Journal of Hematology & Oncology

Relapse during treatment of B-cell acute lymphoblastic leukemia (B-ALL) is a harbinger of poor outcomes. Identifying biomarkers for subsequent relapse risk which are detectable at B-ALL diagnosis remains a priority. Off-target recombination-activating gene (RAG)-mediated structural variants (SVs) generate genomic instability that drives leukemogenesis and may underlie treatment resistance. Leveraging sequencing data in 1,496 pediatric B-ALL patients enriched for relapse status (relapse n=532; non-relapse n=964), we characterized RAG-mediated SVs across B-ALL molecular subtypes and examined their association with patient characteristics and their impact on clinical outcomes. Off-target RAG-mediated SVs were overall frequent, particularly in ETV6::RUNX1, ETV6::RUNX1-like, and Ph-like B-ALL subtypes, while increasing age-at-diagnosis was positively associated with burden of off-target RAG-mediated SVs (P<.001). Off-target RAG-mediated SVs with a recombination signal sequence (RSS) at one breakpoint, a hallmark of off-target RAG activity, were significantly more frequent at diagnosis in patients who subsequently relapsed (P=.001). This association remained significant in multivariable regression analysis (per SV odds ratio [OR]:1.08, 95%CI:1.04-1.12), in minimal residual disease (MRD)-negative patients (OR:1.09, 95%CI:1.04-1.14) and across subtypes. Excluding deletions, MRD-negative ETV6::RUNX1 patients with 3 off-target RAG-mediated SVs had a >3-fold risk of relapse (hazard ratio:3.47, 95%CI:1.86- 6.49). RAG-mediated SVs were also associated with relapse risk in T-cell ALL patients. Off-target RAG-mediated SV burden at diagnosis is a risk factor of relapse in pediatric ALL across molecular subtypes and independent of MRD status.

Impaired cellular differentiation is a defining characteristic of myeloid malignancies and remains a major therapeutic challenge. The BRG1/Brahma-associated factor (BAF) chromatin remodeling complex, through the ATPases SMARCA4 and SMARCA2, maintains the stemness of leukemic blasts and thus represents a promising target for novel differentiation-based therapies. In a phase 1 study in advanced myeloid malignancies, the first-in-class dual SMARCA4/2 inhibitor FHD-286 combined with decitabine (DAC) was tolerated and produced an objective response rate of 12.8% (6/47) compared with no responses with FHD-286 monotherapy. To understand the basis of this activity, we integrated high-dimensional flow cytometry and single-cell genomic analyses of longitudinal bone marrow samples from responders and nonresponders. While FHD-286 monotherapy was predominantly associated with myeloid differentiation, responders to FHD-286+DAC combination therapy exhibited a range of myeloid and erythroid differentiation trajectories. FHD-286 potentiated the transcriptional impact of DAC, driving tumor clones to fully differentiate out of the immunophenotypically and transcriptionally defined blast compartment. Responders had a baseline transcriptional profile similar to that of CEBPA-mutant acute myeloid leukemia and showed further downregulation of CEBPA upon treatment. These findings reinforce tumor cell differentiation as a mechanism of response to pharmacologic SMARCA4/2 inhibition and support further evaluation of FHD-286+DAC in molecularly defined patient subsets.

Approximately 30% of patients with acute graft-versus-host disease (GVHD) develop steroid-refractory disease and have very poor outcomes. Ruxolitinib has become the standard of care for steroid-refractory acute GVHD, but it is unclear which patients derive benefit. The MAGIC Composite Score (MCS), an algorithm that combines clinical symptoms and biomarkers, has been validated to predict outcomes at the start of primary GVHD treatment. Here, we evaluated its performance at the initiation of second-line treatment in 278 patients. MCS stratified patients into three risk groups (MCS1-3), with the majority (88%) classified as intermediate or high risk. Increasing MCS score was associated with progressively higher 1-year non-relapse mortality (NRM) rates (16%, 41%, and 73%; p<0.001), lower 1-year survival (77%, 56%, and 24%; p<0.001), and lower complete response (CR) rates at day 28 (47%, 38%, and 20%, respectively; p<0.01). The area under the receiver operating characteristic curve (AUROC) for 1-year NRM was significantly higher with MCS compared to clinical symptoms alone (0.70 vs. 0.63; p=0.023). Among patients treated with ruxolitinib, higher MCS similarly predicted higher NRM and lower survival and CR rates. Patients classified as MCS2/3 had poor outcomes despite ruxolitinib, underscoring the need for novel therapies in this patient population. In conclusion the MCS is an accurate predictor of outcomes for patients who require second-line treatment and may be of use as an eligibility criterion for future clinical trials in this high-risk population.

DNA methylation-based classification offers a rapid diagnostic complement to conventional molecular workflows in acute leukemia. Existing classifiers are trained on array-derived reference cohorts whose construction favors specimens with adequate tumor content, leaving clinically relevant low-purity specimens underrepresented and classifier robustness in this regime uncharacterized. On held-out low-purity specimens, existing classifiers were concordant with expert pathology in only 7 of 10 (MARLIN) and 5 of 10 (ALMA) cases, motivating a classifier built to maintain accuracy at low tumor purity. We developed MOSAIC (Methylation-Oriented Site Analysis and Information Classifier), a neural network classifier built to maintain accuracy across the full range of tumor purities encountered in clinical practice. MOSAIC is a neural network trained on publicly available array-based methylation data augmented with native methylation calls from Oxford Nanopore sequencing. MOSAIC was evaluated on low-purity specimens held out entirely from training. On these held-out low-blast leukemia specimens, all below 25% blasts and including a case at 1.4%, MOSAIC was concordant with expert pathology in every case, recovering the correct subtype where diluted disease signal would otherwise be mistaken for normal or unrelated tissue. Gradient-based saliency analysis showed that the network relies on a partially distinct set of discriminative CpG probes when classifying low-blast specimens. MOSAIC demonstrates that augmenting training with clinically representative clinical specimens yields methylation-based leukemia classification that maintains effectiveness under the variable tumor purity of real clinical cohorts.

Hyperleukocytosis (white blood cell [WBC] count >100 000/uL) at diagnosis is an important prognostic risk factor in pediatric acute lymphoblastic leukemia (ALL), though its significance with contemporary therapy is unclear. We analyzed 1 826 pediatric ALL patients from a multi-institution cohort to determine whether hyperleukocytosis independently predicts outcomes using multivariable Cox proportional hazard modeling. Hyperleukocytosis occurred in 211 patients (12%), with 121 having B-ALL, and showed no prognostic significance in T-ALL patients. In B-ALL, 5-year event-free survival (EFS) was 65% versus 89% for non-hyperleukocytosis patients, and overall survival (OS) was 78% versus 93%. After adjustment for age, cytogenetic risk, central nervous system disease status, and treatment site, hyperleukocytosis remained an independent predictor of end-of-induction minimal residual disease (MRD) positivity (odds ratio 2.53 [95% confidence interval [CI]: 1.71-3.94; p<0.001]), inferior EFS (hazard ratio [HR] 2.44; 95% CI: 1.77-3.38; p<0.001) and inferior OS (HR 2.00; 95% CI: 1.29-3.12; p=0.002). A continuous dose-response relationship was observed between WBC count and these outcomes. Survival associations persisted across all cytogenetic risk categories and MRD strata. Despite risk-adapted therapy with treatment intensification for high-risk features, hyperleukocytosis identifies an aggressive B-ALL phenotype with persistently inferior outcomes, suggesting these patients may benefit from novel therapeutic approaches.

The PIK3CA gene encodes p110α, the catalytic subunit of phosphoinositide 3-kinase (PI3K), and is among the most frequently mutated oncogenes in multiple cancers, including breast and colorectal cancer (CRC) 1 . Two FDA-approved PI3Kα-specific inhibitors, Alpelisib and Inavolisib, are currently used in combination with fulvestrant to treat hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–), PIK3CA-mutated breast cancer. However, extending PI3Kα-targeted therapy to PIK3CA-mutant CRC requires new and effective combination strategies. Most oncogenic PIK3CA/p110α mutations cluster in two hotspot regions: the helical domain and the kinase domain. Approximately half of all p110α mutations arise in the helical domain, with E545K being the most common recurrent alteration 1 . Our previous work demonstrated that helical-domain mutant p110α aberrantly interacts with insulin receptor substrate 1 (IRS1) while losing its interaction with the regulatory subunit p85β 2 . The disengaged p85β subsequently translocates to the nucleus and stabilizes EZH1/2 3 . We further showed that combining Alpelisib with the EZH1/2 inhibitor Tazemetostat induces regression of xenograft tumors harboring a helical-domain PIK3CA mutation 3 . Here, we report that the combination of Alpelisib and Tazemetostat additively upregulates interleukin-15 (IL15) expression in helical-domain mutant CRC, leading to activation of natural killer (NK) cells, which in turn contributes to robust CRC tumor suppression.

Despite considerable advances, the emergence of treatment resistance to tyrosine kinase inhibitors (TKIs) therapy remains a significant challenge in chronic myeloid leukemia (CML). Here, we report the first clinical case of resistance to combined ponatinib and asciminib therapy in a CML patient who relapsed with B lymphoblastic blast crisis. While at presentation the patient harbored the canonical e13a2 BCR::ABL1 fusion, at relapse his disease harbored the T315I mutation together with a novel e6a3 BCR::ABL1 fusion, arisen by internal deletion in the original translocated allele. Structural modeling and biochemical analyses demonstrated that deletion of exon 2-encoded residues of ABL1 destabilizes the autoinhibited conformation, resulting in a hyperactive kinase with increased propensity for B-cell differentiation. Functional studies revealed that both BCR::ABL1e6a3 and BCR::ABL1e6a3/T315I conferred resistance to ponatinib and asciminib, alone or in combination. BCR::ABL1e6a3 demonstrated enhanced sensitivity to active-state selective inhibitors dasatinib and bosutinib, whereas BCR::ABL1e6a3/T315I remained resistant. Combined drug sensitivity assays showed that axitinib restored inhibitory activity when combined with ponatinib or asciminib. Strikingly, a combination of axitinib and asciminib with low dose ponatinib fully suppressed enzymatic activity of BCR::ABL1e6a3/T315I and cellular proliferation. These data show that treatment with asciminib and ponatinib can select for mutations with notably elevated enzymatic activity, effectively targeted by an axitinib-based triple combination. These data highlight the remarkable mutability of the BCR::ABL1 kinase, including through novel isoforms and provides a strong rationale for the clinical assessment of a triple inhibitor combination as a strategy to overcome resistance to dual ponatinib and asciminib therapy.

Regulatory CD8+ T-cells (CD8+ Treg) are a distinct yet understudied T-cell subset capable of simultaneous immunosuppression and cytolysis. Here, we characterized induced human CD8+ Treg (CD8-iTreg) generated from peripheral blood CD8+CD25- T-cells using anti-CD3e mAb-loaded artificial antigen presenting cells, IL-2, TGF{beta}, and Rapamycin. These CD8-iTreg differentiated into a stable, highly proliferative bifunctional population with suppressive activity comparable to CD4-iTreg while retaining cytolytic capacity similar to conventional CD8 cytotoxic T lymphocytes (CTL). Multi-parameter spectral flow cytometry and single-cell RNA-seq revealed a distinct immunoregulatory signature: a predominantly Treg-like profile marked by tissue-residency marker CD103 with increased canonical Treg markers (FoxP3, HELIOS, CD25, CD39, CTLA-4, CCR4, and IL-10) and reduced pro-inflammatory cytokines. A unique cytotoxic program was marked by elevated Granzyme-K (GzmK) and Thrombospondin-4 (Tsp-4), a thrombospondin family extracellular matrix glycoprotein upregulated in activated CD8+ T-cells. Cytolysis was primarily mediated by Perforin (Prf) and multiple Granzymes packaged into Tsp-4 supramolecular attack particles (SMAPs), with GzmK contributing to both cytotoxic and suppressive functions. After anti-CD19scFv CAR (CAR19) transduction, CAR19+ CD8-iTreg showed superior in vivo anti-tumor efficacy compared with CAR19-CTLs, significantly reducing tumor burden and prolonging survival in a CD19+ Nalm-6 human leukemia xenograft model while maintaining low pro-inflammatory cytokine production. In a xenogeneic graft-versus-host disease (GVHD) model with residual human leukemia, CAR19 CD8-iTreg inhibited GVHD lethality and controlled tumor growth without increasing systemic inflammation. Together, these findings support CD8-iTreg-based CAR therapies as a strategy to retain potent anti-leukemic activity while limiting inflammatory toxicities of conventional CAR T-cells, properties particularly beneficial in treating auto- and allo-immune diseases. One sentence summaryCD8-iTreg drive parallel tumoricidal and immunoregulatory functions mediated by releasing Tsp-4+ SMAPs containing granzyme K.

Haematopoietic stem cells (HSC), while usually quiescent, can rapidly divide following specific stimuli (mobilization). These HSC can seed additional niches, allowing for the swift generation of essential blood cells. However, studies in mice and humans have clearly demonstrated that cycling bone marrow (BM) HSC (cells in the G1/S/G2/M phases) engraft and reconstitute the haematopoietic system poorly compared with HSC in the G0 phase1. This raises the question why mobilized HSC, immediately following 3 or more cell divisions2, efficiently reconstitute the haematopoietic system. We studied this phenomenon in human HSC using scRNAseq analysis. We found that mobilized HSC rapidly start transcribing genes associated with quiescence, specific for the G0 phase of the cell cycle. We hypothesize that this rapid switch from actively dividing to quiescent cells combined with our extensive RNA expression data will allow us to better define pathways involved in this process.

Myelodysplastic neoplasms (MDS) and related myeloid neoplasms such as chronic myelomonocytic leukaemia (CMML) are clonal haematopoietic stem cell disorders characterised by ineffective and dysplastic haematopoiesis. They are associated with peripheral cytopaenias, variable increases in immature blasts, and a risk of progression to acute myeloid leukaemia. Hypomethylating agents (HMA) can improve blood counts and reduce blasts, but responses are usually limited. Epigenetic rewiring of haematopoietic stem and progenitor cells (HSPC) by HMA enhances hematopoietic output but is influenced by clonal mosaicism, which requires tracking of response at the single cell level to achieve full understanding. We developed SCIMETAR-seq for single-cell interrogation of DNA methylation, target amplicons, and mRNA in FACS-indexed HSPC, then deployed SCIMETAR-seq on CD34+ HSPC from longitudinal HMA-treated patient BM in vitro and in vivo. HMA-induced LINE-1 (L1) demethylation was positively correlated with cell cycling; being lowest in quiescent HSC and highest in erythrocyte progenitors. Erythrocyte progenitor frequencies were particularly increased by HMA exposure. SRSF2 p.P95 genotype did not influence HMA-induced L1 demethylation but was enriched into cells with a CMP immunophenotype, which were transcriptionally biased away from MEP towards granulocytic progenitors. Despite a lack of L1 demethylation in quiescent HSC/MPP after 7 days of HMA treatment in vivo, their transcriptomes were enriched for TNF-, TGF{beta}- and WNT-signaling, suggesting that extrinsic factors secreted by other BM cells in response to HMA mediates reprogramming of quiescent HSC during HMA therapy in vivo.

Chimeric Antigen Receptor (CAR) therapy for Acute Myeloid Leukemia (AML) is hindered by disease heterogeneity, antigen overlap with normal hematopoiesis, and the persistence of leukemic stem cells (LSCs). To overcome these barriers, we employed the IF-BETTER strategy to simultaneously target CD33 and the LSC-associated marker TIM-3 by pairing a second-generation CAR with a Cytokine-Costimulatory Receptor (CCR) in two dual CAR configurations (CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR). Both constructs displayed potent antigen-restricted cytotoxicity against AML cell lines and primary blasts, achieving leukemia clearance, while sparing normal immune and hematopoietic cells. Mechanistic studies revealed that the TIM-3.CAR single-chain fragment variable (scFv) recognizes a protein-proximal epitope whose interaction is selectively enhanced by AML-specific hyper-fucosylated and hyper-sialylated N-glycans. Fucosylation blockade reduced TIM-3.CAR avidity and cytotoxicity, confirming a glycosylation-modulated interaction. Integrating this glycosylation-tolerant TIM-3 scFv into a dual CAR framework enables selective targeting of AML cells, providing a rational strategy for safer and more effective AML-directed immunotherapy.

Abstract Mitochondrial DNA (mtDNA) heteroplasmy, the coexistence of multiple mtDNA variants within cells, accumulates with age and is associated with hematological malignancies and mortality. However, whether predicted deleterious heteroplasmies causally contribute to cancer or merely represent passenger mutations remains unresolved. Here, leveraging ~36,000 first-degree relative pairs from the UK Biobank and All of Us Research Program cohorts, we deconvolute overall heteroplasmy metrics into those that are shared across family members (representing inherited variants) and those that are not (representing de novo variants) to establish a Mendelian randomization framework for assessing causality. We show that shared heteroplasmies exhibit strong purifying selection, with reduced predicted deleteriousness compared to not shared variants, and that 90% of an individual's deleterious heteroplasmy burden is somatically acquired. Critically, shared deleterious heteroplasmy burden, fixed at conception and thus temporally upstream of potential confounders, is significantly associated with hematological malignancies (RR=2.81, 95% CI 1.29-6.13), with effect sizes concordant with the not shared heteroplasmy burden. Furthermore, shared deleterious heteroplasmy specifically associates with high-risk clonal hematopoiesis of indeterminate potential (CHIP), particularly spliceosome mutations, suggesting mitochondrial dysfunction promotes clonal expansion of specific CHIP subtypes. Finally, we identify ultra-rare individual mtDNA variants associated with hematological malignancies, a hallmark of driver mutations. These findings establish mtDNA heteroplasmies, including inherited variants, as causal contributors to hematological malignancy risk and demonstrate that most disease-relevant burden is acquired during life, identifying potential opportunities for prevention and therapeutic intervention in individuals at elevated risk for hematological cancer, particularly of myeloid origin.

Chimeric antigen receptor (CAR)-T cell therapy has improved outcomes for patients with multiple myeloma (MM), but its broader use is restricted by manufacturing complexities and treatment-related toxicities. AZD0120 is a dual-targeting B-cell maturation antigen (BCMA)/CD19 CAR-T cell therapy manufactured via the rapid FasTCAR process. We developed a dual-targeting "loop" CAR that incorporates a novel humanized anti-BCMA single-chain variable fragment (scFv), clone SG, and an FMC63-derived anti-CD19 scFv. This AZD0120 CAR preserved functional binding to both antigens and conferred robust in vitro and in vivo cytotoxicity while maintaining single-antigen reactivity. Conventional manufacture of CAR-T cells with the AZD0120 CAR (AZD0120C) yielded cells with minimal tonic signaling, limited responsiveness to soluble BCMA, and preservation of naive/stem cell memory-enriched phenotypes, yet robust cytokine production upon BCMA+ target engagement. AZD0120C demonstrated cytotoxicity comparable to benchmark BCMA CAR-Ts across MM lines in vitro and showed strong in vivo expansion and tumor control in xenograft models. FasTCAR manufacturing - designed to shorten vein-to-vein timelines and enrich less-differentiated phenotypes - further enhanced in vivo performance: AZD0120 consistently achieved superior tumor control and greater CAR-T expansion vs AZD0120C across disseminated MM.1S, NALM-6, and JeKo-1 models, with superior efficacy observed at lower cell doses. Collectively, these data support clinical evaluation of AZD0120 as a differentiated BCMA/CD19 CAR-T cell therapy with the potential to improve disease control and patient access in MM. Key PointsO_LIAZD0120 is a dual-targeting CAR-T that displays a favorable anti-myeloma functional profile and co-targets a source of potential relapse C_LIO_LIThe FasTCAR process yields TN/SCM-rich CAR-T populations, promotes in vivo expansion and achieves potent tumor control in xenograft models C_LI

BackgroundChimeric antigen receptor (CAR)-T cell therapies efficacy in solid tumors remains limited, largely due to the profoundly immunosuppressive tumor microenvironment (TME) which drives CAR-T cells to dysfunction and poor persistence. A comprehensive understanding of the dynamic interplay between CAR-T cells and the TME is therefore critical for the rational design of more effective CAR-T strategies for solid cancers. MethodsHere, we performed single-cell RNA sequencing of tumor samples from immunocompetent mice treated with stroma-targeting EDA-CAR-T cells, profiling CAR-T cell states and TME programs at the peak of antitumor response and during subsequent tumor progression. ResultsOur analysis revealed a marked temporal remodeling of EDA-CAR-T cells within the TME, where early antitumor efficacy is associated with concurrent expansion of cytotoxic effector CD8 CAR-T cells and activation of memory CD4 CAR-T subsets. Moreover, EDA-CAR-T cells effectively engaged the myeloid compartment, resulting in strengthened communication networks involving T cell activation. However, by tumor progression, EDA-CAR-T cells suffered a widespread transcriptional reprogramming towards dysfunction, characterized by loss of effector programs alongside induction of exhaustion and immunoregulatory pathways within the TME, including PD-L1/PD-L2 and TGF{beta} signaling, which impairs sustained immune responses. Notably, early CAR-T cell activation led to increased susceptibility to TME-mediated immunosuppression, revealing EDA-CAR-T-specific soluble galectin-mediated cell-to-cell interaction networks. ConclusionsTogether, this works offers a high-resolution view of CAR-T cell dynamics within the solid TME, uncovering cellular and molecular mechanisms of rapid functional decline and identifying regulatory pathways within the TME that can be exploited to improve CAR-T cell therapy efficacy in solid tumors. KEY MESSAGES OF THE ARTICLEO_ST_ABSWhat is already known on this topicC_ST_ABSThe determinants of CAR-T cell therapeutic efficacy in solid tumors remain poorly defined, largely due to the complexity of the immunosuppressive tumor microenvironment. In this effort, it is necessary to perform comprehensive and detailed mechanistic studies that capture CAR-T cell dynamics within the solid tumor microenvironment to understand treatment failure. What this study addsWe performed single-cell profiling of stroma-targeting EDA-CAR-T cells, revealing their dynamic reprogramming toward dysfunction within the solid tumor microenvironment. We dissected CAR-T cell states and their cell-to-cell interactions with the tumor microenvironment across response and tumor progression and identified mechanisms linking CAR-T cell functionality and therapeutic failure. How this study might affect research, practice or policyThis study provides comprehensive mechanistic insights from an immunocompetent model that can be leveraged to identify shared determinants of CAR-T cell functionality in solid tumors and potentially guide the rational development of improved CAR-T cell therapies.

The biological bases of transient myelomonocytic switch (mmSW) during induction therapy in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) remains largely undefined. Here we integrated single-cell transcriptomic and surface marker profiling with genomic and DNA methylation analyses of pediatric BCP-ALLs, including matched diagnosis (Dx) and Day+15 samples from mmSW-positive (mmSWpos) and mmSW-negative cases. At Dx, mmSWpos leukemia samples were enriched for hematopoietic stem/progenitor-like cell subpopulations. Trajectory and entropy analyses identified a pre-existing "fate-uncertain" cell compartment co-expressing both lymphoid and myeloid programs. Longitudinal single-cell data showed that, in mmSWpos samples, this population undergoes complete transdifferentiation. mmSWpos are enriched for Ras pathway and chromatin regulation mutations and display a specific DNA hypermethylation pattern at Dx. These findings indicate that transient mmSW arises from intrinsic leukemic plasticity, in which immature transcriptomic and distinct epigenetic states at diagnosis enable lineage switching under the pressure of ALL treatment.

Curative-intent immunochemotherapy fails in ~30% of patients with large B-cell lymphoma (LBCL), yet no validated molecular tool enables early identification of high-risk individuals to guide treatment intensification. Using shallow whole genome sequencing (sWGS) of plasma cell-free DNA from 190 LBCL patients, we developed and validated the ACT score (Aberrations, fragment Composition, Terminal motifs), a composite classifier integrating genomic and fragmentomic features from a single post-cycle-1 sample. ACT-positive patients had worse 2-year outcomes versus ACT-negative patients: time-to-progression 29% vs. 83% (HR 4.4, 95% CI 1.9 - 10.0; P = 1.5 x 10 - 4) and overall survival 47% vs. 93% (HR 8.7, 95% CI 3.0 - 25.4; P = 1.8 x 10-6). ACT score was independently prognostic of the International Prognostic Index, and their combination identified the highest-risk patients. Unlike mutation-based approaches, this assay requires neither tumor tissue, germline control nor a baseline plasma sample. Built on open-source tools and sWGS, the ACT score offers a feasible scalable strategy for early risk stratification in aggressive LBCL.

Chimeric antigen receptor (CAR)-T cell therapies achieve remarkable responses in hematologic malignancies but are limited by target heterogeneity and antigen escape; therapeutic antibodies offer flexible targeting yet cannot leverage T-cell effector function. Integrating CAR-T cell potency with antibody targeting adaptability could expand the clinical reach of cellular immunotherapy. We engineered a CAR platform (termed CARFcR) incorporating an Fc receptor (CD16V158) into the CAR extracellular domain, and enabling both CAR-mediated targeting and antibody-dependent recognition. CARFcR-T cells were activated through either pathway, exhibiting robust cytokine secretion, degranulation, and proliferation. Anti-CD19 CARFcR-T cells mediated cytotoxicity comparable to conventional CAR-T cells and eradicated CD19+ leukemia in xenograft models. Importantly, they also exerted antibody-dependent killing of CD19-negative tumor cells when guided by clinically approved antibodies, such as anti-HER2 trastuzumab and anti-CD20 rituximab. Dual engagement further enhanced cytotoxicity and enabled elimination of CD19-negative escape variants. This modular CARFcR architecture was successfully applied to additional CAR targets, including BCMA, CD123, and CD33, maintaining CAR function while allowing redirection with antibodies or CD16-binding bispecific engagers. Overall, CARFcR represents a versatile and clinically adaptable platform that integrates the strengths of CAR-T cells and antibody therapeutics to expand tumor targeting and overcome antigen escape.

Multiple myeloma remains a fatal, incurable disease. Most therapies are targeted to the cancer cell or T cell engagement. Little is known about the supporting myeloma microenvironment and its contribution to tumor fitness. Here, we expand upon the observation of human mast cells in the NSG-hIL6 myeloma patient derived xenograft mouse model to show mast cells decrease time to engraftment, promote increased myeloma engraftment and cause myeloma bone disease. We identify 10 mast cell secreted factors that together improve the survival of patient myeloma cells in vitro. Our results highlight the versatility of the NSG-hIL6 model to study microenvironmental interactions between human bone marrow cells and myeloma and confirm prior suggestions that clinical signs of disease, such as osteolytic lesions, may at least partially be related to non-malignant bone marrow microenvironmental cells, such as mast cells.

Amino acid deprivation with L-asparaginase is a cornerstone of treatment in acute lymphoblastic leukaemia (ALL), but clinical challenges limit its use in adults. Deficiency in the enzyme argininosuccinate synthase (ASS1-low) confers arginine auxotrophy, defining a dependence on extracellular arginine, and represents an analogue metabolic vulnerability that is targetable through arginine deprivation. We analysed transcriptomic data across >550 adult B-ALL cases to establish clinico-biologic characteristics of low ASS1 expression identifying the Philadelphia chromosome-positive (Ph+) ALL subgroup as a stereotypical arginine auxotroph within molecularly diverse B-ALL. Functionally, arginine deprivation with pegargiminase induced robust apoptosis in both Ph+ ALL cell lines and primary samples in an ASS1-dependant manner and was highly effective as a monotherapy treatment in independent Ph+ ALL patient derived xenografts. Mechanistically, arginine deprivation induces endoplasmic reticulum stress mediated apoptosis which was orthogonal to tyrosine kinase inhibition (TKI) mediated outcomes. Using in vitro and in vivo models of non-genetically mediated TKI-resistance, we demonstrate pegargiminase and TKI combinations robustly eradicates TKI-resistant leukaemia. Thus, we establish ASS1 deficiency (ASS1-low) as a therapeutically actionable vulnerability in Ph+ ALL and a strategy to bypass TKI-resistance, supporting clinical evaluation of arginine deprivation as a novel adjunct to chemotherapy-free treatment.

BackgroundVenous thromboembolism is a major cause of morbidity and mortality. Despite identification of risk factors, not all individuals with thrombophilia develop thrombosis. Understanding the multigenic factors modifying this incomplete penetrance would help guide patient care. MethodsThe zebrafish has a conserved hemostatic system and is amenable to large genetic studies. Loss of antithrombin III (At3) in zebrafish leads to an early consumptive coagulopathy and lethality in adulthood. Using this genetic background as a sensitized model we performed a dominant unbiased genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen followed by whole genome sequencing (WGS). We used survival studies, laser-mediated endothelial injury, and ex vivo protein assays to validate hits. ResultsENU-treated at3+/- males were crossed with at3+/- females to produce 4,030 total offspring (1.5x genome coverage). Four permanent lines transmitting a survival benefit beyond 7 months were identified and sequenced. A candidate screen of 63 known coagulation-related loci revealed a missense mutation, C504F, in a highly conserved residue of the prothrombin (F2) heavy chain, which was validated through genetic and biochemical studies. Evaluation of UK Biobank electronic health record (EHR) data was underpowered to detect interactions between F2 and AT3 due to minmal deleterious mutations. Mutations produced through genome editing revealed that heterozygosity for factor X and plasminogen also modified at3-/-, resulting in reduced lethality. The three remaining lines had no coagulation-related variants segregating with survival, suggesting the presence of novel modifier loci. ConclusionsUnbiased genome-wide screening identified a modifier of thrombosis. This demonstrated that re-balancing of hemostasis to mitigate thrombosis is conserved in zebrafish, including an unexpected role for fibrinolysis. This interaction was not detected even in a large human dataset, establishing the continued benefit of the zebrafish model. Finally, we found evidence for novel loci outside of the canonical coagulation cascade that may be new targets for diagnosis or treatment.