Haematologica

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.

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.

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.

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.

Replication stress (RS) can act as an input to differentiation: in erythroid and myeloid cells it is sufficient to accelerate commitment. Yet genotoxic chemotherapy, which imposes replication stress on the bone marrow can cause anemia; a frequent dose-limiting toxicity whose deficit of mature red cells is often attributed to progenitor depletion rather than to any effect on differentiation itself. Here, we ask how RS-induced accelerated commitment gives rise to impaired red cell output, using in vitro erythroid differentiation of cord-blood CD34+ progenitors perturbed with clinically relevant chemotherapy agents. We find that treated erythroblasts differentiate faster than controls but fail to mature, measured by lack of enucleated cells. Inhibition of ATR, but not ATM or DNA-PK, reproduces this phenotype. Mechanistically, single-cell profiling of drug-treated cells shows convergence on a transcriptional program, marked by loss of MYB and MYC activity. Treated cultures also accumulate aberrant late erythroblasts depleted for globin and heme programs and enriched for p53 and proteotoxic stress signatures. Together, these results propose that defective maturation is an alternative mechanism for chemotherapy-induced anemia, in addition to depletion of progenitors.

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.

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.

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy caused by a severe deficiency of the ADAMTS13 protease. We performed the first large-scale genome-wide association study (GWAS) of TTP in 1,344 individuals of Han Chinese ancestry, comprising a discovery cohort (n = 913) and a validation cohort (n = 431). We identified two independent susceptibility loci, including the known locus 6p21 and the novel locus 14q32. At the known locus 6p21, the lead signal rs2187668 is near HLA-DQA1 and strongly associated with TTP risk (P = 5.35e-10; Odds Ratio [OR] = 3.94). At the novel locus 14q32, the lead SNP rs1024350 (P = 1.01e-10; OR = 1.90) is located between LINC00221 and MIR5195. Fine-mapping prioritized rs2187668 as the leading signal at 6p21, whereas HLA imputation and stepwise conditional analysis further pinpointed amino acid position 74 of HLA-DRB1 as the primary driver of the broader MHC association (P = 2.28e-10), superseding classical allele-based models. Structural analysis revealed that position 74 lines pocket 4 of the HLA-DRB1 peptide-binding groove; the risk-conferring Leu/Arg residues at this position carried by HLA-DRB1*08:03 and DRB1*03 may favour presentation of ADAMTS13-derived autoantigenic peptides, whereas the common Ala residue is protective. In summary, we reconstructed the genetic architecture of TTP in East Asians and identified structural gatekeepers of autoimmunity, providing novel targets for precision medicine and ethnic-specific risk stratification.

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.

BACKGROUND: Severe influenza pneumonia with secondary bacterial infection is complicated by progressive pulmonary thrombus exacerbation, a key contributor to respiratory failure, yet anticoagulant therapies show limited efficacy and bleeding risks. Although platelet-monocyte crosstalk initiates thrombosis, whether and how it drives thrombus exacerbation via procoagulant platelets and monocyte efferocytosis remains unclear. METHODS: Clinical samples, mouse models, and isolated platelets challenged with influenza A virus followed by methicillin-resistant Staphylococcus aureus (MRSA) were analyzed. Procoagulant platelet, platelet programmed cell death and monocyte efferocytosis were assessed, and pharmacological inhibition, platelet depletion and systemic/platelet-specific Gsdmd knockout were used. Platelet proteomics and exogenous C1qa supplementation identified C1qa as a key mediator. RESULTS: We showed that elevated procoagulant platelet-driven thrombus exacerbation, rather than initial thrombus formation, was a critical driver of disease progression in influenza pneumonia with secondary bacterial infection, whereas influenza alone caused milder illness. Procoagulant platelet formation was induced via MLKL-mediated necroptosis and GSDMD-mediated pyroptosis. These platelets exacerbated pulmonary thrombosis and lung injury by inhibiting monocyte efferocytosis via complement C1qa. Platelet depletion reduced monocyte efferocytosis and worsened pneumonia, while pharmacological inhibition or platelet-specific Gsdmd knockout decreased procoagulant platelet levels, restored monocyte efferocytosis, and alleviated thrombotic and pulmonary injury. Mechanistically, C1qa impaired efferocytosis both by directly suppressing monocyte function and by reducing the proportion of reparative (M2-like) monocytes. Clinical relevance was confirmed by detection of MLKL/GSDMD-dependent procoagulant platelets and reduced efferocytosis receptor levels on monocytes in bronchoalveolar lavage fluid from severe pneumonia patients. CONCLUSIONS: Necroptosis/pyroptosis-driven procoagulant platelets exacerbate pulmonary thrombosis by suppressing monocyte efferocytosis in a C1qa-dependent manner. These findings extend platelet-monocyte crosstalk from thrombus initiation to thrombus exacerbation, identifying modulation of the interaction between procoagulant platelets and monocyte efferocytosis as a potential therapeutic strategy for thrombus-exacerbating diseases, especially in subpopulations of patients with severe pneumonia and progressive pulmonary thrombosis.

The human T cell repertoire is generated through recombination of a multitude of T cell receptor gene segments, yielding a complex array of T cell clones. T cell receptor beta (TRB) V gene segment defined, T cell clonal frequency when organized in correspondence with the respective V gene segment positions on the TRB genomic locus yields a periodic, undulating curve in the spatial domain. Using the genomic distance from the TRB-D1 segment to the TRB-V1-29 segments, spectral analysis was performed utilizing Lomb-Scargle periodogram (modified Fourier analysis) to obtain spectral power curves quantifying the TRB V clonal frequencies from 12 allogeneic stem cell transplant donors (baseline) and recipients (<100 days or b/w 100-365) using a variety of analytic software. Patients either underwent HLA matched related bone marrow or HLA matched unrelated blood stem cell transplantation, using PT-CY for the former and ATG for the latter. Spectral Power curves revealed dominant spectral peaks at wavelengths ranging from 113-335 millicycles/kb in the 12 donors, with consistent frequency domain spectral patterns, supporting similar use of V segments across healthy individuals. PT-CY recipients had power spectra closely corresponding to the respective donors, however the ATG recipients had relatively dispersed spectra, with spectral centroid shifted towards higher frequencies compared to donors and a reciprocal decline in the low frequency indices. Spectral power was concentrated in the <3 kb and 3-12 kb wavelengths in both groups, consistent with the periodicity observed in the relative V gene segment usage across the repertoire. The analyses reported here demonstrate that the healthy SCT donors have a spectral signature occupying short to intermediate wavelegnths in the frequency domain, PT CY recipients recover with donor-like spectra on the average, however ATG recipients tend to shift towards higher frequencies. These findings are consistent with a normal organized distribution of TRB V segment usage in healthy individuals, with PT-CY preserving this organization more consistently than ATG. Spectral (Fourier) analysis of TRB sequencing data provides a repertoire wide summary of T cell clonal distribution.

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.

Background: Barth syndrome (BTHS) is an X-linked mitochondrial disorder caused by loss-of-function mutations in TAFAZZIN (TAZ), resulting in defective cardiolipin (CL) remodeling, cardiomyopathy, and premature death. The mechanisms linking TAZ deficiency to impaired mitochondrial quality control remain incompletely understood, and there are no disease-modifying therapies available for BTHS. Here, we investigated the role of Acyl-CoA Lysocardiolipin Acyltransferase-1 (ALCAT1), a stress-inducible phospholipid-remodeling enzyme, in BTHS cardiomyopathy. Methods: Inducible TAZ knockdown (TAZKD) and cardiomyocyte-specific TAZ knockout (TAZcKO) mice were used to determine the effects of genetic ALCAT1 deletion or pharmacological inhibition with Juvenatin, a highly selective small molecule ALCAT1 inhibitor. Cardiac function, exercise capacity, mitochondrial function, lipid remodeling, lysosomal function, and mitophagic flux were assessed. Results: ALCAT1 was markedly upregulated in TAZ-deficient hearts. Genetic ALCAT1 deletion attenuated cardiac dysfunction in TAZKD mice. Juvenatin similarly improved cardiac function in TAZKD mice and reversed established cardiomyopathy in TAZcKO mice. Genetic or pharmacological ALCAT1 inhibition improved mitochondrial ultrastructure and respiration and reduced mitochondrial oxidative stress. These benefits occurred without correcting the characteristic CL abnormalities caused by TAZ deficiency. Instead, ALCAT1 deletion or inhibition corrected pathological phosphatidylinositol remodeling, normalized phosphoinositide signaling, and restored lysosomal function and mitophagic flux, leading to improved mitochondrial quality control in TAZ-deficient cells. Conclusions: ALCAT1 contributes to BTHS cardiomyopathy through a CL-independent pathway involving dysregulated phosphatidylinositol/phosphoinositide signaling, lysosomal dysfunction, and impaired mitophagy. Genetic and pharmacological targeting of ALCAT1 restores mitochondrial and cardiac function despite persistent CL abnormalities, establishing ALCAT1 as a promising disease-modifying therapeutic target for BTHS.

Acute myeloid leukemia (AML) remains limited by high relapse rates and a scarcity of biomarker-directed therapies, underscoring the need to identify actionable vulnerabilities and mechanisms of therapeutic resistance. O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that directly reverses mutagenic O6-alkylguanine lesions, is epigenetically silenced in multiple cancers. Although MGMT silencing is predictive of temozolomide (TMZ) response in glioblastoma with significant survival advantage, prior clinical trials of TMZ in AML have shown only modest responses. Therefore, the prevalence and therapeutic relevance of MGMT silencing, as well as genetic factors that modify its therapeutic response in AML, remain to be comprehensively investigated. Here, we first profiled MGMT status across a diverse cohort of 23 de novo and 16 relapsed/refractory (R/R) primary AMLs using an integrated analysis of MGMT mRNA expression, promoter methylation, and protein expression. We found that approximately 25-30% of AMLs harbor MGMT silencing, in contrast to consistent MGMT expression in healthy CD34+ hematopoietic stem and progenitor cells. We further identified frequent loss of mismatch repair (MMR) proteins in these AML cohorts, a known resistance mechanism against TMZ in glioblastoma. Using CRISPR knockout screening and genetically engineered AML models, we found that MMR loss also drives both upfront and acquired TMZ resistance in MGMT-silenced AML. These findings identify MMR deficiency as an important and previously underappreciated genetic contributor to the modest responses observed in prior trials of TMZ in AML. To therapeutically exploit MGMT silencing while bypassing genetic determinants of resistance, including MMR deficiency, we evaluated clinically and preclinically characterized MGMT-dependent DNA-alkylating agents and identified a fluoroethylating analog of TMZ, N3-(2-fluoroethyl) imidazotetrazine (KL50), with pronounced and selective activity in MGMT-silenced AML. KL50 retained antileukemic activity irrespective of MMR status, significantly prolonging survival in humanized MISTRG6 mice harboring primary AML patient-derived xenografts. Mechanistically, KL50 induced DNA damage through the time-dependent formation of interstrand DNA crosslinks, bypassing MMR dependence and triggering a replication stress response dominated by ATR signaling. Pharmacologic ATR inhibition synergized with KL50, producing marked antileukemic activity and significantly extending survival across AML models without compromising hematologic safety. Together, these findings establish MGMT silencing as a prevalent and therapeutically actionable biomarker in AML, define MMR status as a key determinant of TMZ response in AML but not KL50 sensitivity, and provide a translational rationale for combining low-dose O6-fluoroethylating imidazotetrazines with ATR inhibitors to target both de novo and R/R AML across diverse genetic backgrounds.

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.

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.

Sphingolipids are bioactive lipids that regulate key signaling pathways both directly as ligands and through membrane re-organization. Ceramide sits at the center of this network and is considered pro-death in many contexts, making ceramide accumulation an attractive therapeutic strategy. Given the wide-ranging regulation of cellular responses this network exerts, better understanding ceramide metabolism may promote therapeutic efficacy of sphingolipid-based therapeutics. Ceramide glycosylation, catalyzed by glucosylceramide synthase (GCS; UGCG), is in turn widely regarded as a detoxification route thus limiting efficacy of ceramide- based therapeutics. Here we show the opposite. Delivery of short-chain C6-ceramide via a ceramide nanoliposome (CNL) induced organelle stress and cell death in chronic lymphocytic leukemia (CLL) via the accumulation of glycosphingolipids (GSLs), rather than through ceramide itself. Pharmacologic and genetic blockade of GCS protected B-cell leukemia, breast carcinoma, glioblastoma, lung adenocarcinoma, and non- malignant embryonic kidney cells from CNL-induced death. Conversely, exogenous C8-glucosylceramide was sufficient to kill cells that cannot degrade it. We show that GSL accumulation drives an ordered organelle response beginning with lysosomal deacidification, endoplasmic reticulum stress, followed by mitochondrial respiratory capacity decline, each attenuated by inhibition of GSL synthesis. These effects were accompanied by MLKL phosphorylation, increased activity of the stress sensor JNK and CHOP induction, with JNK inhibition partially protecting from death. These findings invert the prevailing view of ceramide glycosylation as a resistance mechanism and identify glycosphingolipid flux as a required effector arm of ceramide-directed therapy.