Despite great progress in understanding the genomic basis of immature T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL) and acute leukemias of ambiguous lineage (ALAL), there are still cases that lack defining genetic markers, complicating risk stratification and limiting targeted therapeutic options. Recent studies have shown that enhancer hijacking drives oncogene activation in approximately half of T-ALL cases, with the BCL11B enhancer frequently involved. Here, we describe a subtype of leukemia with a distinct gene expression signature, and immunophenotype characterized by positivity for immature (CD38), myeloid (CD13), T-lymphoid (cytoplasmic (c)CD3, CD7), and B-lymphoid markers (CD19, CD79a, CD10). This subtype is defined by the t(14;16)(q32;q24) translocation, which places the FOXF1 gene and its antisense long noncoding RNA gene FENDRR under the regulatory control of the BCL11B enhancer, leading to their ectopic transcriptional activation. Common concomitant genetic lesions are loss-of-function alterations of GATA3, CDKN2A/CDKN2B deletion and activating JAK/STAT and NOTCH1 pathway mutations. Patients were predominantly children and adolescents/young adults (AYA) and experienced poor treatment outcome. High-throughput drug screening of 176 compounds demonstrated efficacy of combined BCL2-family proteins and JAK/STAT signaling inhibitors. Additionally, the clinical use of tyrosine kinase inhibitors in some of these cases showed therapeutic efficacy. Collectively, these findings identify BCL11B-enhancer mediated deregulation of FOXF1/FENDRR as a hallmark of a subtype of high-risk lineage ambiguous leukemia that is potentially amenable to targeted therapeutic intervention.

The Bcl2 inhibitor venetoclax in combination with the hypomethylating agents azacitidine (ven/aza) has become increasingly utilized clinically for the treatment of many hematological malignancies. Whilst its effects on malignant cells have been extensively studied, its impact to the surrounding bone marrow microenvironment (BME) remains unexplored. In this study, we report that ven/aza therapy causes significant damage to the BME of mice. Comparatively high Bcl2 expression in the sinusoidal endothelial cell compartment (SEC) amongst all stromal subtypes, results in high sensitivity to ven/aza treatment, causing selective depletion of SECs and breakdown in cell-cell communication pathways in the endothelial cell (EC) network, leading to vascular leakiness in the BM. Furthermore, our detailed transcriptomic and imaging studies reveals significant downregulation of essential adhesion molecules in residual SECs, leading to significant defects in human hematopoietic stem/progenitor cell (HSPC) homing and engraftment of hematopoietic stem cells (HSCs) after ven/aza treatment. To conclude, our study showcases that maintaining SEC integrity in response to ven/aza therapy may play a key factor in achieving effective engraftment of donor derived HSCs.

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.

Long telomere length (TL) extends replicative capacity in vitro, and predisposes to clonal hematopoiesis. We characterized the cancer phenotype in 51 individuals from 24 families with mutant POT1, a negative regulator of telomerase elongation (median age 51, range 5-94). Hematologic malignancies were second in prevalence after melanoma (27%), and lymphoid subsets were more common. They clustered with history of sarcoma, thyroid cancer and chronic myeloproliferative neoplasms. UKB participants with pathogenic POT1 variants had long TL and higher lymphoid malignancy rates (45% by age 80, Hazard ratio 8.28, 95% CI, 5.29-13.0). Across cohorts, diagnoses encompassed acute lymphoblastic leukemia and Hodgkin lymphoma in children/young adults, and chronic lymphocytic leukemia/multiple myeloma in adults. They clustered in families manifesting as autosomal dominant pan-lymphoma with genetic anticipation at times. Lymphocyte TL was longer than granulocytes at baseline (age-adjusted mean +1 kb, P<0.0001), and was preserved longitudinally with aging. Ultra-long lymphocyte TL >99th percentile was more sensitive for identifying pathogenic variants (58% vs. 38% for granulocytes). Among asymptomatic POT1 variant carriers, 60% (12 of 20) had immunophenotype-detected B and/or T cell clonality with complete penetrance after age 65 (7 of 7). IGH CDR3 sequencing supported age-dependent pruning of the B cell repertoire, and cytogenetic and next-generation analyses uncovered preclinical clonal lymphoma-associated changes in nearly all POT1 variant carriers older than 60 (9 of 10). Our data identify extended cellular longevity due to long TL as an inherited risk factor for lymphoma explaining its syndromic association with solid tumors, and in some cases, myeloproliferative neoplasms.

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.

Recessively inherited loss-of-function mutations in Excision Repair Cross-Complementing 6 like 2 (ERCC6L2) cause a bone marrow failure (BMF) syndrome characterized by moderate cytopenias, frequent somatic TP53 mutations, and a propensity to develop myeloid malignancies. The pathophysiology and molecular mechanisms underlying the BMF syndrome as well as its association with TP53-mutant clonal hematopoiesis (CH) and myeloid malignancies have remained poorly understood. Using novel preclinical in vitro and in vivo model systems, we demonstrate that Ercc6l2 maintains the competitive fitness of hematopoietic stem and progenitor cells (HSPCs) by mitigating replication stress. Sustained replication stress and DNA damage in Ercc6l2-deficient HSPCs cause p53 pathway activation followed by cell cycle arrest and apoptosis. Moreover, Ercc6l2 deficiency results in decreased expression of master hematopoietic regulators Runx1 and Gata1 in HSPCs. Altogether, loss of Ercc6l2 leads to reduced HSPC numbers, bone marrow hypocellularity, and cytopenias. Notably, somatic Trp53 mutations restore cellular fitness of Ercc6l2-deficient HSPCs by abrogating p53 pathway activation and restoring Runx1 and Gata1 expression, thereby correcting the BMF phenotype. However, p53 loss fails to normalize replication stress, allowing for the accumulation of DNA damage over time which increases the likelihood for leukemic transformation. Our data uncover the pathogenesis of ERCC6L2 disease and provide a prototypic example of clonal compensation in BMF syndromes, where somatic mutations in leukemia-associated genes - in this case TP53 - transiently improve blood cell production at, however, the expense of increasing leukemogenic potential.

Circadian rhythms orchestrate immune activation and effector function, yet whether within-day timing influences chimeric antigen receptor (CAR) T-cell therapy outcomes remains unknown. We conducted an international, multicenter retrospective study of 1052 adults with relapsed or refractory large B-cell lymphoma treated with CD19-directed CAR T-cell therapy across 7 centers (2017-2025). The median infusion time was 11:48 am (interquartile range, 11:06 am to 12:45 pm). Each hour later in infusion time was associated with an increased risk of progression, relapse, or death (hazard ratio, 1.11; 95% confidence interval, 1.03-1.20; P = .004) after adjustment for center, product, and key clinical variables. One-year progression-free survival (PFS) was 51.4% for early (before 12:00 noon) infusion vs 35.2% for late (at or after 12:00 noon) infusion, whereas overall survival was similar between groups. The PFS benefit was driven by lower relapse and higher complete response rates in the early infusion group. Although no differences were observed in immune toxicities, late infusion correlated with higher peak inflammatory markers and reduced day 7 CAR T-cell expansion. Together, these findings suggest that the timing of CAR T-cell infusion may influence therapeutic efficacy and support prospective evaluation of circadian-informed delivery strategies.

Intestinal Enterococcus domination has been associated with an increased risk of mortality from acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT), a curative-intent treatment for patients with hematologic malignancies. In this study, we investigated interactions between Enterococcus and the intestinal epithelium as a mechanism to aggravate GVHD. We observed that endogenous intestinal Enterococcus outgrowth was associated with increased GVHD mortality and major histocompatibility complex class II (MHC-II) expression by intestinal epithelial cells in the colon in an MHC-disparate mouse model of GVHD. Monocolonization of nontransplanted gnotobiotic mice with Enterococcus faecalis was sufficient to induce colonic MHC-II expression. Conversely, select species within the genus Enterococcus, as well as a consortium of 4 anaerobic commensal bacteria including Blautia producta, did not affect colonic MHC-II expression in gnotobiotic mice. In addition, E faecalis colonization induced inflammatory responses in CD4+ T cells and natural killer cells from the colonic lamina propria, the 2 main sources of interferon gamma production that drives MHC-II expression in nonprofessional antigen-presenting cells. We further explored the potential therapeutic benefit of establishing colonization resistance against E faecalis through administration of a lantibiotic-producing B producta strain after allo-HCT. Colonization of transplanted mice with a consortium of commensal bacteria containing the lantibiotic-producing B producta strain prevented intestinal Enterococcus domination after transplantation and improved GVHD survival. Our results demonstrate a potential mechanism by which Enterococcus aggravates GVHD through increased MHC-II expression in the intestinal epithelium. Targeting the Enterococcus-epithelium-MHC-II axis thus presents a therapeutic opportunity to prevent lethal GVHD.

High levels of circulating tumor cells (CTC) are a powerful predictor of poor outcomes in newly diagnosed multiple myeloma, yet the mechanistic underpinnings of this correlation remain unknown. To investigate whether CTC-related pathobiology is driven by a specific CTC subset, paired bone marrow and blood samples from patients with newly diagnosed multiple myeloma were analyzed by single-cell transcriptomics and whole-genome sequencing. This revealed that down to the individual clone level, CTC and paired bone marrow cells are transcriptionally similar, without evidence for a distinct circulating population. In contrast, bone marrow myeloma cells from patients with high CTC levels had increased proliferation and unbalanced primary genetic events, including enrichment for MAF and CCND translocations. To investigate the impact of heterogenic genomic events on CTC levels, whole-exome and bulk RNA sequencing from the Multiple Myeloma Research Foundation CoMMpass data set were analyzed and validated in our in-house data sets. Bone marrow tumor cells from patients with high CTC levels were uniformly characterized by transcriptomic signatures of proliferation. Furthermore, CTC levels were uniquely dependent on primary genomic events and high-risk secondary genomic events, including amplification 1q, deletion 1p, deletion 13q, biallelic TP53 mutations, and increased apolipoprotein B editing complex-induced mutations even in patients without MAF translocations. Finally, we developed a model that predicts the impact of genetic alterations and tumor burden on CTC levels. In sum, we reveal that CTC are the net result of tumor burden, primary translocations, and secondary genomic events, making CTC a powerful biomarker for genomics-driven high-risk disease in patients with newly diagnosed myeloma.

Sickle cell disease (SCD) and β-thalassemia are devastating genetic disorders resulting from defects in the β-globin subunit of adult hemoglobin. Both disorders are ameliorated by the induction of γ-globin, a component of fetal hemoglobin (HbF). Therefore, the development of safe, effective, and widely available inducers of HbF is needed. Here, we discovered that slow cycling erythroid cells exhibit increased γ-globin expression. To understand the molecular basis of this, we screened all cyclin-dependent kinase inhibitors (CDKIs) for their ability to induce HbF using CRISPR activation. We found that overexpression of CDKN1B, which encodes p27Kip1 (but not overexpression of other CDKIs), induces γ-globin expression at the transcriptional level. CDKN1B mutants expressing proteins unable to bind/inhibit CDKs and/or cyclins revealed that γ-globin induction by p27Kip1 depends largely on the domains involved in its cell cycle function. Pharmacological inhibition and genetic reduction of CDK4/6 also result in increased HbF. In genetic rescue experiments, we show that p27Kip1 induces HbF by inhibiting CDK4/6, through a mechanism that is likely BCL11A and ZBTB7A independent. Furthermore, palbociclib, an oral CDK4/6 inhibitor, significantly increases HbF in a murine SCD model at doses that are well tolerated. Moreover, we show that HbF induction by hydroxyurea, a drug currently in use to treat SCD, may be mediated in part by CDK4/6 inhibition. Overall, our findings establish a causal relationship between CDK4/6 activity and γ-globin production and suggest that single or dual CDK4/6 inhibitors might be therapeutically beneficial for SCD and β-thalassemia.

Infections remain a challenge during treatment of patients with multiple myeloma (MM) with anti-B-cell maturation antigen (BCMA) and anti-G protein-coupled receptor class C group 5 member D (GPRC5D) bispecific antibodies (bsAbs). However, the mechanism underlying different rates and severity of infections induced by the 2 bsAbs remains poorly understood. Single-cell RNA sequencing of bone marrow (BM) aspirates of 11 patients with MM and 8 healthy donors revealed BCMA expression on mature B cells and, surprisingly, in small pre-B cells within B-cell precursors. GPRC5D expression was restricted to malignant and less to normal plasma cells (PCs). Next-generation flow cytometry immune profiling showed that anti-BCMA bsAbs severely depleted BM mature B cells, from 4.9% to 0% (P< .001), and normal PCs, from 0.17% to <0.0002% (P< .001), during treatment of 62 patients with relapsed MM. This was observed throughout therapy. Additional flow cytometry (n = 31) and single-cell RNA sequencing studies (n = 8) demonstrated that, in contrast to anti-GPRC5D, anti-BCMA bsAbs also depleted immature and small pre-B cells. The MIcγ1 mouse model was used as a negative control of BCMA expression in all stages of the B-cell lineage, confirming no depletion of any B-cell subset after anti-BCMA treatment. In conclusion, we show that although GPRC5D bsAbs selectively target PCs, anti-BCMA bsAbs target both PCs and B cells from the small pre-B stage onward. Our study provides mechanistic insight into the increased infection risk with anti-BCMA therapy and supports individualized bsAb strategies in MM. Moreover, dual targeting of B cells and PCs may have therapeutic potential in other B-cell malignancies or autoimmune diseases.

Marstacimab, a monoclonal antibody that inhibits tissue factor pathway inhibitor, is approved for prophylactic use in individuals with hemophilia A or B without inhibitors. We present efficacy and safety for individuals with inhibitors. The open-label, single-arm, phase 3 study evaluated once-weekly subcutaneous flat-dose marstacimab in males aged 12 to <75 years with severe hemophilia A or moderately severe to severe hemophilia B. Participants with inhibitors received bypassing agents (on-demand or routine prophylaxis) during a 6-month observational phase (OP) before entering a 12-month active treatment phase (ATP) with marstacimab. Primary end points were annualized bleeding rate (ABR) of treated bleeds and safety. Of 60 participants with inhibitors in the OP, 51 entered the ATP and received marstacimab. In the on-demand group (n = 48), mean estimated ABR declined from 19.78 (95% confidence interval [CI], 16.12-24.27) in the OP to 1.39 (95% CI, 0.85-2.29) during the ATP (ABR ratio, 0.07 [95% CI, 0.042-0.118]; 2-sided P< .0001). Results were consistent by hemophilia type (ABR ratio, 0.05 [hemophilia A, n = 40]; 0.13 [hemophilia B, n = 8]). Participants reported significant improvements in health-related quality of life. Adverse events were common but mostly mild; 1 treatment-related grade 3 skin rash led to discontinuation. Antidrug antibodies were detected in 19.6% of participants, with no apparent effect on efficacy or safety. In participants with inhibitors, marstacimab was associated with reduced bleeding rates and an acceptable safety profile, with no thromboembolic events. Marstacimab may be a viable treatment option for people with hemophilia A or B with inhibitors. This trial was registered at www.clinicaltrials.gov as #NCT03938792. ClinicalTrials.gov identifier: NCT03938792.

We assessed the overall survival (OS) impact of time to relapse (TTR) in multinational cohorts with independent observational and randomized validation. Patients with nMTCL with frontline complete response were assigned to TTR12 (≤12 months) or without TTR12 based on time to progression or next therapy. OS analyses included modified landmark (m-LM), standard landmark (s-LM), and time-dependent Cox (td-Cox), adjusting for age, histology, and prognostic index for T-cell lymphoma (PIT) score. Across 452 patients, 165 (36.5%) had TTR12, 181 (40%) relapsed at ≥12 months, and 106 (23.5%) remained relapse-free. TTR12 conferred worse OS using m-LM (hazard ratio [HR], 2.14; 95% confidence interval [CI], 1.58-2.90; P< .001), s-LM (HR, 1.92; 95% CI, 1.39-2.66; P< .001); and td-Cox (HR, 5.81; 95% CI, 2.94-11.46; P< .001). Results were consistent in the independent validation cohorts. TTR12 consistently conferred worse OS irrespective of frontline stem cell transplantation or PIT score, in peripheral T-cell lymphoma, not otherwise specified (m-LM: HR, 2.32; 95% CI, 1.51-3.55; P< .001; s-LM: HR, 2.10; 95% CI, 1.33-3.31; P = .001), anaplastic large cell lymphoma (m-LM: HR, 3.34; 95% CI, 1.18-9.50; P = .023; s-LM: HR, 2.96; 95% CI, 1.02-8.81; P = .046), and angioimmunoblastic T-cell/T follicular helper cell lymphoma (m-LM only: HR, 1.92; 95% CI, 1.15-3.21; P = .013). Second-line novel therapies improved OS (second-line start to death) vs chemotherapy in TTR12 only (HR, 0.60; 95% CI, 0.37-0.97; P = .038; without TTR12: HR, 0.82; 95% CI, 0.51-1.32; P = .407). TTR12 serves as a prognostic and potential OS surrogate marker, supporting stratification of new risk groups and need for their differential treatment.

We show that somatic genetic rescue is frequent in telomere biology disorders (TBDs) caused by germ line ZCCHC8 variants. Our results highlight the critical intrinsic role of ZCCHC8 in human hematopoiesis and a potential mechanism for disease modification in TBDs.

Sickle cell disease (SCD) and β-thalassemia, caused by mutations or deletions in the β-globin gene, are among the most prevalent genetic disorders worldwide, significantly affecting global health and mortality. Recently, reactivation of δ-globin gene expression has been proposed as a potential therapeutic strategy for these conditions. In this study, we found that interferon gamma (IFN-γ) and IFN-β significantly enhance δ-globin expression and activate the JAK/STAT signaling pathway in erythroid cells, with IFN-γ exerting a stronger effect than IFN-β. In erythroid cells derived from CD34+ progenitors, IFN-γ not only increased δ-globin expression but also promoted differentiation, as confirmed by quantitative polymerase chain reaction, western blotting, high-performance liquid chromatography, and flow cytometry. Inhibition of the JAK/STAT pathway, either through a JAK1/2 inhibitor (AZD1840 or ruxolitinib) or via small interfering RNAs targeting JAK1, JAK2, STAT1, or STAT3, significantly decreased both basal and IFN-γ-induced δ-globin expression in HBD-HiBiT knockin HUDEP2 cells. Mutation or removal of the putative IRF-1/STAT2 binding site (-265 to -242) and the adjacent STAT binding site (-243 to -231) in the δ-globin promoter impaired IFN-γ-induced δ-promoter activity. Chromatin immunoprecipitation assays confirmed enhanced binding of interferon regulatory factor 1 (IRF-1) and STAT1 upon IFN-γ treatment. Our elucidation of the mechanism by which a specific molecule induces δ-globin expression suggests that IFN-γ may hold therapeutic potential for patients with SCD, and that screening for compounds that can induce δ-globin could offer a novel pharmaceutical strategy for treating β-hemoglobinopathies.