state in Gata1s progenitors. Furthermore, chromosome 21-encoded miR-125b blocked megakaryocytic differentiation of Gata1s progenitors lacking either Stag2 or Ezh2 alone, but drove full transformation and expansion of CD150+Sca-1+c-Kit+ leukemic stem cell-like populations only upon concurrent loss of both Stag2 and Ezh2, leading to acute megakaryoblastic leukemia in mice. These findings reveal that cohesin and PRC2 insufficiencies converge on PRC2 dysfunction while exerting distinct epigenetic effects, and synergize with trisomy 21 and GATA1s to remodel the epigenetic landscape, driving progression from a preleukemic state to overt leukemia.

Hemostasis and thrombosis are strongly dependent on the unique ability of platelets to rapidly activate integrin receptors and to firmly adhere to sites of injury under shear stress conditions. Central to integrin activation is the small GTPase RAP1, which itself is activated by guanine nucleotide exchange factors (GEFs). CalDAG-GEFI (RASGRP2) is the highest expressed and functionally dominant platelet RAP-GEF. However, a genome-wide association study also suggested a significant role for RAPGEF2 (PDZ-GEFI), a low expressed RAP-GEF, in human platelet aggregation. Here, we used mice deficient in RAPGEF2 (megakaryocyte-specific, Rapgef2mKO), CalDAG-GEFI (Caldaggef1-/-), or both RAPGEF2 and CalDAG-GEFI (DKO) to characterize the contribution of RAPGEF2 signaling to platelet function, hemostasis and thrombosis. RAPGEF2 protein was detected in murine and human platelets. Compared to control or Caldaggef1-/- platelets, both RAP1 activation and integrin aIIbb3-mediated aggregation were significantly diminished in DKO platelets. When compared to controls, Rapgef2mKO platelets exhibited reduced integrin activation, a more reversible aggregation response, and impaired adhesion under conditions of shear stress ex vivo and in vivo. Mechanistic studies strongly suggest that RAPGEF2 operates downstream of receptors coupled to the heterotrimeric G protein, G13 (GNA13), such as aIIbb3 and the thromboxane receptor. Together, our studies provide genetic evidence that RAPGEF2 in platelets operates downstream of G13 as an important regulator of RAP1 signaling and integrin activation, especially under conditions of elevated shear stress. These findings markedly improve our understanding of G protein signaling and integrin function in platelets, with potential implications for the development of improved platelet-targeted therapies for cardiovascular disease.

Allergic transfusion reactions (ATR) are potentially lethal adverse events that occur in approximately 0.3%-6% of plasma or platelet transfusions. The main mechanism described is an IgE-mediated response to donor proteins, such as IgA and haptoglobin in deficient patients. Recently, in a nationwide retrospective observational study, we reported that group O recipients of group B or AB platelet concentrates or plasma were at significantly higher risk of severe ATR than recipients with other ABO combinations. Given the antigenic proximity between group B determinant and the galactose-α-1,3-galactose (α-Gal) epitope, we hypothesize a role for α-gal IgE-mediated sensitization in these ATRs. In this report, we investigated the presence of anti- α-Gal IgE in a cohort of 59 patients with severe ATR. We found that group O recipients of group B or AB blood components (plasma or platelet concentrates) were substantially more likely to exhibit high titers of group B cross-reactive anti-α-gal IgE than other patients, supporting our hypothesis that α-gal sensitization underlies these severe ATRs. These findings suggest that α-gal sensitization is an additional mechanism responsible for ATRs in major ABO-incompatible transfusion and should be systematically explored in group O patients having received group B/AB plasma or platelets.

NPM1-mutated AML is driven by aberrant HOX/MEIS1 expression, whose mechanistic basis remained unresolved for years. Recent paradigm-shifting studies show that mutant NPM1 organizes phase-separated nuclear condensates that concentrate transcriptional regulators at active chromatin, directly sustaining the pathogenic HOX/MEIS1 transcriptional program. This framework explains the activity of Menin-KMT2A inhibitors, recently approved by the FDA, in this AML subtype and positions disruption of these assemblies as a precision strategy to eliminate oncogenic transcription.

Aberrant enhancer usage is a defining feature of oncogenic transcriptional reprogramming. Therapeutic strategies that disrupt enhancer-driven gene regulation may offer new treatment avenues. MYB is a key hematopoietic transcription factor that is frequently dysregulated in a broad range of cancers and plays a critical role in sustaining malignant cell states, including in aggressive leukemia subtypes such as KMT2A-rearranged (KMT2A-r) leukemias. The molecular mechanisms by which it maintains oncogenic transcription remain incompletely understood. Here, we investigate the role of MYB in directing pathological enhancer activity to drive oncogene expression in leukemia. Using high-resolution Micro-Capture-C, we show that upon MYB degradation, highly defined enhancer-promoter interactions at MYB binding sites are lost, correlating with significant downregulation of target gene expression. When anchored to a gene desert region, the Myb transactivation domain (MybTA) is sufficient and necessary for nucleation of an enhancer-like region. Critically, long-range chromatin interactions are established up to 400 kb away from where MybTA is anchored. This results in the activation of transcription from distal cryptic elements, which is reduced or abolished in the presence of point mutations that disrupt its interaction with the co-activators P300/CBP. Together, these results indicate that MYB activity alone is sufficient to generate an enhancer, inducing transcription through precise enhancer-promoter crosstalk, and identify the MYB-P300/CBP axis as a therapeutically actionable vulnerability in enhancer-driven malignancies.

The increasing availability of genomic and transcriptomic sequencing has uncovered diverse genomic alterations and distinct gene expression profiles driving hematologic diseases, yet a data integration and sharing platform dedicated to hematology remains lacking. We developed the American Society of Hematology (ASH) HematOmics Program (ASHOP, ashop.hematology.org), a resource for exploring somatic alterations and gene fusions, transcriptomic results, and clinical data from 5,960 patients spanning B-cell precursor and T-cell acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic syndromes, and chronic lymphocytic leukemia. Users can explore genomic alteration landscapes and co-mutation patterns via lollipop and matrix plots, and analyze significantly altered genes in user-defined sub-cohorts. Transcriptomes can be explored through interactive UMAPs, clustering, differential expression, and pathway enrichment. Genomic, transcriptomic features, and clinical outcomes can be correlated in a user-driven manner, or combined to precisely define study cohorts. We illustrate four use cases of ASHOP: (1) stratification of DUX4-rearranged B-cell leukemias into Early/Multipotent and Committed subgroups with distinct outcomes, (2) characterization of HOXA/HOXB expression patterns in acute myeloid leukemias, (3) correlating mutational burden with mismatch repair deficiency and mutational signatures, (4) investigation of TP53 alteration landscape. ASHOP is an open-access resource to inform genomic and transcriptomic data interpretation for hematologic malignancies, and will expand to support additional diseases and data modalities from the ASH community.

Dynamin-related protein 1 (Drp1) is an abundant platelet protein best known for its function in mitochondrial fission. Yet little is known about how Drp1 is controlled during platelet activation. While evaluating signaling pathways leading to phosphorylation of Drp1 in platelets, we identified a phosphorylation network wherein activation of G protein-coupled receptors or ITAM/hemITAM receptors resulted in phosphorylation of Ser616-Drp1 via p38. These signaling mechanisms were reinforced by ADP- and TxA2-mediated amplification pathways. In contrast, exposure of platelets to pacifying agents such as PGE1 or nitric oxide resulted in Ser637-Drp1 phosphorylation by cyclic nucleotide-dependent protein kinases. This unique circuitry was leveraged to develop immune-based platelet function assays, enabling ELISA and lateral flow assay formats. Compared to standard platelet function assays, Drp1 phosphorylation remained robust in whole blood samples following agitation or extended incubation and samples could be frozen for batching. As proof-of-principle for antiplatelet testing, phospho-Drp1 measurements were obtained at baseline, during a week of aspirin or clopidogrel exposure, and during a week of washout. Arachidonic acid-induced Ser616-Drp1 phosphorylation following aspirin ingestion demonstrated an enhanced dynamic range with improved linearity relative to light transmission aggregometry and improved signal to noise relative to the VerifyNow™ aspirin test. Ser637-Drp1 phosphorylation enabled sensitive detection of clopidogrel ingestion. These studies elucidate the unique signaling circuit controlling Drp1 phosphorylation in platelets and validate the approach of using detailed knowledge of platelet signaling pathways to develop high-fidelity immune-based assays to monitor platelet function.

In 82 patients with classic Hodgkin lymphoma, stable gonadal hormone levels were observed up to 24 months after nivolumab and AVD (N-AVD) first-line treatment in the NIVAHL trial. These findings suggest preserved gonadal function and fertility following 4×N-AVD. NCT03004833.