Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms with an increased risk of progression to secondary acute myeloid leukemia (sAML). This study investigates the genomic correlates of disease progression in MDS by profiling active genomic regulatory regions and their transcriptional impact through H3K27ac ChIP-seq and RNA-seq analysis on CD34+ bone marrow progenitors cells isolated from a prospective cohort of 86 and 357 patients, respectively. Our analysis revealed distinct patterns of genomic region activation and transcriptional regulation across different disease stages (low-risk MDS, high-risk MDS and sAML). Unexpectedly, unsupervised clustering revealed a subset of low-risk MDS patients displaying regulatory and transcriptional profiles similar to those of high-risk MDS and sAML, highlighting early molecular events that may predispose patients to disease progression. This subset is characterized by PU.1 genomic occupancy in regions linked to immune and inflammatory responses, increased T-cell and NK activation, and a higher frequency of SRSF2 mutations. Clinically, patients in this group exhibit greater susceptibility to infections and cardiovascular events, along with an elevated risk of disease progression, resulting in a significantly reduced overall survival. Functional studies demonstrate that PU.1 inhibition suppresses MDS cell proliferation and clonogenicity, as impaired PU.1 binding inhibits the activation of key transcriptional programs involved in disease advancement. Collectively, these findings identify epigenetic factors that predispose low-risk MDS patients to progression into high-risk MDS and, ultimately, sAML.

ANKRD26-related thrombocytopenia (THC2) is a rare inherited platelet disorder caused by germline variants in the 5' untranslated region (UTR) of ANKRD26. While prior studies using in vitro models or isolated case reports have suggested impaired megakaryopoiesis as a central mechanism, detailed insights have remained elusive-primarily due to the rarity, fragility, and heterogeneity of megakaryocytes. Here, we present a comprehensive, cross-validated analysis of bone marrow samples from four independent THC2 patients, integrating single-cell transcriptomics and ex vivo functional profiling. Across all patients, we analyzed CD34⁺ hematopoietic stem and progenitor cells (HSPCs) (47,281 THC2-HSPCs vs. 51,907 control cells) and primary megakaryocytes (pMKs) (7,309 THC2-pMKs vs. 5,077 controls), uncovering a consistent pattern of megakaryocyte progenitors (MkP) expansion and a marked reduction in polyploid megakaryocytes-indicating a conserved pathophysiologic phenotype. In our index patient, we identified the 5'UTR single-nucleotide variant in ANKRD26 that led to significantly elevated expression across four megakaryocyte-lineage subsets-spanning multipotent progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, and MkPs-as well as in terminally enriched pMKs. Spatial transcriptomics and confocal imaging localized ANKRD26 to the centrosome, implicating it in mitotic regulation during megakaryocyte maturation. Mechanistically, we discovered that elevated ANKRD26 induces apoptosis in polyploid megakaryocytes via JUNB-mediated transcriptional activation of CDKN1A (p21)-operating independently of the canonical p53-PIDDosome axis. This multi-patient study provides the most comprehensive cellular and molecular portrait of ANKRD26-driven thrombocytopenia to date, offering novel insights into defective megakaryopoiesis and identifying candidate therapeutic targets to restore platelet production.

The Pediatric Hodgkin Consortium hypothesized that by increasing chemotherapeutic dose density for Hodgkin lymphoma (HL) they could increase the complete response (CR) rate among patients with favorable-risk HL after 8 weeks of Stanford V (vinblastine, doxorubicin, vincristine, bleomycin, mechlorethamine, etoposide and prednisone) compared with 8 weeks of VAMP (vinblastine, Adriamycin [doxorubicin], methotrexate, and prednisone). This would translate to a decrease in patients who required radiation therapy (RT) to achieve a cure. The HOD08 study was a phase 2 multicenter, investigator-initiated single-arm trial for patients aged ≤21 years with previously untreated stage 1A or 2A HL without mediastinal bulk or extranodal disease extension and <3 sites of disease. Treatment consisted of a modified 8-week Stanford V regimen. Modified, tailored, field RT was administered only to disease sites achieving less than a CR. The primary objective was to increase CR rate after 8 weeks of chemotherapy by at least 20% (from an estimated 44% to 64%) compared with patients treated on a previous trial (HOD99). HOD08 enrolled 85 patients with HL and 72 were evaluable for the primary objective, of whom 55 (76.4%) achieved a CR at all sites and did not receive RT. The 5-year event-free survival and overall survival rates for the entire cohort were 87.4% (95% confidence interval [CI], 80.4-95.0) and 98.7% (95% CI, 96.2-100), respectively. A dose-dense modified Stanford V regimen reduced the proportion of pediatric patients with low-risk HL who received RT while maintaining excellent outcomes. This trial was registered at www.clinicaltrials.gov as #NCT00846742.

Chronic lymphocytic leukemia (CLL) and clonal hematopoiesis (CH) both commonly occur in older individuals. To characterize CH in CLL, 620 patients were analyzed (CLL12 [ibrutinib vs placebo] and CLL14 [venetoclax-obinutuzumab (Ven-Obi) vs chlorambucil-obinutuzumab (Clb-Obi)]) using error-corrected next-generation sequencing with a variant allele frequency (VAF) threshold of 0.5%. Median follow-up was 76.1 months, and median age was 68 years. CH was detected in 58.2% of patients, most commonly affecting DNMT3A, TET2, TP53, and ASXL1. Longitudinal analysis in CLL14 revealed persistence of the majority of CH clones during follow-up, whereas in more than half of the patients, additional CH mutations were detected. BAX- and U2AF1-mutated CH emerged during Ven-Obi exposure, and PPM1D-mutated CH emerged during Clb-Obi exposure, highlighting treatment gene-specific selection. Clonal fitness analyses revealed accelerated CH clone expansion during therapy, followed by slower growth after treatment. In vitro, genetically modified CD34+ hematopoietic stem/progenitor cells harboring BAX mutations showed enhanced survival and decreased apoptosis. CH was associated with neutropenia, and all patients with Richter transformation (RT; n = 11) had CH. Whole-exome sequencing delineated the contribution of CH mutations in 2 of 4 investigated patients with RT. Large CH clone size (>10% VAF) was independently associated with shorter overall survival with placebo (P = .049) and shorter progression-free survival with Clb-Obi after adjusting for age, immunoglobulin heavy chain variable status, and del(17p). In contrast, CH had no prognostic impact in patients receiving targeted therapies. This study demonstrates the high prevalence of CH, highlights its differential impact across CLL therapies, and underscores its adverse influence on patient outcomes.

Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent β-thalassemia (TDT) involves autologous transplantation of hematopoietic stem and progenitor cells transduced with a modified β-globin gene to produce functional adult hemoglobin (HbAT87Q). Sixty-three participants with TDT (median [range] age: 17 [4-35] years) received beti-cel in phase 1/2 (n = 22) or phase 3 (n = 41) studies and enrolled in the long-term follow-up LTF-303 study (clinicaltrials.gov/NCT02633943; median [range] follow-up: 5.9 [2.9-10.1] years). Manufacturing refinements in phase 3 increased transduction efficiency, resulting in higher drug product vector copy number and HbAT87Q levels, which translated into higher hemoglobin and transfusion independence (TI) rates compared with phase 1/2. TI was achieved by 68.2% (15/22) of phase 1/2 participants (median weighted average Hb during TI, 10.2 g/dL) and 90.2% (37/41) of phase 3 participants (median, 11.2 g/dL) and was sustained through last follow-up. Treatment efficacy was similar across ages and TDT genotypes. Among participants achieving TI, 73% (38/52) had discontinued iron chelation at last follow-up, with no increase in liver iron concentration. Markers of ineffective erythropoiesis, including serum transferrin receptor and erythropoietin, improved with restoration of iron homeostasis. Health-related quality-of-life assessment scores showed durable improvements. No malignancies, insertional oncogenesis, or vector-derived replication-competent lentivirus were reported. These findings establish beti-cel as a durable, one-time therapy that achieves TI, restores iron balance, and improves quality of life, offering a potentially curative treatment option for people with TDT.

Reactivating the fetal globin genes HBG1 and HBG2 in adult erythroid cells represents a validated therapeutic approach for hemoglobinopathies. Central mediators of the fetal-to-adult hemoglobin transition include the direct transcriptional HBG1/2 repressors BCL11A1,2, LRF3, and NFIA/X4. Limited-scale screens have attempted to expand the regulatory circuity surrounding fetal globin silencing, but systematic genome-wide dissection of such pathways is lacking. We employed a two-tiered genetic screening strategy - a novel CRISPR-Cas12a-based screening platform followed by a domain-focused CRISPR-Cas9 screen - to interrogate all known human coding genes for their impact on HBG1/2 regulation and erythroid cellular fitness, generating a comprehensive resource for the field. Among the top hits was PTPA, an activator of the serine-threonine phosphatase PP2A whose loss elevates HBG1/2 levels while preserving erythroid differentiation. Phenotypic rescue experiments revealed that PTPA silences HBG1/2 expression primarily by regulating BCL11A expression. To our knowledge, this study represents the most comprehensive CRISPR dissection of HBG regulation to date, highlighting the power of Cas12a-based genome-scale screening for uncovering disease-relevant pathways.

Clonal hematopoiesis of indeterminate potential (CHIP) is driven by hematopoietic stem cells (HSCs) carrying leukemia-associated mutations that expand in the bone marrow. Several prior studies have revealed that the spatial organization of hematopoietic cells in the bone marrow impacts clonal behaviors. Specifically, leukemic blasts have been found to expand almost exclusively in a subset of marrow cavities that are undergoing active bone remodeling, but whether these cavities also support the expansion of non-malignant mutant clones has never been visualized. Although it is widely appreciated that systemic inflammation promotes the selection of mutant clones, this view has emerged without considering the potential heterogeneity in the inflammatory landscape shaped by local bone remodeling. Leveraging intravital imaging and a murine model of CHIP (Tet2+/-), we demonstrated transcriptional and functional compartmentalization of the marrow microenvironment. Macrophages within non-resorptive cavities are inherently anti-inflammatory, which suppresses disease-initiating Tet2+/- cells while preserving the healthy counterpart. Time-lapse imaging further revealed non-transient association between Tet2+/- clones and CD206+ macrophages. Spatially resolved single-cell transcriptomic profiling and functional assessment revealed that physiological bone remodeling influences CD206+ macrophage plasticity and cytokine secretion which regulate the clonal burden. Additionally, anti-tumor immunity alteration within the microenvironment occurred as early as the formation of initial clones. Suppressing bone remodeling with zoledronate or targeting macrophage-associated niche factors mitigated clonal development. Collectively, our study reveals a previously unrecognized inflammatory landscape shaped by local bone remodeling. The finding presents targetable mechanisms and warrants further studies on the use and precautions of bone-modulating management in clonal blood disorders.

Janus kinase (JAK) inhibitors have changed the treatment landscape of myeloproliferative neoplasms (MPNs), graft-versus-host disease, and several autoimmune conditions. Although approved JAK inhibitors generally target the JAK2 kinase domain, and several also target the JAK1 kinase domain in active form (type I inhibition), new inhibitors that either exhibit a type II mechanism of inhibition of the kinase domain in an inactive state or that target the pseudokinase domain with potential preference or specificity for the JAK2 V617F mutant have progressed to clinical trials. This is the most prevalent mutation in MPNs. An ideal inhibitor would target persistently activated JAK2 in MPNs, eradicate the clone or induce deep molecular remission in addition to clinical and hematologic remission, and spare wild-type JAK2 that is critical for hematopoiesis and immune response. We discuss perspectives of these and other modes of JAK inhibition, as well as primary and secondary/exploratory study end points in clinical trial design, along with potential biomarker correlates to evaluate the potential efficacy of next-generation vs conventional JAK inhibitors.

Acute graft-versus-host disease (aGVHD) is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT) and patients with steroid-refractory aGVHD have a dismal prognosis. We have previously shown that the enteroendocrine hormone glucagon-like peptide-2 (GLP-2) has tissue regenerative activity in the lower GI in mice and patients with steroid-refractory aGVHD. Here we explored the tissue protective effect of the enteroendocrine hormone gastrin for aGVHD of the stomach. We observed that aGVHD caused a loss of gastrin-producing G-cells and parietal cells (PCs) and an increase of pH in the stomach, while allogeneic T cells infiltrated the stomach wall. Pentagastrin treatment of aGVHD mice rescued the loss of PCs, normalized the pH in the stomach, increased stomach stem cell marker expression and abundance of LGR5+ cells, and changes in the stomach microbiome. Gastrin also increased the viability of stomach and small intestine organoids in vitro. Gast-/- mice experienced more severe aGVHD in the intestine and liver compared to WT mice, which was rescued by pentagastrin-treatment. In patients developing aGVHD, low gastrin levels in stomach biopsies were connected to reduced survival. Moreover, gastrin expression in the stomach correlated with aGVHD severity and tissue damage scores in independent patient cohorts. This study delineates the protective role of gastrin in aGVHD of the stomach in mice and patients and provides a rationale for therapeutic use of pentagastrin in a clinical trial for patients with aGVHD.

Organ transplantation is a pivotal treatment for patients with organ failure. ABO-incompatible (ABOi) transplantation, developed to expand the donor pool, presents significant clinical challenges due to pre-existing antibodies targeting ABO antigens on donor organs. Current therapies employing broad B-cell depletion, such as rituximab, effectively reduce antibody-mediated rejection but increase infection risks. Therefore, there is a critical need of targeted methods to specifically eliminate ABO-responsive B cells. Here, we developed a novel bispecific antibody-ligand conjugate (BiALC) platform designed to selectively target ABO-responsive B cells. Utilizing synthetic trisaccharide A antigens conjugated to T-cell-recruiting Fab fragments, our optimized hexameric construct, (A3-peg)2-αCD3, demonstrated enhanced affinity and potent cytotoxicity specifically against type A antigen-responsive B cells. Notably, BiALC maintained robust efficacy even in the presence of circulating anti-A antibodies. In murine models, (A3-peg)2-αCD3 selectively depleted A-responsive B cells without broadly affecting total IgM+ and IgG+ B cell populations, preserving overall immune competence. Similarly, the human-compatible BiALC, (A3-peg)2-αhCD3, effectively and selectively depleted type A-responsive B cells from human PBMCs, with potency comparable to rituximab, while sparing total antibody-secreting cells. Overall, the BiALC strategy offers a promising antigen-specific approach to reduce rejection risks in ABOi transplantation without inducing broad immunosuppression through nonspecific pan-B cell depletion, supporting its potential for clinical translation.

Secondary central nervous system (CNS) large B-cell lymphoma (SCNSL) occurs in the de novo setting, as a CNS-isolated relapse, or synchronous (concomitant CNS and systemic) relapse. SCNSL is a devastating event without therapeutic consensus. Thus, we aimed to evaluate treatment outcomes in an international cohort. Progression-free survival (PFS), overall survival (OS) and cumulative incidence of relapse (CIR, estimated using competing-risk models) were reported. Prognostic factors were identified in a 6-month landmark multivariate analysis. Outcomes following thiotepa autologous stem cell transplant (ASCT) and chimeric antigen receptor T-cell therapy (CAR-T) delivered at relapse were compared following propensity score matching (PSM). A total of 1139 patients were included in the analysis (de novo: 537; relapsed SCNSL: 602). 2-year PFS estimates were 40.4%, 43.9% and 16.2% for de novo SCNSL, CNS-isolated relapse, and synchronous relapse respectively. Patients with CNS-isolated relapse demonstrated low rates of systemic recurrence (24-month CIR 6%). Thiotepa-ASCT correlated with longer survival in de novo SCNSL (PFS: HR=0.57; P=0.005; and OS: HR=0.62; P=0.023) and CNS-isolated relapses (PFS: HR=0.55; P=0.002; and OS: HR=0.39; P<.0001) in 6-month multivariable landmark analysis. ASCT (thiotepa or non-thiotepa) also associated with improved survival in synchronous relapses (PFS: HR=0.57; P=0.023; and OS: HR=0.48; P=0.019). Higher survival with thiotepa-ASCT compared to CAR-T was observed in survival analyses following PSM (PFS: HR=0.45; P=0.005 and OS: HR=0.41; P=0.014). These data support thiotepa-ASCT in eligible patients, particularly de novo disease and CNS-isolated relapses. CNS-isolated relapse was infrequently associated with systemic recurrence, supporting treatment regimens adopted from primary CNS lymphoma.

Core-binding factor acute myeloid leukemia (CBF-AML) is associated with KIT mutations and deregulated expression of KIT.

The contribution of clonal hematopoiesis (CH) and disease-initiating precursors in B-cell non-Hodgkin lymphomas (B-NHLs) remains underexplored. Such precursors may drive clonal evolution, contributing to disease progression and relapse. Here, we systematically profiled genetic precursor lesions in 43 B-NHL patients using complementary whole exome, targeted, and single-cell sequencing approaches. CH-associated mutations with a variant allele frequency of ≥1% were detected in the peripheral blood of 55% of patients, with significantly higher frequencies in indolent compared to aggressive B-NHL (p=0.03). Quantification of allele burden in flow-sorted cell populations revealed a B-cell-skewed expansion of CH clones - contrasting the myeloid differentiation bias reported in individuals without hematologic malignancies. Gene-specific expansion patterns were evident among the most frequent CH lesions, with DNMT3A-mutant clones exhibiting impaired hematopoietic differentiation and TET2-mutant clones showing multi-lineage propagation. Notably, identical CH clones were detected in 41% of corresponding lymphomas, displaying distinct clonal dynamics: tumor-promoting CH (expansion in B-NHL; 10/16 clones; mainly TP53) and tumor-infiltrating CH (no expansion; mainly DNMT3A). Moreover, we identified lymphoma-associated mutations in flow-sorted hematopoietic progenitors from patients with indolent but not aggressive B-NHL and observed a stepwise accumulation of mutations along the lymphoid differentiation path. Single-cell genotyping confirmed the presence of mutated progenitors in 3 follicular, 2 mantle cell and 2 marginal zone lymphoma patients, providing direct evidence of a pre-neoplastic state in disease pathogenesis. Our findings offer novel insight into the cellular origin of nodal B-NHLs and highlight a previously underappreciated role for early clonal events involving the stem/progenitor cell compartment.