Idecabtagene vicleucel (ide-cel) induces deep responses in relapsed/refractory multiple myeloma (RRMM), yet more than half of patients relapse within one year. The intrinsic features of CAR-T products that distinguish durable from non-durable responders are poorly defined, particularly at single-cell resolution, and defining drivers of durable response is critical to guide patient counseling and to inform strategies for optimizing CAR-T manufacturing and efficacy. To address this need, 40 ide-cel infusion products (184,398 cells) were profiled using single-cell RNA sequencing. These analyses revealed that a transcriptional program in CD4 CAR-T cells that led to durable responses is characterized by NF-κB signaling, pro-survival circuits, tonic/chemokine signaling, and elevated CAR transgene expression. These features were associated with prolonged progression-free and overall survival irrespective of baseline clinical characteristics. Further, analysis of paired apheresis and tumor microenvironment samples showed that elevated NF-κB activity is an intrinsic hallmark of T-cell fitness that is characterized by a central memory phenotype and the lack of checkpoint receptorligand expression, and that these features were manifest in marrow-derived and peripheral blood T cells prior to CAR-T manufacturing. Finally, validating functional relevance, pharmacologic inhibition of NF-κB abrogated CAR-T cytotoxicity and cytokine production in vitro. Our results support that NFKB in the ide-cel product marks a signaling axis impacting CAR-T function and NFKB activity represents a global marker of T cell fitness present prior to CAR-T manufacture.

Pharmacological plasminogen reduction enhanced liver regeneration experimentally and clinically. siRNA-induced plasminogen deficiency promoted hepatocyte proliferation after partial hepatectomy in mice, contrasting genetic deficiency models. In the HeLiX trial, tranexamic acid reduced post-hepatectomy liver failure odds, suggesting a novel therapeutic strategy.

Thrombosis is a major contributor to global morbidity and mortality. Current standards of care target the extrinsic and/or common pathways of coagulation, effectively inhibiting thrombosis but also increasing bleeding risk, highlighting the unmet need for additional treatment options. Genetic deficiency in factor XI (FXI), a component of the intrinsic pathway, reduces thrombosis risk without spontaneous bleeding. We generated 2 FXI monoclonal antibodies (mAbs) with distinct profiles to provide new approaches to anticoagulation. Cenvacibart (REGN7508Cat) targets the catalytic domain to completely block FXI activity (induced by FXIIa or FXIa in the intrinsic pathway or thrombin in an intrinsic/common pathway amplification loop), thereby maximizing anticoagulation; amrecibart (REGN9933A2) targets the apple 2 domain of FXI/FXIa to specifically prevent FXI activity induced by FXIIa-delivering perhaps less anticoagulation but with potentially lower bleeding risk. We evaluated the anticoagulant effects of both mAbs in vitro in human/non-human primate plasma, in vivo in non-human primates, and healthy volunteers. Both mAbs inhibited intrinsic pathway-triggered coagulation, assessed by activated partial thromboplastin time (aPTT); cenvacibart exhibited a greater increase in aPTT versus amrecibart or other FXI-targeted inhibitors. Neither amrecibart nor cenvacibart affected the extrinsic pathway, assessed by prothrombin time (PT). In non-human primates, both mAbs prevented thrombosis without increasing bleeding. In first-in-human studies, both mAbs were generally well tolerated and dose-dependently inhibited intrinsic pathway-triggered coagulation, with durable aPTT prolongation without affecting PT. Amrecibart and cenvacibart may offer tailored therapies for patients with different bleeding risk profiles. The trials are registered at www.clinicaltrials.gov as #NCT05102136 and #NCT05603195.

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, human leukocyte antigen (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 (NCT05066022) enrolled five patients (four with relapsed and refractory multiple myeloma [RRMM] and one with primary plasma cell leukemia [pPCL]). CT0590 was well-tolerated and caused no dose-limiting toxicities, treatment-related death, or graft-versus-host disease. Three patients achieved confirmed responses, including two 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 (uCAR) T cells (Cmax > 280,000 copies/µg gDNA) 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.

NPM1-mutated acute myeloid leukemia (AML) is genetically well-defined, but clinical outcomes remain heterogeneous, suggesting that quantitative clonal features may refine current risk stratification. We analyzed 688 intensively treated NPM1-mutated AML integrating variant allele frequency (VAF), mutation order, and clonal architecture inferred by PyClone and ClonEvol. Co-mutations were present in 97% of patients (median = 3 per case), dominated by DNMT3A (49%), FLT3-ITD (46%), TET2 (22%), and IDH2 (20%). Prognostic modelling of NPM1 VAF identified an optimal cut-off of 31.44%, defining NPM1high and NPM1low groups. NPM1low correlated with splicing-related alterations and independently predicted inferior overall (HR = 1.46; p = 0.037) and relapse-free survival (HR = 1.40; p = 0.036). Gene-specific VAF analyses revealed divergent effects across partners, high DNMT3A, FLT3-OTHER, KRAS, and PTPN11 burdens were adverse, whereas high IDH2 VAF was protective. Combined models showed that patients with NPM1high and favorable co-mutation VAFs had the best outcomes, while dual unfavorable burdens conferred the poorest survival. Mutation ordering inferred from VAFs positioned NPM1 after epigenetic and splicing lesions but before signaling and transcription-factor mutations. Non-canonical orders, such as early FLT3-OTHER/TKD or WT1 prior to NPM1, significantly stratified outcomes. Clonal reconstruction revealed predominantly linear evolutionary trajectories (84.3%), with increased mutational burden and clonal diversity associating with inferior survival. Notably, intra-clonal co-localization of NPM1 with IDH1 or TET2 was associated with improved outcomes, whereas co-localization with WT1 predicted dismal prognosis. These results demonstrate that quantitative and structural dimensions of clonality refine the biological and prognostic landscape of NPM1-mutated AML beyond mutational status alone.

N7-methylguanosine (m7G), a prevalent modification in tRNAs, is primarily catalyzed by the methyltransferase METTL1. While growing evidence supports a role for METTL1 in various tumors, its therapeutic potential and precise function in leukemia stem cell (LSC) homeostasis remain largely unexplored. Here, we identify METTL1 as a key regulator of LSC self-renewal and homing within bone marrow (BM) microenvironment through catalyzing m7G formation on a specific tRNA, tRNAPheGAA, thereby driving leukemogenesis. Mechanistically, METTL1 loss significantly reduces m7G abundance and steady-state levels of tRNAPheGAA, leading to translation suppression and degradation of transcripts enriched with tRNAPheGAA-related codons, such as tyrosine-protein kinase HCK. Decreased HCK expression disrupts CXCR4 signaling, impairing LSC self-renewal and BM homing. Therapeutically, we characterize a small-molecule METTL1 inhibitor (M1i; NSC137443), through high throughput screening. Pharmacological inhibition of METTL1 demonstrates potent anti-tumor efficacy by reduction of tRNA m7G levels and disrupting the tRNAPheGAA/HCK/CXCR4 cascade. Notably, targeting METTL1 significantly reduces LSC frequency, delays leukemogenesis, and prolongs survival in multiple acute myeloid leukemia models. Our findings establish a previously unrecognized role for METTL1 and its target tRNAPheGAA in LSC homeostasis and provide compelling proof-of-concept evidence that METTL1 is a druggable epitranscriptomic target for anti-leukemia therapy.

Drug resistance is a major challenge in cancer therapy, especially in hematologic malignancies where kinase inhibitors have transformed treatment yet are frequently undermined by drug resistance. While targeted protein degradation (TPD) offers a mechanistically distinct mode of action compared to inhibition-based therapeutic therapies, the potential value of TPD in drug-resistant blood cancer remains unclear. Here, we report the discovery of cereblon-recruiting molecular glue degraders (MGDs) targeting LCK, an oncogenic kinase in T-cell acute lymphoblastic leukemia (T-ALL). By high-throughput screening and medicinal chemistry optimization, we developed a series of MGDs that induced CRBN-dependent degradation of LCK as well as potent cytotoxicity in T-ALL in vitro. Structure-activity relationship analysis and ternary complex modeling revealed a non-canonical degron at the LCK-CRBN interface involving the G-loop, whose mutation disrupts this interaction. Unlike inhibitors and inhibitor-based PROTACs, these MGDs engage LCK in regions distal to the ATP binding site and thus their activities in T-ALL are not affected by gate-keeper LCK mutations that drive resistance to inhibitor-based therapeutics. Taken together, our data highlights the potential of LCK-targeting MGDs as a strategy to overcome kinase inhibitor resistance in T-ALL, offering a framework for targeting kinase dependencies in drug-refractory hematologic malignancies more broadly.

Chimeric antigen receptor (CAR) T cells specific for myeloid-associated antigens expressed on the cell surface of acute myeloid leukemia (AML) can cause depletion of normal myeloid progenitor cells. We developed a CAR specific for a human Leucocyte Histocompatibility Antigen (HLA)-A*02:01-restricted peptide of the myeloid-restricted cathepsin-G protein. Cathepsin-G-specific CAR (CG1.CAR) T cells were further engineered to increase their functional avidity. Specifically, we developed CG1.CAR-T cells co-expressing the lymphocyte-specific protein tyrosine kinase (LCK) and duplicated CD3ζ chain, which allows the functional recognition of the CG1 peptide as low as 0.025 mM. Optimized CG1.CAR T cells displayed antileukemia effects in vitro and in vivo in AML patient-derived-xenotransplant (PDX) mouse models and did not cause hematopoietic toxicity in colony assays and humanized mice. Mechanistically, LCK overexpression in CG1.CAR-T cells caused transcriptional modifications characterized by the overexpression of mitochondrial-encoded electron transport chain components that were correlated with increased mitochondrial mass and improved respiratory capacity. Based on these data, CG1.CAR-T cells hold clinical potential for the treatment of AML.