VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is an autoinflammatory disorder caused by acquired somatic ubiquitin like modifier activating enzyme 1 (UBA1) mutations in hematopoietic stem cells, affecting peripheral myeloid and natural killer (NK) cells. Given the high rate of severe infections in patients with VEXAS, we hypothesized that NK-cell dysfunction contributes to this susceptibility. We conducted a comprehensive immune characterization of peripheral NK cells in patients with VEXAS (n = 40), patients with autoinflammatory diseases without UBA1 mutations (n = 22), and older sex-matched healthy controls (n = 16). Multiparameter phenotyping used cytometry by time-of-flight, single-cell RNA sequencing (scRNA-seq), whole-blood stimulation assays, and in vitro NK-cell cytotoxic assay. Peripheral NK cells in VEXAS were quantitatively and qualitatively impaired. Mass cytometry revealed reduced frequencies of mature cytotoxic CD56dim NK cells and expansion of the CD56high CD16dim subset. NK cells exhibited exhaustion features, including increased programmed cell death protein 1 expression, and reduced cytotoxic markers such as NKp46 and CD8α. scRNA-seq analysis showed decreased signatures of cytotoxicity and interleukin-2 (IL-2) and interferon gamma (IFN-γ) production, alongside increased inflammatory signatures. Whole-blood stimulation assays confirmed impaired IL-2, IFN-γ, and granzyme B production following Toll-like receptor 3 (TLR3), TLR4, and TLR7/TLR8 agonist stimulation. Extended NK phenotyping by flow cytometry confirmed reduced activating receptors' expression and impaired IFN-γ production in VEXAS syndrome. Moreover, in vitro UBA1 inhibitors impaired NK-cell cytotoxic capacity and promote cell death. Finally, reduced NK-cell frequencies were independently associated with an increased risk of severe infections. These findings suggest that NK-cell dysfunction in VEXAS syndrome contributes to increased susceptibility to severe infections.

Throughout thrombopoiesis megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Casein kinase 1α (CK1α) is a major player and thus, an important therapeutic target in several hematological malignancies. This study aimed to define the role of CK1α for the essential steps of thrombopoiesis and to dissect potential mechanisms of thrombocytopenia. MK-specific CK1α-deficiency resulted in a macrothrombocytopenia. Ck1αPf4Δ/Pf4Δ mice displayed a substantial BM hyperplasia with pivotal changes in MK nuclear lobulation and reduced contact to BM sinusoids. Ck1αPf4Δ/Pf4Δ MKs displayed a defective cytoskeleton organization reflected by a decreased amount of polymerized filamentous actin and disturbed microtubule dynamics due to p21/p53 accumulation and impaired Rho-associated protein kinase (ROCK)/LIM domain kinase (LIMK)/cofilin signaling. Further, pronounced defects in DMS (demarcation membrane system) polarization and proplatelet formation of Ck1αPf4Δ/Pf4Δ MKs, unraveled CK1α as a prerequisite for thrombopoiesis. Our findings could be translated into a human approach, because a CRISPR/Cas9-mediated genetic deletion of CSNK1A1 in MKs derived from human CD34+ progenitor cells resulted in a substantial defect in human MK maturation and platelet production. The present observations elucidated CK1α as an important signaling molecule in MK cytoskeletal dynamics and polarization, proplatelet formation, and polyploidization, thus highlighting the crucial role of CK1α in platelet biogenesis.

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain underexplored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single-cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. By generating high-resolution Micro Capture-C data in primary patient samples, we identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may be a mechanism that drives transcriptional heterogeneity in cancer more generally.

The purpose of this study was to explore and determine the optimal landmark for defining complete remission after intensive induction therapy that best correlates with long-term survival outcome among patients with newly diagnosed acute myeloid leukemia.

This phase 2 trial assessed CD7 chimeric antigen receptor (CAR) T cells derived from previous transplant or newly HLA-matched donors for relapsed/refractory T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). Early termination from departmental closure yielded 55 treated patients out of 70 planned. Within 3 months, 89% of treated patients achieved best overall response of partial remission or better. A total of 19 received stem cell transplantation at a median of 1.3 (range, 1.0-10.6) months. After a 26.3-month median follow-up, median event-free survival was 5.0 months (95% confidence interval [4.1-8.4]), with median 8.5-month overall survival (95% confidence interval [6.1-15.6]). No deaths occurred within 30 days; adverse events included cytokine release syndrome in 87% at grades 1 to 2 and 11% at grade 3 and neurotoxicity in 9% at grade 1. In addition, graft-versus-host disease was in 38% at grades 1 to 2 and 2% at grade 3. Grades 1 and 2 infections occurred in 29%. Cytopenias occurred in 4% at grade 2 and 96% at grades 3 and 4. After 30 days, grades 3 to 5 adverse events included cytopenias (grade 3 in 24%; grade 4 in 67%), infections (grade 3 in 9%; grade 4 in 5%; grade 5 in 9%), graft-versus-host disease (grade 3 in 4%; grade 5 in 4%), thrombotic microangiopathy (grade 5 in 4%), and hepatic failure (grade 5 in 2%). Furthermore, 11 encountered nonrelapse mortality after 30 days, representing 20% of treated patients and 35% of responders without consolidatory transplantation. Although effective at inducing remission, death in remission beyond 30 days is a concern. This trial was registered at www.clinicaltrials.gov as #NCT04689659.

Immunoglobulin light-chain (AL) amyloidosis is a plasma cell disorder characterized by progressive organ dysfunction secondary to deposition of organized immunoglobulin light-chain aggregates. Achievement of rapid and deep normalization of involved immunoglobulin free light chains is necessary to maximize chances of reversibility of organ dysfunction, which, in turn, results in improved quality and length of life. There are no US Food and Drug Administration (FDA)-approved therapies for patients with relapsed AL amyloidosis. B-cell maturation antigen-targeting (BCMA)-bispecific T-cell engagers teclistamab and elranatamab have shown high activity and acceptable safety profile in patients with relapsed and/or refractory multiple myeloma, leading to their FDA approval. Herein, we report on safety and efficacy of elranatamab for patients with relapsed and/or refractory AL amyloidosis. We treated 9 consecutive patients with advanced-stage AL amyloidosis with single-agent elranatamab, observing a 100% overall response and 67% complete response rate, including minimal residual disease negativity, with expected toxicities. Median time to hematological response was 9 days (range, 6-24), with deep suppression in involved free light chains observed within 1 cycle of therapy, translating in cardiac and renal responses at 3 to 6 months. These data support prospective studies exploring elranatamab for patients with relapsed AL amyloidosis.

Clinical resistance or intolerance to tyrosine kinase inhibitors remains challenging for the treatment of chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) with central nervous system (CNS) relapse. Therapeutic options are currently limited for patients who develop the gatekeeper mutations or compound mutations. Herein we describe the preclinical profile of TGRX-678, an allosteric, specifically targeting the ABL myristoyl pocket (STAMP) inhibitor, with potent antiproliferative activity against most adenosine triphosphate (ATP) site mutants of BCR::ABL1 and minimal off-target cytotoxicity. When combined with ponatinib, TGRX-678 synergistically resensitizes the highly resistant compound mutants and T315M to growth inhibition at clinically achievable concentrations. TGRX-678 exhibits relatively high cell permeability and is not a substrate of drug efflux transporters, namely ATP-binding cassette B1 (ABCB1) and ABCG2. It also demonstrates a markedly improved in vivo pharmacokinetic profile and higher oral bioavailability compared with asciminib. Importantly, TGRX-678 penetrates the blood-brain barrier and exhibits in vivo efficacy in a murine model of CNS blast crisis leukemia. Collectively, these findings suggest that TGRX-678 is a novel BCR::ABL1 allosteric inhibitor with high selectivity, potency, and unique pharmacologic features, which has the potential to treat relapse or refractory CML and Ph+ ALL, even with CNS involvement.

Leukemias with NUP98 rearrangements exhibit heterogeneous phenotypes such as acute myeloid leukemia, T-cell acute lymphoblastic leukemia (T-ALL), or myelodysplastic syndrome/neoplasms associated with fusion partners, whereas the mechanism responsible for this heterogeneity is poorly understood. Through genome-wide mutational and transcriptional analyses of 177 NUP98-rearranged leukemias, we show that cooperating alterations are associated with differentiation status even among leukemias sharing the same NUP98 fusions, such as NUP98::KDM5A acute megakaryocytic leukemia with RB1 loss or T-ALL with NOTCH1 mutations. CUT&RUN profiling of in vitro cord blood CD34+ cell (cbCD34) models of major NUP98 fusions revealed that NUP98-fusion oncoproteins (FOs) directly regulate differentiation-related genes contributing to the disease phenotypes, represented by NUP98::KDM5A binding to MEIS2 or GFI1B for megakaryocyte (MK) differentiation. In patient samples, NUP98-FO binding patterns are heterogeneous, potentially shaped by somatic mutations and differentiation status. Using cbCD34 models and CRISPR/Cas9 gene editing, we show that RB1 loss cooperates with NUP98::KDM5A by blocking terminal differentiation toward platelets and expanding MK-like cells, whereas WT1 frameshift mutations skew differentiation toward dormant lymphoid-myeloid primed progenitor cells and cycling granulocyte-monocyte progenitor cells, providing evidence for NUP98-rearranged leukemia phenotypes affected by cooperating alterations. NUP98::KDM5A cbCD34 models with RB1 or WT1 alterations have different sensitivities to menin inhibition, suggesting that cellular differentiation provides stage-specific menin dependencies and resistance mechanisms that can be leveraged for future treatment strategies for NUP98-rearranged leukemia.

Targeting metabolic dependencies and "starving" malignant cells have long been considered potential strategies to treat cancer. However, with rare exceptions, the implementation of these maneuvers has been fraught with limited activity and lack of specificity. Multiple cytoplasmic and mitochondrial transaminases catalyze reactions that lead to amino acid catabolism. These enzymes use α-ketoglutarate (αKG) as a nitrogen acceptor, and accumulation of the competitive inhibitor metabolite D-2-hydroxyglutarate perturbs their function. We postulated that exogenous αKG supplementation would influence the directionality of these reactions and deplete amino acids in cancer cells. Using B-cell lymphoma as a model system, we found that αKG mediates a rapid and sustained amino acid depletion, principally of aspartate and branched-chain leucine, valine, and isoleucine. The decrease in leucine levels influenced mammalian target of rapamycin complex 1 (mTORC1) subcellular movement, suppressed its activity, and associated with inhibition of B-cell lymphoma growth in vitro and in vivo. Increasing import of aspartate or leucine levels in the lymphoma cells, genetically forcing mTORC1 lysosomal localization or blocking leucine catabolism through branched-chain amino acid transaminase 2 deletion, all blunted the antilymphoma effects of αKG. In addition, long-term dietary supplementation of αKG, a toxicity-free strategy, significantly hindered lymphoma development in Eμ-Myc mice, in association with amino acid perturbation and impaired energy generation. We posit that αKG supplementation, which has been shown to improve health and life span in mice, also encodes marked anticancer properties.

During thrombopoiesis, megakaryocytes (MKs) transform their cytoplasm into proplatelets through complex cytoskeletal rearrangements. The shear force of blood flow releases newly formed platelets from the proplatelets into the bloodstream. Defects at any phase of this process can impair platelet production. Although various noncoding RNAs have been identified as regulators of platelet production, the regulatory mechanisms of thrombopoiesis remain to be further investigated. Despite the high abundance of circular RNAs (circRNAs) in platelets, their role in platelet production is unclear. In this study, using RNA sequencing and bioinformatics analysis, we identified a circular RNA from the FUT8 gene (circFUT8) as a novel circRNA that increases as hematopoietic stem cells from human umbilical cord blood differentiate into mature MKs, showing high expression in these mature cells. Knockdown of circFUT8 led to diminished proplatelet formation (PPF) and abnormal demarcation membrane system formation in human cultured MKs. In addition, inhibition of circFut8 in vivo decreased murine platelet counts. circFut8 deficiency reduced the number of MKs in contact with sinusoids. Mechanistically, we revealed that circFUT8 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 2 to stabilize tensin-1 (TNS1) mRNA in an m6A-dependent manner. In human cultured MKs, TNS1 knockdown resulted in defective filamentous actin polymerization and assembly, impaired spreading on extracellular matrix proteins, and decreased PPF. Taken together, our research reveals the crucial functions of circRNAs in platelet production and has significant implications for the development of therapeutic strategies for thrombocytopenia and bleeding disorders.

Bruton tyrosine kinase inhibitors (BTKis) and cell therapy have successfully been used to treat mantle cell lymphoma (MCL). However, therapy resistance inevitably emerges. Cancer cells can progressively develop stable resistance by traversing through a transient drug-tolerant persister (DTP) state. The mechanisms enabling DTP cells to reversibly adapt to therapies and evolve to acquire heterogeneity remain poorly understood, and characterizing DTP cells in MCL continues to pose a challenge for clinic translation. Here, using pirtobrutinib, a recently US Food and Drug Administration-approved noncovalent BTKi, we identified pirtobrutinib-tolerant persister cells exhibiting morphological variability by presenting a unique population of enlarged cells (giant cells) with reversible fate transitions. During treatment, giant cells enter a nonproliferative, dedifferentiated state, addicted to an activated cytosolic tricarboxylic acid (TCA) cycle coupled with the malate-aspartate shuttle to engage in biosynthesis. Upon drug removal, the TCA cycle shifts to oxidative catabolism, promoting giant cells to differentiate into regular-sized cells. Throughout the transition, acetyl coenzyme A modulates cell fate by fine-tuning stemness. Our biphasic model demonstrates that the metabolic switch governs the phenotypic plasticity of DTP cells in MCL, resulting in a dynamic presence of DTP cells across various developmental states in response to systemic therapies. Targeting giant cells before their differentiation offers a promising strategy to overcoming therapy resistance in MCL.

Front-line pharmaceutical interventions for treating acute graft-versus-host disease (GVHD) are not uniformly effective and have toxic side effects. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent in vitro and in vivo immunosuppressive functions. Clinical translation of in vitro-generated MDSCs has been limited because of requirements for multiple, high infusion doses, the relatively low yield from peripheral blood-sourced MDSCs (PB-MDSCs), and inconsistent product quality. To circumvent these obstacles, we developed a methodology to generate MDSCs using human induced pluripotent stem cell (iPSC)-derived CD34+ cells. Compared with PB-MDSCs, iPSC-derived MDSCs (iMDSCs) shared similar morphology, phenotype, and suppressive function. We found that the CD14+ iMDSC subset possessed the highest suppressor function. In previous studies, we reported that MDSCs transferred into mice with GVHD lost suppressor function because of inflammasome activation and immature myeloid cell maturation. In striking contrast to human PB-MDSCs, we show herein that iMDSCs retained 95% of suppressor function in vitro despite exposure to lipopolysaccharide (LPS) plus adenosine triphosphate (ATP), which are stimuli that activate the inflammasome via danger-associated molecular patterns released during early posttransplant conditioning and GVHD-induced injury. In an in vivo xenogenic GVHD model with PB mononuclear cells, iMDSCs significantly increased recipient survival without loss of antileukemia effects. iMDSC RNA sequencing and gene knockdown studies revealed that the maintenance of the purine metabolizing enzyme, phosphoglycerate dehydrogenase, during LPS plus ATP treatment, was linked to iMDSC inflammasome resistance. Taken together, these findings provide a platform for translating in vitro-generated, off-the-shelf iMDSCs into the clinic for suppressing a spectrum of adverse immune responses, including GVHD.