Blood

Sickle cell disease (SCD) results from homozygosity or compound heterozygosity for a single point mutation in the beta hemoglobin gene. Despite carrying the same underlying causal mutation, patients exhibit highly heterogeneous clinical outcomes. Acute chest syndrome (ACS) is one of the leading causes of mortality in SCD. In this study, we aimed to identify genetic variants associated with ACS occurrence and recurrence (>5 episodes) in two SCD cohorts (OMG-SCD and Walk-PHaSST) of predominantly African ancestry and characterize their downstream molecular consequences. Although none of the SNPs reached the genome-wide threshold in the occurrence meta-analysis, we identified 40 independent genetic loci with p <10-5. However, the recurrence GWAS in OMG-SCD, which compared individuals with no prior history of ACS to individuals with a history of >5 episodes, identified one genome-wide significant locus along with 38 other independent loci with p <10-5. Survival analysis suggested lower survival probability among young ACS recurrence cases compared to controls or occurrence cases. Furthermore, molecular QTL analysis of ACS recurrence identified 42 SNP-protein and eight SNP-metabolite associations with p <10-3. Pathway and enrichment analysis identified secretory, platelet/hemostatic, and complement-mediated pathways, confirming the complex and heterogeneous pathophysiology of ACS. This study highlights the importance of a well-defined phenotype for identification of risk loci and elucidates the complex interplay of hemolytic, inflammatory, and cellular organization pathways that warrants further evaluation through large-scale multi-omic studies of SCD patients. Ultimately, the more extreme phenotype of ACS recurrence may be a more informative phenotype for analysis, risk prediction, and therapeutic targeting.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive HTLV-1-associated malignancy with poor responses to chemotherapy. Although constitutive NF-kB activation is a hallmark of ATL, its persistence in tax-negative (Type I) ATL, where Tax is irreversibly silenced, remains unresolved. Here, we identify Fra-2 as a coactivator of NF-kB p65 that redirects p65's transcriptional activity toward inflammatory genes. Mechanistically, Fra-2 interacts with the Rel homology domain of p65 via its bZIP domain, enhancing p65 DNA-binding activity without altering nuclear localization. The HTLV-1 antisense protein HBZ disrupts Fra-2/JunD AP-1 complexes while stabilizing Fra-2/p65, as shown by co-immunoprecipitation, BiFC, and PICO assays. This rewiring selectively enhances NF-kB-dependent transcription of IL-1b, which is elevated in ATL patient sera and cells independently of proviral load. Chromatin immunoprecipitation revealed Fra-2 recruitment to NF-kB response elements in the IL1b promoter, an effect amplified by HBZ. Pharmacological inhibition of IL-1 signaling with anakinra or anti-IL-1b antibodies restored doxorubicin sensitivity in primary ATL cells. Together, these findings establish the Fra-2-NF-kB-IL-1b axis as a driver of chemoresistance in tax-negative ATL and a therapeutically targetable vulnerability.

Germline mutations in the RNA helicase DDX41 are the most common genetic predisposition to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), representing 5-7% of AML. Over 70% of patients acquire a somatic mutation at specific hotspots (R525H/G530D) in the helicase domain of DDX41 in trans, which is linked to disease progression. DDX41 has diverse cellular functions, including RNA splicing, ribosome biogenesis, R-loop resolution and inflammation. However, the mechanisms by which somatic DDX41 mutations drive MDS/AML pathogenesis are yet to be determined. Here, we systematically evaluated the function of pathogenic, missense germline and somatic DDX41 mutations. We report that DDX41 somatic mutations are gain of function alleles and fail to rescue AML cell growth, distinct from germline DDX41 mutations. DDX41 somatic mutations R525H and G530D drive widespread splicing changes in isogenic AML cell lines, consistent in genetically engineered murine bone marrow HSPCs and CD34+ cells from DDX41-mutant MDS/AML patients. Mechanistically, DDX41-R525H exhibits increased RNA binding affinity compared to wildtype, with aberrantly spliced RNA targets overlapping with enriched binding. Strikingly, aberrant splicing of SEPTIN7 is enriched exclusively in CD34+ HSPCs of patients harboring both germline and somatic DDX41 mutations but absent in samples with germline mutation alone or lacking DDX41 mutations altogether. SEPTIN7 mis-splicing introduces a premature stop codon resulting in its downregulation at the protein level. SEPTIN7 is a known regulator of cytokinesis, HSC polarity and repopulation capacity. Our results identify a novel mis-spliced candidate, SEPTIN7, whose deregulation implicates a mechanism by which the DDX41 somatic mutations promote dysplastic hematopoiesis.

Dual PARP1/2 inhibitors (PARPi) selectively eliminate BRCA1/2-deficient cancers and represent the first targeted therapy for homologous recombination (HR)-deficient cancers. However, their use in maintenance therapy is limited by severe anemia and an increased risk for therapy-related leukemia. These toxicities are unexpected because PARP1 loss, which eliminates most DNA-damage-induced PARylation, does not cause anemia in mice. In contrast, PARP2 loss or catalytic inactivation causes anemia, motivating the development of PARP1-selective inhibitors. Using wild-type (WT), Parp1-/- and Parp2-/- mice, we show that hematopoietic toxicity of FDA-approved PARPi is driven primarily by inactive PARP1 rather than PARP2 inhibition. Accordingly, PARP1-selective inhibitors also cause PARP1-dependent anemia. Somatic expression of catalytically inactive Parp1 (Parp1E988A) causes lethal bone marrow failure, not found with somatic deletion of both Parp1&2. Mechanistically, inactive PARP1 obstructs the repair of diverse DNA lesions, including gaps, nicks, and Top1-cc, in contrast to the nick-selectivity of Parp2. In cells, inactive PARP1 compromises PARP2 recruitment to DNA lesions and causes severe genomic instability and mitotic bridges absent in Parp1&2-null cells. Thus, PARPi-induced hematopoietic toxicity is driven primarily by PARP1 inactivation, informing the design and use of next-generation PARP inhibitors.

Many studies unraveled the reciprocal regulation between hematopoietic stem cells (HSC) and the bone marrow (BM) niche by using transgenic knock-out or reporter mice, often overlooking the pathophysiological relevance and the existence of compensatory mechanisms. Here, we exploit the th3 murine model of a genetic anemia, beta-thalassemia (BThal), to investigate the dual impact of chronically reduced thrombopoietin (TPO) production on HSC and megakaryocytic BM niche. Decreased systemic TPO downregulates stemness programs in HSC and impairs their commitment towards megakaryocytes (Mk). We uncover dysmegakaryopoiesis in the BM from BThal patients and mice with defective Mk maturation and reduced production of niche factors, altering the HSC-niche crosstalk. Notably, in vivo stimulation of the TPO signaling restores HSC quiescence, Mk maturation and niche support. These findings demonstrate that TPO regulates HSC through both direct and niche-mediated routes, underscoring the value of using a genetic disease model to disentangle general molecular mechanisms.

TNF is the primary age-related cytokine responsible for hyperreactive platelet formation. In mouse models, increased TNF induced abnormal megakaryocyte development and platelet activity. Here, we extend these findings to demonstrate that TNF drives this thrombotic phenotype through expansion of the hematopoietic stem and progenitor cell (HSPCs) compartment. Using HSPC-specific TNF receptor labeling and single cell RNA sequencing, we found that TNF receptors are absent from megakaryocytes and their progenitors (MkPs) indicating that TNF does not directly act on these cells. Chronic TNF exposure expanded HSPCs in the bone marrow and extramedullary tissues - and these expanded HSPCs retained functional repopulation capacity. Using species-specificity TNF receptor activation, we further demonstrate that TNFR1 signaling is sufficient to induce platelet hyperreactivity independent of HSPC expansion. Unlike emergency hematopoiesis, chronic TNF promoted megakaryopoiesis through the canonical hematopoietic hierarchy as demonstrated by lineage-tracing studies. Mechanistically, chronic TNF induced a distinct transcriptional program in single cell RNA sequencing of HSPCs and megakaryocytes. Together, these findings establish that chronic TNF promotes hyperreactive platelet formation not through direct effects on megakaryocytes or MkPs but by expanding and transcriptionally reprogramming HSPCs, thereby imprinting a TNF dependent program that persists through megakaryopoiesis and ultimately produces hyperreactive platelets.

Glucocorticoids (GCs) are foundational to B-cell acute lymphoblastic leukemia (B-ALL) therapy, yet how glucocorticoid receptor (GR) activation interacts with lineage regulatory circuitry to shape therapeutic response remains incompletely understood. Using LEF1 to interrogate canonical Wnt regulatory circuitry, we show that GR activation reorganizes GR+LEF1 elements into recruitment, retention, and dissociation states. LEF1 dissociates from Wnt-responsive regulatory elements while being recruited to GRE-rich GR-associated elements that progressively acquire chromatin accessibility, H3K27ac, BRD4 and BRG1. RUNX1 and AP-1 show class-dependent redistribution across LEF1-defined states, extending organization beyond LEF1. GR+LEF1 reorganization is observed in patient-derived B-ALL. GR+LEF1 elements lose accessibility in GC-resistant B-ALL and during diagnosis-to-relapse evolution, with recruitment elements enriched for remodeling. Perturbation of resistance-associated GR+LEF1 elements at BIM and BMF alters GC-induced apoptotic signaling. Together, these findings identify Wnt/LEF1 circuitry as an interface through which therapeutic GR activation reorganizes the B-ALL regulatory landscape into states associated with glucocorticoid response and resistance.

Background: The clinical course of acute leukemias and related hematologic neoplasms is defined by aggressive clonal proliferation, severe hematopoietic disruption, and recurrent molecular alterations. Although serial bone marrow biopsies, multiparameter flow cytometry, and next-generation sequencing remain standard surveillance modalities, procedural invasiveness, high costs, and operational turnaround times limit their frequency. Minimally invasive, scalable biomarkers integrating routine hematologic indices, targeted gene expression, circulating oncoprotein levels, and patient compliance metrics may provide an accessible framework for dynamic monitoring. Methods: In this longitudinal cohort study of 66 patients across 272 serial encounters (discrete follow-up intervals: days 0, 14, 41, 71, and 78), we assessed the prognostic value of peripheral complete blood counts (white blood cell count [WBC], hemoglobin [Hb]), molecular indicators (FLT3, NPM1, and DNMT3A transcript levels; targeted soluble FLT3 quantification), and medication adherence. The primary clinical endpoint was longitudinal progression to critical deterioration or disease-related mortality. Generalized linear mixed-effects models (GLMM) with patient-specific random intercepts were constructed to account for intra-subject repeated measures. Predictors were standardized per 1-SD increment. Model performance was assessed via the Akaike Information Criterion (AIC) and the Concordance index (C-index). Results: Leukocyte expansion correlated inversely with hemoglobin concentration (r=-0.877, p<0.001), reflecting bone marrow infiltration and secondary erythroid suppression. Multivariable mixed-effects modeling confirmed that elevated WBC count (adjusted odds ratio [aOR] = 1.52 per 1-SD increase; 95% CI: 1.24-1.86; p<0.001) and reduced medication adherence (aOR = 0.58; 95% CI: 0.44-0.76; p<0.001) independently predicted adverse clinical transition. Integrating targeted molecular markers (FLT3 expression and soluble protein levels) with routine hematologic indices and adherence demonstrated superior calibration and discrimination over single-domain models ({Delta}AIC[&ge;]116; Harrells C-index = 0.81). Conclusions: Readily accessible peripheral hematologic indices, targeted molecular quantification, and medication adherence provide complementary prognostic information regarding disease activity. Integrating these low-burden parameters into clinical workflows offers an efficient, scalable paradigm for early risk stratification in hematologic malignancies.

Clonal hematopoiesis (CH) can predict treatment outcomes in solid cancers, but the prevalence and clinical impact of CH in previously untreated, aggressive large B-cell lymphomas (LBCL) remains poorly characterized. Moreover, CH may be detected in cell-free DNA (cfDNA)-based biomarker assays, but the longitudinal dynamics of CH mutations during and after immunochemotherapy in LBCL remain unknown. Hence, we characterized the spectrum of CH mutations in 247 patients with high-risk primary LBCL from three phase II clinical trials. We identified CH mutations in 23% of patients. Compared with healthy controls, TET2 mutations were enriched in peripheral blood (PB) samples of LBCL patients (P=0.01). CH clone size showed high concordance between PB and cfDNA (R2=0.59, P<0.0001) but was not associated with circulating tumor DNA (ctDNA) concentration (P-values 0.25-0.86). In contrast with prior reports in the context of recurrent lymphoma, CH mutation status was not associated with overall survival (OS) (P=0.78) or progression-free survival (PFS) (P=0.89) in our primary LBCL cohort. CH clones showed variable longitudinal dynamics during immunochemotherapy, with 18% of evaluable patients developing mutant CH clones, mostly with TP53/PPM1D mutations. Together, CH mutations can be readily detected in cfDNA of lymphoma patients, but they do not predict survival in primary high-risk LBCL.

Background: Relapse remains the principal cause of treatment failure in pediatric acute leukemia. Clinically validated minimal residual disease (MRD) assays - multiparameter flow cytometry, immunoglobulin/T-cell-receptor rearrangement sequencing, and targeted next-generation sequencing panels -- are highly sensitive but read out predefined features and do not reconstruct clonal architecture or link a cell's genotype to its functional state. We systematically reviewed what single-cell sequencing has revealed about clonal evolution, chemoresistance, the immune microenvironment, and post-therapeutic immune dynamics in this disease. Methods: This review followed PRISMA 2020. PubMed/MEDLINE, the Cochrane Library, and Google Scholar were searched to September 2026 for primary studies applying single-cell DNA, RNA, chromatin-accessibility, surface-protein, or immune-repertoire sequencing to pediatric acute leukemia samples or pediatric-derived models. Risk of bias in non-randomized studies was assessed with ROBINS-I. Because designs, platforms, and outcomes were heterogeneous, findings were synthesized narratively by biologic theme without meta-analysis, following the SWiM guideline. Results: Of 63 records identified, 10 studies were included (published 2016-2026; 6-159 patients or samples per study; up to ~700,000 cells profiled). Single-cell DNA sequencing traced minor NOTCH1-mutant diagnostic subclones into PTEN- or TP53-mutant relapse clones that bulk sequencing missed. Rare dormant, label-retaining cells and developmental-state-dependent drug sensitivity defined chemoresistant compartments. Remodeling of the bone marrow immune microenvironment identified patients with adverse outcome among those classified as MRD-low, and pre-infusion CAR T-cell type 2 functionality and post-transplant cytotoxic memory CD8+ T-cell subsets were associated with durable remission. Of the seven studies assessed with ROBINS-I, four were at moderate and three at serious overall risk of bias, driven chiefly by small, selected cohorts. Conclusions: Single-cell sequencing yields mechanistic insight that complements, rather than replaces, clinically validated MRD assays, and nominates candidate biomarkers for risk stratification, treatment selection, cell-therapy manufacturing, and post-transplant monitoring. The evidence remains preliminary; adequately powered, prospective, and harmonized studies are required before clinical adoption.

CBFA2T3-GLIS2 (C/G) defines a high-risk pediatric AML subtype in which de novo enhancer rewiring sustains an aggressive leukemic transcriptional state. The early epigenomic mechanisms reinforcing this program and linking it to leukemic survival remain incompletely defined. Here, we identify enhancer-proximal hypermethylation as a noncanonical, transcription-permissive feature of C/G AML. Using primary C/G-positive AML samples and a developmentally relevant C/G-transformed cord blood hematopoietic stem/progenitor cell model, we integrated DNA methylation, chromatin state, fusion occupancy, and gene expression across early and leukemia-stage contexts. Early C/G-associated hypermethylated regions localized near H3K27ac/H3K4me1-marked active or primed cis-regulatory elements, were enriched for ERG/ETS-like motifs, and showed related architecture in leukemia-stage contexts. DNMT3B was prominently increased in C/G contexts, and its loss selectively altered methylation-associated gene expression without measurably changing global 5mC. Genetic DNMT3B loss increased venetoclax sensitivity in vitro, while pharmacologic DNMT3B inhibition with Nanaomycin-A similarly enhanced venetoclax response. During venetoclax treatment, DNMT3B-deficient xenografts showed reduced disease burden and prolonged survival. To assess whether this interaction extended to clinically used hypomethylating agents, azacitidine or decitabine plus venetoclax induced apoptosis-related transcriptional programs and mitochondrial depolarization. These findings identify hypermethylated active cis-regulatory elements as C/G-associated states linked to transcriptional dysregulation and venetoclax response.

Stem cell factor (SCF) from Leptin Receptor-expressing (LepR+) mesenchymal stromal cells and endothelial cells is required for hematopoietic stem and progenitor cell maintenance in bone marrow. Consistent with this, Scf is highly expressed by LepR+ cells and at lower levels by endothelial cells in bone marrow. However, endothelial cells outside the bone marrow produce circulating SCF, raising the question of whether the SCF that promotes stem and progenitor cell maintenance is produced inside or outside bone marrow. We found that Scf deletion in spleen and liver endothelial cells reduces SCF levels in the blood without affecting stem or progenitor numbers in the bone marrow. Moreover, deletion of Scf from mesenchymal cells in the long bones, but not vertebrae, of Prx1Cre; Scf fl/fl mice depletes stem and progenitor cells in long bones but not vertebrae. SCF thus acts locally within the bones in which it is made to promote stem and progenitor cell maintenance.

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

Cancer genes are classically categorized as oncogenes or tumor suppressors based on whether gain or loss of function promotes malignancy. Here we show that oncogene-imposed dosage constraints can produce mutations that mimic tumor suppressor loss yet mark essential genes. In acute promyelocytic leukemia (APL), the transcription factor EBF3 gene harbors recurrent heterozygous loss-of-function mutations, a tumor suppressor signature, and is essential for APL survival. We resolve this paradox by showing that while PML::RARA, the fusion oncoprotein defining APL, drives EBF3 expression, excess EBF3 suppresses proliferation. Patient mutations reduce EBF3 activity through heterozygous inactivation, optimizing its expression within a corridor bounded by fitness constraints at both extremes. PML::RARA activates EBF3 via a p300-dependent intronic enhancer, and p300 inhibition exploits this constraint by suppressing EBF3 below the viability threshold. Our findings reveal that oncogenes can impose dosage constraints that drive adaptive mutations conventionally misclassified as tumor suppressor loss, with implications for interpreting cancer genomes.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy, yet how donor alleles shape red blood cell (RBC) storage, transfusion performance, and recipient physiology remains unresolved. Across 13,091 donors, a recalled cohort, donor-recipient data, humanized mice, and a prospective sickle cell disease cohort, variants reduced G6PD abundance, constrained pentose phosphate pathway flux and redox metabolism, and accelerated age-dependent functional decay. Murine A-; RBCs recovered poorly; A-; and Mediterranean RBCs impaired renal oxygenation, especially the latter, which caused greater plasma hemolysis. In humans, A-; units yielded smaller hemoglobin increments, while deficient units reduced allogeneic 51Cr post-transfusion recovery and four-week survival by 8 and 12-percentage-points. NADP;-bound cryo-EM resolved mechanisms: remodeling of the catalytic NADP;-binding region and dimer interface underlies A-; kinetic fragility, whereas Mediterranean S188F exhibits temperature-dependent oligomerization and conformational instability. These findings define an allelic series linking genotype to molecular instability, RBC quality, transfusion performance, and recipient physiology.

Quantitative measurement of fetal hemoglobin (HbF) using high-performance liquid chromatography (HPLC) is standard but expensive and widely unavailable in low-income settings. The newer Gazelle method, which employs single-use alkaline electrophoresis cartridges, provides a portable and relatively inexpensive potential alternative. In the current study, we tested 3055 venous blood samples collected from 1780 children with sickle cell anemia who were enrolled into the H-PRIME trial by both Gazelle and the Bio-Rad D-10 HPLC instrument, before and during hydroxyurea treatment. Data reinterpretation was required to quantify accurately high HbF values derived using the D-10. Mean (standard deviation) baseline HbF values by HPLC and Gazelle (N=2042) were 11.4% (SD 5.9%) versus 16.3% (SD 7.5%), respectively, while mean on-treatment HbF values (N=1013) were 18.9% (SD 9.0%) versus 25.4% (SD 11.5%), respectively. While overall agreement was good (Lins concordance 0.76, 95% CI 0.75-0.78), HbF values derived using Gazelle were modestly higher than HPLC values (Bland-Altman agreement mean +5.4%, 95%CI -2.4% to +13.2%), with the difference between measures increasing at higher HbF percentages. With strong concordance and more easily interpretable HbF values, Gazelle represents an attractive and inexpensive alternative to HPLC for HbF quantification in the low-income settings where most patients with sickle cell anemia live.

MLL/KMT2A amplifications and rearrangements are prevalent in infant, adult, and therapy-induced leukemia; however, the molecular contributors controlling these alterations remain elusive. Here, we reveal that rapid CTCF degradation generates local genome structure alterations at KMT2A and copy gains and rearrangements. We then established a conserved, coordinated interplay between DNA and histone methylation pathways that control CTCF occupancy, and in turn, KMT2A locus stability. For example, DNMT1 overexpression promotes increased H3K9 methylation, reduced CTCF occupancy, and causes KMT2A alterations. However, DNMT1 inhibition suppresses these events and KMT2A alterations caused by topoisomerase II inhibition. Locus-specific epigenome targeting demonstrated that DNA or H3K9 methylation promotes KMT2A copy gains and rearrangement, whereas targeted TET activity suppresses these events upon methylation perturbation or doxorubicin treatment. These findings identify a conserved, coordinated DNA-histone methylation axis governing KMT2A amplifications and rearrangement susceptibility, revealing biomarkers and therapeutic targets to predict and intercept these events in cancer.

Venetoclax and azacitidine (ven-aza) is a standard of care therapy for newly diagnosed acute myeloid leukemia (AML) patients unfit to receive intensive therapy, and may also be appropriate for certain fit patients. Models predicting response to ven-aza currently focus predominantly on genetics. Here, we present two multimodal fusion models: a custom ResNet-34-based model and a pathology foundation model fine-tuned for bone marrow smear (BMS) images that combine clinical and genetic variables with whole slide images. Training on 193 newly diagnosed AML patients who received ven-aza, we obtained an area-under-the-receiver-operating-characteristic (AUROC) of 0.79 and 0.82 for the ResNet-based and fine-tuned foundation models in a heldout test set, and 0.76 and 0.73 in an external validation set, predicting best response to this therapy. Sensitivity analysis showed that fusing image data with clinical and genetic variables outperformed training on either modality alone. In univariable and multivariable analyses, image-based classification was an independent predictive marker, second only in effect size to IDH1/2 mutation status, outperforming other genetic alterations. Image-based explainability showed high model attention on myeloblasts, suggesting previously unrecognized cell-specific patterns associated with response. Our findings suggest BMS images complement genetic biomarkers to refine outcome prediction in ven-aza treatment and incentivize AI-supported outcome prediction, including bone marrow morphology.

Background: Metabolic plasticity contributes to therapeutic resistance in T-cell lymphoma, but the metabolic dependencies underlying tyrosine kinase inhibitor (TKI) resistance remain poorly understood. ATP citrate lyase (ACLY), which converts citrate to cytosolic acetyl-CoA, links glucose metabolism to lipogenesis and acetylation-dependent regulation. We hypothesized that ACLY represents an actionable metabolic vulnerability in lymphoma. Methods: Structure-guided drug discovery and biochemical screening were used to identify the novel ACLY inhibitor VCB1. VCB1 was evaluated against recombinant human ACLY and in TKI-sensitive and TKI-resistant lymphoma models. Antitumor activity was assessed by proliferation, cell-cycle analysis, apoptosis, and immunoblotting, followed by integrated transcriptomic, metabolomic, and lipidomic profiling. Results: Our xenograft study showed that ACLY dependence in ALK+ALCL. We discovered novel ACLY inhibitor VCB1 directly inhibited recombinant ACLY with uM potency (IC, 0.9 uM) and strongly suppressed lymphoma cell growth. In ALK inhibitor-sensitive SUPM2, VCB1 showed an IC of 0.299 uM and retained potency in lorlatinib-resistant SUPM2-LR1000 cells (1.62 uM), whereas BMS-303141 was substantially less active (17.46 and 22.07 uM). VCB1 similarly inhibited BTK inhibitor sensitive and resistant mantle cell lymphoma cell growth (IC, 0.28 and 0.278 uM), compared with BMS-303141 (30.5 and 37.01 uM). Multi-omics profiling revealed suppression of de novo lipogenesis, including ACACA, FASN, and PPARGC1B, together with extensive remodeling of triglyceride, phospholipid, cardiolipin, ceramide, and acylcarnitine species and increased expression of fatty-acid oxidation genes. VCB1 activated the integrated stress response (ISR), inducing ATF4, PPP1R15A, DDIT3, DDIT4 and SESN2, while disrupting purine and pyrimidine metabolism and reducing nucleotide biosynthetic genes, including CAD, DHODH, UCK2, and ATIC. VCB1 further activated an ACLY/ISR/CHAC1 axis associated with reduced GCLC/GCLM expression, glutathione depletion, and a decreased GSH/GSSG ratio, indicating impaired redox homeostasis. TKI-resistant cells exhibited distinct lipid-metabolic remodeling consistent with metabolic plasticity. VCB1 also altered immune-regulatory programs involving PDCD1, TIGIT, CD247, CD226, NFATC2, and inflammatory cytokines. Functionally, VCB1 induced cell cycle arrest and apoptosis. Conclusions: ACLY represents an integrated metabolic and signaling vulnerability in lymphoma. VCB1 suppresses lymphoma growth across TKI-sensitive and resistant models by disrupting lipid and nucleotide metabolism, metabolic adaptation and ISR and redox homeostasis supporting ACLY inhibition as a therapeutic strategy for TKI-resistant lymphoma.

CD8 T cells are key mediators of anti-tumor immunity, yet their differentiation states and clonal architecture within the acute myeloid leukemia (AML) bone marrow microenvironment remain incompletely defined. We performed high dimensional immune profiling of bone marrow samples from 46 newly diagnosed AML patients using mass cytometry to probe the microenvironment and T cell states. We used single-cell RNA and T cell receptor (TCR) sequencing to define CD8 T cell phenotypes, transcriptional states, and clonal architecture. AML-associated CD8 T cells were skewed toward terminally differentiated effector memory cells re-expressing CD45RA (TEMRAs), while canonically exhausted T cell populations were rare. Functional studies revealed that TEMRA CD8 T cells robustly produced IFN{gamma} and TNF upon in vitro stimulation. Clonal expansion was concentrated within the TEMRA compartment. Comparison of TCR sequences with known viral epitopes identified a small subset of expanded viral-specific clonotypes, while the majority of highly expanded clonotypes had unknown specificity and may include AML-specific T cells. Differential gene expression analysis revealed shared cytotoxic effector programs during clonal expansion but diverging activation signatures: viral-specific clones upregulated canonical T cell receptor signaling genes, whereas expanded clones of unknown specificity exhibited enrichment of natural killer (NK) receptor-associated genes. These genes comprised the leukemia-associated NK-immune (LaNKi) score which was elevated in an independent AML dataset compared to pan cancer tumor infiltrating lymphocytes. These findings define the CD8 T cell differentiation landscape and clonal architecture in AML marrow at diagnosis and suggest that T cell dysfunction in AML diverges from classical activation and exhaustion paradigms.