Glanzmann thrombasthenia (GT) is characterized by absent platelet aggregation in response to all agonists except ristocetin and is caused by recessive inactivating variants in ITGA2B or ITGB3. Although patients with GT typically are described as having normal platelet counts, autosomal dominant activating variants in ITGA2B or ITGB3 cause macrothrombocytopenia. Interestingly, in our cohort of 16 patients with GT, 8 consistently exhibited platelet counts at the lower end of the normal range. We studied the role of integrin a IIb R 3 in platelet formation using megakaryocytes (MKs) derived from genetically modified immortalized MK cell lines (imMKCLs), focusing on 2 modifications of ITGB3: ITGB3 -/- (inactivating) and ITGB3 WT/D673_E713del (activating). In static differentiation culbut reduced proplatelet formation. Platelet production was also impaired in a 3-dimensional silk-based bone marrow system and in shaking cultures, confirming a quantitative role for ITGB3 in platelet production independent of the type of variant. Although thrombin receptor activating protein-activated, in vitro-generated platelets lacking a IIb R 3 failed to bind the activation-dependent PAC-1 antibody, ITGB3 WT/D673_E713del platelets bound PAC-1 before activation, mimicking the patient's phenotype. Transcriptome profiling and metabolomic analyses of integrin a IIb R 3 -deficient MKs revealed impaired serine metabolism and downregulation of SLC3A2 (CD98hc), an amino acid transporter chaperon known to interact with the R 3 subunit. Flow cytometry confirmed decreased CD98hc in mutant MKs, whereas reexpression of wild-type ITGB3 in ITGB3 -/- MKs restored a IIb R 3 and CD98hc expression, normalized proplatelet formation, and enhanced serine uptake. These results uncover a previously unrecognized role of integrin a IIb R 3 in coupling serine metabolism to platelet biogenesis.
Host-versus-graft reaction (HVGR) is a major challenge in allogeneic chimeric antigen receptor (CAR) T-cell therapy. To counter host natural killer (NK) cell attacks, we armored allogeneic, HLA-I-deficient, B-cell maturation antigen (BCMA)-targeting CAR T cells with an NKG2A CAR. In vitro and animal studies demonstrated that allogeneic CAR-NKG2A T cells effectively resisted host NK cell-mediated killing. BCMA and NKG2A dual-targeting allogeneic CAR T cells (CT0590) resisted killing by NK cells and showed robust antitumor activity in preclinical in vivo models. On the basis of these data, a first-in-human study enrolled 5 patients (4 with relapsed and refractory multiple myeloma [RRMM] and 1 with primary plasma cell leukemia [pPCL]). CT0590 was well tolerated and caused no doselimiting toxicities, treatment-related death, or graft-versus-host disease. Three patients achieved confirmed responses, including 2 with stringent complete response (sCR). Notably, sCR in the patient with RRMM was still ongoing (duration of response >23 months) at the time of data cutoff, and sCR in the patient with pPCL lasted for 20 months. Both patients showed robust expansion of universal CAR T cells (maximum concentration of >280 000 copies per mu g genomic DNA) and higher baseline NKG2A expression on NK cells than nonresponders. These results suggest that CAR-NKG2A technology may overcome HVGR, especially in patients with elevated NKG2A expression on NK cells. Further studies of CT0590 in RRMM and pPCL are warranted. This trial was registered at www.clinicaltrials.gov as NCT05066022.
Abstract Antibodies directed against platelet factor 4 (PF4) are increasingly recognized as key mediators of immunothrombosis. In heparin-induced thrombocytopenia (HIT), these antibodies emerge following exposure to the polyanion heparin, whereas in vaccine-induced immune thrombocytopenia and thrombosis (VITT), they arise in response to vector-based vaccination or (adeno)viral infections. In both settings, anti-PF4 antibodies promote platelet activation and enhance inflammatory and thrombotic processes. PF4 itself has also been implicated in atherosclerosis and cardiovascular disease (CVD), although clinical studies investigating this link have yielded inconsistent results. We review the current knowledge on the roles of PF4 and anti-PF4 antibodies in atherosclerosis and CVD and discuss preclinical and clinical evidence for their relevance in the perioperative setting, for disease progression and antiplatelet therapy.
Myelodysplastic neoplasms (MDS) feature hematopoietic deficits driven, in part, by transcript splicing abnormalities. To date, 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. In this study, we characterize an MDS-associated isoform of methyl-binding-domain 1 (MBD1; 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. Over-expression of MBD1-L in healthy human hematopoietic stem and progenitor cells recapitulated archetypal MDS defects, including reduced terminal glycophorin A + 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 the resulting changes in chromatin accessibility, histone mark deposition, and transcriptional changes, revealed that these defects arise from an isoform-specific switch in MBD1 binding behavior. The MBD1-L isoform refocuses heterochromatin-promoting activity of MBD1-L from methylated to unme thylated CpGs, thus enacting broad downregulation of CpG-rich promoters and secondary epigenetic effects mediated through its downstream target, BCOR. Remarkably, we also found that directly reversing abnormal MBD1 splicing across a broad range of primary human MDS samples using nanoparticle-encapsulated antisense RNA oligonucleotides (ASO) enhanced in vitro erythroid differentiation, supporting the therapeutic use of ASO-based therapies for MDS treatment. Thus, our findings demonstrate MBD1-L to be a global disease-driving splice variant in MDS and illustrate the potential of RNA-based therapies for the broad treatment of MDS.
The transcription factor GATA1 has pleiotropic hematopoietic functions, particularly in erythroid and megakaryocytic ontogeny. Although mechanistic investigations have uncovered many facets of GATA1 biology, how GATA1 coregulates divergent cell fates remains incompletely characterized. We previously described that loss of Stag2, a member of the cohesin complex and a recurrent mutational target in myelodysplastic syndrome (MDS) and myeloid leukemia of Down syndrome, results in altered chromatin accessibility, transcription factor function, and cell differentiation. Therefore, we hypothesized that chromatin accessibility determines GATA1 cistrome specificity and lineage fate decisions. To understand the connection between chromatin accessibility and GATA1, we comprehensively studied erythropoiesis in Stag2 Delta mice. Defects in Stag2-deficient hematopoiesis included reduced numbers of erythroid progenitors (EryPs), impaired terminal erythroid differentiation, increased number of megakaryocyte progenitors, and increased megakaryocytes. RNA-and assay for transposase-accessible chromatin-sequencing of EryPs revealed altered patterns of Gata1 target gene expression with altered accessibility in conjunction with loss of expression of erythroid targets and gain of megakaryocyte targets. Despite unchanged Gata1 expression, Gata1 occupancy was reprogrammed from erythroid to megakaryocyte targets with Fli1 motifs enriched at Stag2 Delta Gata1 binding sites. Functionally, we observed that Stag2-deficient EryPs have a diminished erythroid output and augmented megakaryocyte output in orthogonal differentiation assays, which was partially reversed with Fli1 knockdown. Human models and patients with primary MDS recapitulated the essential phenotypic and molecular features of our in vivo murine MDS model. Collectively, this study establishes chromatin accessibility as a determinant of transcription factor binding specificity, revealing an accessibility-driven Gata1 retargeting mechanism underlying MDS dyserythropoiesis.
