Blood

Germline variants in DDX41 are the most frequent genetic predisposition to adult hematologic malignancies. The most common variants are truncating, implicating loss of function in the pathogenesis. However, non-truncating variants account for 30-40% of cases, and their impact on essential DDX41 functions remains unknown. We utilized a genetic complementation assay to assess the functionality of 10 recurrent germline non-truncating variants of DDX41. All variants restored viability to Ddx41-deficient hematopoietic progenitor cells at exogenous expression levels. In contrast, the hotspot mutant p.R525H, which is somatically acquired at disease onset in >50% of patients, failed to restore viability. CRISPR-based modeling in cell lines and mice revealed heterogeneity: some variants were non-functional at endogenous expression levels whereas others maintained complete functionality, supporting normal cell proliferation and even lifelong hematopoiesis in a homozygous setting. Notably, co-expression of p.R525H with some variants caused impaired hematopoietic progenitor cell viability, indicating a dominant-negative effect of p.R525H. In contrast, other variants, all classified as variants of unknown significance, were unaffected by the presence of p.R525H. A screen of 100 disease-associated variants confirmed that many non-truncating germline variants are susceptible to p.R525H-mediated dominant-negative effects, whereas wild-type DDX41 is not. These findings indicate that DDX41 variant curation is complicated by variable effects on functionality and variant-specific interactions with somatically-acquired DDX41 mutations. The dominant-negative effect of p.R525H provides a mechanistic basis for the conclusion of recent patient cohort analyses that co-occurrence with a somatic hotspot mutation is a reliable indicator of DDX41-driven disease in carriers of non-truncating variants.

Therapy-driven genomic changes in multiple myeloma (MM) remain poorly defined. We analyzed whole-genome sequencing (WGS) data from relapsed/refractory MM (rrMM, N=386) and identified regional 1p31.1-p12 (hereafter 1pCEN, a region proximal to the centromere) loss-of-heterozygosity (LOH) as the only enriched aberration showing strong therapy-associated clonal selection (clonal timing rank fold-change = 3.7, P<2.2x10-16). This event showed enriched co-occurrence with 1qGain (OR = 2.3 (1.5-3.8), P=2x10-4) forming a recurrent "double-hit" in rrMM. To validate the clonal selection process, we examined three longitudinal cohorts (180 patients, 390 samples) and confirmed clonal expansion of 1pCEN and consistent prevalence of the 1pCEN+1q double-hit (20-24%). Survival analyses demonstrated significantly reduced progression-free survival in rrMM patients with this double-hit compared with those without. Comparison with a large newly diagnosed MM (ndMM) cohort confirmed previously-described 1p32 LOH is the prognostic locus at baseline, whereas 1pCEN is therapy-selected and largely independent of the 1p32 locus. Thus, 1pCEN+1q represents a recurrent double-hit event that clonally emerges in rrMM, conferring selective advantage under drug exposure and is distinct from the ndMM high-risk markers defined by current consensus guidelines. These findings nominate 1pCEN as a new genomic biomarker in rrMM and 1pCEN+1q may help patient stratification for therapeutic monitoring. Key PointsA therapy-driven common genomic double-hit (1p31.1-p12 LOH with 1q gain) clonally emerges in relapsed/refractory myeloma.

The JAK2V617F (JAK2VF) driver mutation is found in 95% of patients with polycythemia vera (PV), a progressive myeloproliferative neoplasm. Current treatments suppress excessive hematopoiesis but lack specificity for targeting JAK2VF cells, are unable to deplete mutant stem/progenitor cells and ultimately result in drug resistance. We discovered that the FDA-approved antibiotic, linezolid (LZD), ameliorates the PV phenotype across multiple model systems. LZD suppressed cell proliferation and STAT5 signaling, altered the cell cycle, and increased apoptosis of JAK2VF-harboring human erythroleukemia cells, but not in wild-type acute leukemia cells. Computational modelling indicated that LZD interacts specifically with mutant JAK2VF but not with wild-type JAK2 protein. We further showed that, in JAK2VF mice that faithfully recapitulate human PV, LZD mitigates disease burden by selectively targeting JAK2VF stem cells thereby normalizing spleen size and blood counts. LZD also inhibited hematopoietic colony formation by patient-derived peripheral blood mononuclear cells, with the more primitive progenitors being preferred targets. Importantly, LZD selectively decreased JAK2VF+ colony numbers, without impacting wild-type JAK2 colonies. In all, the data provide a firm foundation for evaluating LZD-like molecules as an effective therapy for PV and other myeloproliferative neoplasms. Key pointsO_LILinezolid acts as a JAK2V617FIZselective inhibitor in PV mouse models and PV patient samples while sparing wildIZtype hematopoiesis. C_LIO_LILinezolid acts directly on JAK2V617F hematopoietic stem cells. C_LI

Fetal hemoglobin (HbF) prevents the polymerization of sickle hemoglobin (HbS). HbF, measured usually as a percent of total hemoglobin (%HbF), is inversely associated with the severity of sickle cell disease (SCD) but fails to capture the distribution of HbF concentrations within red blood cells (RBCs). The relative proportion of HbF and HbS within a RBC is reflected by the HbF:HbS ratio whereas HbF/F-cell quantifies the absolute amount of HbF/RBC. While correlated, HbF:HbS ratio and HbF/F-cell are not interchangeable. In the context of mean corpuscular hemoglobin (MCH), HbF/F-cell approximates whether sufficient HbF is present to inhibit HbS polymerization. We examined the association of mean HbF/F-cell with sub-phenotypes of sickle cell disease in three independent cohorts. Both %HbF and HbF/F-cell were significantly associated with multiple clinical and laboratory features of SCD; however, HbF/F-cell demonstrated stronger associations with clinical severity measures across cohorts. Higher HbF/F-cell was associated with fewer clinical events, reduced hemolysis, and mortality. Changes in HbF/F-cell after hydroxyurea treatment were associated with ~11-13% reduction in acute events in patients with <1 pg increase and >60% reduction with a >5 pg increase in HbF/F-cell. For each pg increase in HbF/F-cell there was ~6% reduction in the rate of acute events. As a surrogate for the distribution of HbF concentrations among F-cells, HbF/F-cell adds physiologically relevant insights that could guide prognosis and treatment

Despite extensive sequencing, the genetic etiology of sporadic angiosarcoma remains poorly defined (1-3). Maffucci syndrome, characterized by vascular tumors and elevated cancer risk, is driven by mosaic gain-of-function mutations in IDH1/2 (4,5), though these have not been reported in sporadic angiosarcoma. We identify recurrent, low-variant allele frequency hotspot mutations in IDH1/2 in over half of sporadic angiosarcomas. Mutations were validated by Sanger sequencing and immunohistochemistry. Mutant IDH1 endothelial cells promote tumorigenesis through non-cell-autonomous mechanisms, secreting 2-hydroxyglutarate (2-HG) to increase growth factor and endothelial-to-mesenchymal transition gene expression, activate pAkt/pERK signaling, induce DNA methylation changes, and promote anchorage-independent growth, which are reversed by the mutant IDH1 inhibitor ivosidenib. Patients with mosaic IDH1 mutations show reduced serum 2-HG and marked tumor regression following ivosidenib treatment. The clinical efficacy of ivosidenib in vascular tumors with subclonal IDH1 mutations suggests that low VAF IDH1/2 mutations may be a targetable vulnerability in sporadic angiosarcoma. (6,7) Statement of SignificanceWe identify recurrent, low-VAF IDH1/2 mutations in angiosarcoma and provide evidence that these subclonal mutations promote tumorigenesis through non-cell-autonomous mechanisms. Vascular tumors driven by subclonal IDH1 mutations responded dramatically to ivosidenib, thus revealing a novel treatment for a subset of vascular tumors.

Measurable (or minimal) residual disease (MRD) predicts relapse in patients with acute myeloid leukemia (AML). However, the biological and spatial characteristics of the AML bone marrow (BM) microenvironment (BMME) in which MRD cells survive remain largely unexplored; in particular, little is known of the BMME in TP53 mutant (TP53mut) AML. Here, we applied sequential immunofluorescence to whole BM biopsy specimens obtained from patients with TP53 wild-type (TP53WT) AML and TP53mut AML at diagnosis and in morphological complete remission (CR) to generate a comprehensive spatial map of the hematopoietic and BMME components. We identified TP53mut leukemia cells based on high p53 expression and delineated their spatial organization relative to stromal and immune niches. Biopsy-based cell composition analysis revealed marked B-cell depletion and an increased abundance of regulatory T-cells (Tregs) in TP53mut BM at CR. Unlike TP53WT BM, TP53mut BM at CR exhibited persistent TP53mut erythroid and immature leukemia cell clusters, spatially segregated from T-cell clusters, in perisinusoidal niches, suggesting niche-level immune evasion. Spatial profiling further revealed that Tregs characterized by FOXP3 upregulation were enriched near TP53mut MRD cells, indicating a locally enhanced immunosuppressive activity. Single-cell RNA sequencing-based cell-cell communication analysis identified erythroid-T-cell interactions mediated by the GDF15-CD48 axis as a potential mechanism of T-cell suppression, suggesting that the erythroid differentiation of TP53mut AML cells enhances local immunosuppression. Collectively, our results show a spatially organized immunosuppressive BMME in TP53mut AML and highlight the potential of spatial proteomics to identify actionable MRD niches in leukemias. Key pointsO_LITP53 mutant erythroid and immature leukemia cells form spatial clusters segregated from T-cells in complete remission. C_LIO_LIAn erythroblast-centered immunosuppressive niche characterizes TP53 mutant leukemia cells. C_LI

Pediatric platelet disorders are commonly classified according to specific structural or functional abnormalities, yet it remains unclear how well these diagnoses capture overall hemostatic phenotype. Here, we combined quantitative single-cell platelet measurements with spatially resolved plasma clotting analysis to characterize pediatric patients with dense granule deficiency, platelet function defects, immune thrombocytopenia, and other inherited platelet disorders. Quantitative fluorescence microscopy revealed reduced dense granule abundance not only in dense granule deficiency but also in several patients from other diagnostic groups. Measurements of platelet adhesion, spreading, and calcium signaling identified substantial functional diversity, with individual patients exhibiting distinct combinations of abnormalities that were not predicted by diagnostic category. Unexpectedly, plasma clotting analysis frequently revealed hypercoagulable behavior, including accelerated fibrin clot growth and spontaneous fibrin formation, despite clinical diagnoses associated with platelet-related bleeding disorders. Hypercoagulable phenotypes occurred across multiple diagnostic groups and did not show a simple relationship with platelet functional abnormalities. Together, these findings reveal previously unrecognized complexity in pediatric platelet disorders and suggest that platelet and plasma pathways contribute independently to hemostatic variability. These findings argue that pediatric platelet disorders are best viewed as multidimensional functional phenotypes rather than isolated platelet defects and motivate broader integration of platelet and coagulation measurements in future studies.

Tyrosine kinase inhibitors (TKIs) are the first-line therapy for chronic myeloid leukemia (CML), yet fail to eliminate quiescent CML cells residing in the bone marrow (BM). While transcriptome adaptation and metabolic rewiring have been recognized as mechanisms enabling CML survival, the contribution of RNA processing, known to expand the repertoire of isoforms and directly mediate therapy resistance in leukemia, is poorly characterized. We previously found that a subset of alternative splicing (AS) changes detected in CML cells surviving months of therapy are initiated within hours of treatment onset. Here, we developed a humanized BM stromal niche in vivo to investigate the influence of the human BM microenvironment on gene expression and AS in CML cells. Stroma-facilitated transcriptome adaptation targeted transcription regulation, transmembrane transport, lipid metabolism, respiration and energy production. We identified RNA-binding protein TIAR (T-cell intracellular antigen-related protein) as a key mediator of CML survival and determined that TIAR-dependent post-transcriptional regulation coordinates RNA processing-metabolism program induced by stromal interaction. Quantitative nascent proteome analysis confirmed that TIAR is a translational regulator of metabolic enzymes and proteins involved in imatinib-induced erythroid differentiation. In hypoxic co-culture with BM stromal cells, TIAR knockdown reduced the viable erythroid-differentiated cell population in association with increased lipid peroxidation and decreased redox potential. Taken together, these findings identify TIAR-dependent RNA processing within the BM niche as a previously unrecognized mechanism of CML therapy resistance and potential therapeutic vulnerability.

Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are caused by genetic variants in the WAS gene. How WAS variants lead to clinical disease remains unsolved in many cases. We expressed human WASp using a spider silk inspired solubility tag (NT*-tag) and inserted patients variants. Native mass spectrometry and pyrene actin assays showed that five variants (L270P, F271S, S272P, I290T, I294T) predicted to cause XLN led to open protein conformation and high actin polymerization rate in the absence of the WASp activator, Cdc42. One previously reported XLN variant (R268W), two loss-of-function WAS variants (A236G, D485N), and one variant of unknown significance (R431W) behaved similarly to wildtype WASp in terms of structural conformation and actin polymerization. Patient CD4+ T cells were used for analysis of WASp expression and phosphorylation, actin polymerization, anti-CD3 induced proliferation capacity, and upregulation of high affinity LFA-1, distinguishing loss-of-function and gain-of-function variants from benign WAS variants. This systematic approach reveals how WAS genetic variants cause severe human disease and stratify variants to guide clinical decision for definitive therapy. Key PointsO_LIGain-of-function WASp variant has extended protein conformation probed by native mass spectrometry and raised pyrene actin polymerization. C_LIO_LIFunctional analysis of patients CD4+ T cells classifies WASp variants as loss-of-function, reduced-function, gain-of-function, and benign. C_LI

Richter transformation of Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) into classic Hodgkin lymphoma (CHL-RT) is rare and remains incompletely understood. Two histologic subtypes are recognized: type 1 (CLL/SLL with scattered Hodgkin/Reed-Sternberg (HRS) cells) and type 2 (HRS cells within a polymorphous inflammatory background). In this multi institutional study of 77 patients with CHL-RT (27 type 1 and 50 type 2), we characterized immune evasion markers, PD-L1/PD-L2 copy number alterations, tumor microenvironment, and performed targeted next-generation sequencing on 37 CLL/SLL samples. HRS cells in CHL-RT displayed immune evasion phenotypes similar to de novo CHL, though PD-L1 expression was lower in type 1 cases. PD-L1/PD-L2 gain/polysomy were frequent (83.3%). CLL/SLL with CHL-RT harbored increased mutations in XPO1, FBXW7, BIRC3, TRAF3, and HLA-A versus reference CLL/SLL. Similar mutational profiles, demographics, and survival outcomes support a biological continuum between type 1 and type 2 CHL-RT, with distinct genetic features in CLL/SLL predisposing to CHL transformation.

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.

ObjectiveHereditary hemorrhagic telangiectasia (HHT) is a vascular genetic disorder caused by endothelial cell dysfunction and characterized by telangiectasias and arteriovenous malformations (AVMs). HHT results primarily from loss-of-function mutations affecting components of the BMP9-ALK1-ENG-SMAD signaling cascade, a pathway essential for endothelial quiescence and vascular homeostasis, and currently lacks a cure. Here, we investigated whether nitazoxanide, an orally bioavailable drug with extensive clinical use, can modulate endothelial signaling relevant to HHT. Approach and ResultsNitazoxanide treatment activated SMAD1/5/8 signaling and increased expression of the downstream target ID1 in endothelial cells, while concurrently inhibiting mTOR signaling, indicating a dual modulatory effect on pathways implicated in HHT pathogenesis. In vivo, nitazoxanide activated SMAD signaling in BMP9/10-immunoblocked mice and significantly reduced AVM formation and hypervascularization. Importantly, nitazoxanide restored SMAD1/5/8 activation and ID1 expression in patient-derived blood outgrowth endothelial cells harboring loss-of-function mutations in ALK1 or SMAD4, which exhibit impaired BMP signaling. ConclusionThese findings identify nitazoxanide as a pharmacological modulator capable of activating BMP-SMAD signaling while restraining mTOR activity, thereby overcoming key signaling defects in HHT endothelial cells. Collectively, our results highlight nitazoxanide as a promising therapeutic candidate to target endothelial dysfunction in HHT.

Background: Modern therapy for childhood and adolescent leukemia requires accurate risk classification of genomic subtype. Although short-read next-generation sequencing (NGS)- based approaches provide comprehensive clinical diagnostics in limited, highly resourced settings, they remain expensive, slow, and inaccessible to most children worldwide. Transformative approaches are needed to improve diagnostic classification for leukemia globally. Methods: We simultaneously continued to develop an analytical pipeline NASVar (Nanopore variant calling for adaptive sampling), and conducted a multicenter, type-two hybrid clinical validation study of an Oxford Nanopore Technologies (ONT) adaptive-sampling whole-genome sequencing (asWGS) assay across hospitals with varying diagnostic resources. In preparation for implementation, a global panel developed a leukemia-based standardized gene set and consensus laboratory-developed test (LDT) validation guidelines. Measures of assay effectiveness compared to both conventional and orthogonal NGS methods, where available, were simultaneously collected with data to measure the implementation outcomes of feasibility, fidelity, appropriateness, and cost. Results: All four centers successfully completed the LDT validation, with minimal adaptations required for regulatory compliance. A total of 457 specimens were sequenced (331 B-ALL, 83 AML, 43 T-ALL). For the 210 B-ALL cases with locally resolved genomic subtypes defined by DNA alterations, asWGS was 100% concordant (210/210). Cases locally defined as B-other were resolved via asWGS with disease-defining DNA alterations in 47% (49/105) of cases. An additional 41% (43/105) of locally defined B-other cases were classified by incorporation of DNA methylation, and all 16 B-ALL patient-derived xenograft controls were correct, for a total of 96% (318/331) of all B-ALL cases in the cohort resolved with single assay asWGS. For AML, 97% (56/58) of cases with locally resolved genomic subtypes were identified by automated asWGS analysis, while an additional two cases were identified after targeted manual review. At Indus Hospital in Pakistan, the B-ALL and AML diagnostic genomic subtype yield increased from 28% with local standard of care diagnostic testing, to 84% with asWGS. The cost of reagents and consumables in the United States, assuming pooled three-plexing, was $343/sample. Based on the combined hybrid validation results, all centers are independently preparing for clinical return of results. Conclusions: ONT asWGS was successfully validated as a clinical assay in four diverse hospital settings. As a single, multi-omic platform that delivers value across the continuum of high-resource to resource-limited contexts, the approach offers a disruptive solution to address the global equity gap in cancer diagnostics.

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.

In AL amyloidosis, monoclonal immunoglobulin light chains (LCs) aggregate as amyloid fibrils in tissues. In synergy with the intrinsic aggregation propensity of specific LC sequences, microenvironment factors may be involved in tuning the disease pathophysiology, in particular proteolytic LC remodelling and heterotypic interactions in the extracellular milieu. Accounting for extrinsic modulators is critical for understanding the phenotypic variability of AL, usually imputed mainly to the LC diversity. We investigated the effects of apolipoprotein E (allele 3, apoE3) and clusterin (CLU), two amyloid-signature proteins involved in extracellular proteostasis, on the fibrillogenesis kinetics of amyloidogenic LC fragments from patient-derived sequences, as well as on aggregate composition, fibril morphology and thermodynamic stability. We show that apoE3 and CLU act as heterotypic interactors of prefibrillar and fibrillar LCs, significantly modulating LC amyloidogenesis, with complex and non-monotypic effects that range from anti- to pro-amyloidogenic depending on their concentration and on the LCs intrinsic amyloidogenicity. ApoE3 and CLU also influence fibril morphology, possibly by modifying protofilament association, and alter their thermodynamic properties. LC interactors may play a significant and insofar underappreciated role in the AL pathophysiology in vivo, likely contributing to phenotypic variability and structural polymorphisms and, possibly, to fibril resilience to amyloid reabsorption strategies.

The mechanistic target of Rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to nutrient availability. mTORC1 recruitment to lysosomes by the Rag dimer complex (RagA/B and RagC/D) is a crucial step in mTORC1 signaling. Substrates of the MiT/TFE transcription factor family, like TFEB and TFE3, directly interact with the Rag complex when activated by folliculin (FLCN). This selective recruitment and subsequent phosphorylation of MiT/TFE substrates leads to a non-canonical mTORC1 signaling pathway regulating lysosomal biogenesis and autophagy. Recently, we showed that compound heterozygous mutations in VPS41 specifically impair non-canonical mTORC1 signaling. VPS41, as part of the HOPS complex, is required for fusion of lysosomes with endosomes and autophagosomes. Here we addressed the mechanism by which VPS41/HOPS complex regulates mTORC1 activity. We show that multiple HOPS subunits interact with the Rag dimers as well as with FLCN. Depletion of HOPS subunits results in reduced lysosomal localization of both Rags and FLCN, and the nuclear translocation of TFE3/TFEB. The VPS41-Rag interactions required the RING domain of VPS41, but were independent of RHEB, Rag activity or presence of other HOPS components. We conclude that HOPS is necessary for the recruitment of crucial components of the non-canonical mTORC1 signaling pathway onto lysosomal membranes. These data further our molecular understanding of disease-causing VPS41/HOPS mutations and indicate a crucial role for HOPS in connecting lysosomal trafficking to lysosomal signaling.

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.

BackgroundPatients with systemic lupus erythematosus (SLE) face markedly increased cardiovascular disease (CVD) risk driven by mechanisms beyond traditional risk factors. Thoracic aortic perivascular adipose tissue (tPVAT) is dysfunctional in lupus and exacerbates endothelial dysfunction, yet the molecular basis of this dysfunction remains poorly defined. MethodsIntegrated multi-omics profiling, including bulk RNA-seq, untargeted proteomics, lipidomics, and high-dimensional spectral flow cytometry, was performed on tPVAT from 15-week-old MRL/lpr mice (active lupus, n = 4-6) and MRL control mice (n = 5-6). Adipogenic differentiation capacity of tPVAT adipose stromal and progenitor cells (ASPCs) from MRL/lpr was assessed by Oil Red O staining at 5 (pre-dieasea) and 15 weeks (active disease), with subcutaneous ASPCs used as depot controls. ResultsTranscriptomic profiling of tPVAT from MRL/lpr mice identified 2,742 upregulated and 1,494 downregulated genes (adjusted p < 0.001, |log2FC| > 1), with strong activation of interferon, IL6-JAK-STAT3, and TNFA signaling pathways together with suppression of fatty acid metabolism, oxidative phosphorylation, and adipogenic pathways. Proteomic and lipidomic analyses were concordant, revealing broad downregulation of mitochondrial bioenergetic machinery, depletion of cardiolipin and acylcarnitines, and enrichment of ceramide phosphoinositols and lysophosphatidylcholines. Cardiolipin strongly correlated with the mitochondrial/metabolic protein module (r = 0.95) and inversely with the immune/inflammatory protein module (r = -0.92). Spectral flow cytometry confirmed marked CD45+ leukocyte infiltration dominated by T cells, together with a significantly reduced Treg/CD4+ ratio indicating loss of local immunoregulatory balance. ASPCs derived from PVAT of 15-week-old MRL/lpr mice exhibited impaired white and beige adipogenic differentiation, while APCs from PVAT of 5-week-old MRL/lpr mice, and from subcutaneous adipose tissues of 15-week-old MRL/lpr mice, had normal white and beige differentiation, consistent with an acquired, depot-specific, disease-stage-dependent progenitor defect in PVAT of MRL/lpr mice. ConclusionsLupus tPVAT undergoes a concordant cross-platform molecular reprogramming of mitochondrial bioenergetic genes coupled with establishment of an interferon-dominant immune niche and acquired loss of ASPC adipogenic capacity. These findings provide a molecular framework for lupus PVAT dysfunction and identify restoration of mitochondrial function, suppression of interferon-driven inflammation, and renewal of progenitor differentiation as potential therapeutic strategies for lupus vasculopathy.

Global mRNA translation is a defining functional property of hematopoietic stem cells (HSCs) and is increasingly recognized as a critical axis of dysregulation in myelodysplastic syndromes (MDS) and other clonal hematopoietic disorders. Yet the quantitative measurement of protein synthesis at single-cell resolution across phenotypically defined HSPC subpopulations, in parallel with apoptotic state, is technically challenging. Here we describe and validate a single-tube flow cytometry protocol that simultaneously quantifies global protein synthesis by O-propargyl-puromycin (OP-Puro) incorporation and intracellular cleaved Caspase-3 with cell immunophenotyping across the canonical CD34+ HSPC hierarchy in cryopreserved human cord blood (CB) CD34+ cells. The protocol enables quantitative assessment of key dynamic cell processes in defined subsets of primary hematopoietic cells on a standard flow cytometer. We apply this assay to a four-condition factor-omission analysis of the canonical SR1 + UM729 + dmPGE2 ex vivo expansion cocktail across three independent CB donors. The analysis assigns each compound a distinct functional profile: UM729 constrains protein synthesis and supports apoptotic priming across the hierarchy; SR1 maintains a pro-survival state without modulating translation; and dmPGE2 promotes HSC cycling and progressive exit from the primitive state, with minimal direct effect on the translation or apoptotic axes measured here. This analysis resolves three mechanistically distinct small-molecule signatures using a protocol directly transferable to clinical biobank specimens. We propose it as a functional-state analytic platform that may be useful for patient-derived CD34+ cells from MDS and other myeloid neoplasms in which translational dysregulation is a recognized pathological feature.

Sex-specific genetic effects on platelet aggregation may contribute to differences in thrombotic risk between women and men, yet the underlying genetic mechanisms remain poorly defined. We performed whole-genome sequencing-based genome-wide association studies (GWAS) of 19 harmonized platelet aggregation phenotypes in response to ADP, epinephrine (EPI), and collagen across three independent cohorts: GeneSTAR, the Framingham Heart Study and the Old Order Amish. Our meta-analysis identified a cluster of low-frequency variants within a 20 kb region on chromosome 10 showing strong sex-specific associations with platelet aggregation in response to low-dose EPI. The lead variant, rs116725046, exhibited a genome-wide significant sex interaction (p = 5.2e-9), with opposite phenotypic effects in women and men. Female carriers demonstrated substantially increased platelet aggregation, whereas male carriers showed decreased aggregation, consistent across cohorts. Several additional variants in tight linkage disequilibrium yielded comparable interaction signals for low-dose EPI, including five SNPs driving the lowest meta-analysis p-value (p = 8.3e-9). The associated variants reside within an intronic region of the long noncoding gene LINC00702, with FUMA annotations indicating regulatory chromatin states. Megakaryocyte epigenome data also indicates potential regulatory activity in platelet precursor cells near the lead variants. eQTL analyses suggested sex-differentiated genetic regulation of LINC00702 in multiple tissues, with reduced expression in male heterozygotes only. An ARE motif was identified upstream of LINC00702, supporting a potential hormone-responsive regulatory mechanism. Together, these findings identify a novel sex-specific regulatory locus influencing platelet reactivity and highlight LINC00702 as a biologically plausible mediator of sexually dimorphic platelet responses.