Haematologica

The Src homology 2 (SH2) domain-containing non-transmembrane protein-tyrosine phosphatases 1 and 2 (Shp1 and Shp2) have been implicated in regulating signaling from a variety of receptors and cell types, including the thrombopoietin (Tpo) receptor Mpl in megakaryocytes (MKs) and platelets. We previously showed that deletion of Shp1 and Shp2 in the MK/platelet lineage in mice using the Pf4-Cre transgene/loxP system impairs megakaryopoiesis and thrombopoiesis. However, we also observed unexpected phenotypes including a motheaten-like phenotype in Shp1-deficient mice and severe myelofibrosis in mice lacking both phosphatases. To determine whether these were lineage-specific effects, we utilized the Gp1ba-Cre transgenic mouse to delete loxP-flanked Shp1 and Shp2 in mice. Bone marrow-derived MKs from these mice expressed approximately 20-25% of Shp1 and Shp2, whereas platelets contain 5-10% of each phosphatase compared with controls. Minor MK/platelet defects were observed in mice lacking either Shp1 or Shp2 alone, however mice lacking both Shp1 and Shp2 exhibited macrothrombocytopenia, mild bleeding following tail injury, and impaired GPVI-mediated platelet aggregation and Syk phosphorylation, associated with reduction GPVI and integrin 2 subunit expression. Reduced Shp1 and Shp2 expression resulting in a significant reduction in ploidy, a block in MK maturation and proplatelet-producing MKs. Tpo-mediated Ras/MAPK signaling was reduced in Shp1/2-deficient MKs. Treatment of MKs with structurally distinct Shp2 allosteric inhibitors recapitulated key aspects of the Shp2-deficient phenotype, including aberrant megakaryopoiesis and reduced Mpl signaling. Our study highlights the synergistic functions of Shp1 and Shp2 in the MK/platelet lineage, and identifies Shp2 as a potential therapeutic target in myeloproliferative neoplasms. Key PointsO_LIDeletion of Shp1 and Shp2 in the MK/platelet lineage in mice results in macrothrombocytopenia and minor effects on platelet function. C_LIO_LIDefects can be partially explained by reduced Mpl signaling and aberrant megakaryopoiesis in the absence of Shp2 activity. C_LI

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by hemolysis due to the loss of GPI-anchored complement regulators. While terminal complement inhibitors improve survival, the precise intracellular mechanisms driving the destruction of PNH erythrocytes remain controversial. A recently proposed model suggests PNH cells undergo an inflammatory programmed cell death ("spectosis") driven by an NLRP3-Caspase-8 signaling cascade. Here, we use a whole packed cell lysis approach to map the cytoskeletal degradation of primary erythrocytes across a 22-patient PNH cohort. Our data show that membrane attack complex (MAC) pore formation drives targeted {beta}-spectrin fragmentation, which correlates with rapid intracellular potassium (K+) efflux. Notably, when probing these primary patient samples, we detected a complete absence of the NLRP3 protein and found no functional evidence of Caspase-8 activation during MAC pore formation. Furthermore, caspase inhibition did not alter cytoskeletal degradation or K+ efflux. Instead, our data demonstrate that MAC-induced membrane perforation permits a rapid influx of calcium, which activates calpain, the dominant calcium-dependent protease in erythrocytes. Rather than an inflammatory cascade, this calcium-dependent calpain activity executes the degradation of {beta}-spectrin. These findings challenge current models of PNH hemolysis. We show that the destruction of PNH erythrocytes is a consequence of the MAC-calcium-calpain axis, rather than an inflammatory programmed cell death event. Consequently, therapeutic strategies aimed at targeting the inflammasome or caspase signaling will likely offer no clinical benefit for PNH patients.

LZTR1 negatively regulates RAS family proteins via proteasomal degradation. Germline loss-of-function variants cause Noonan syndrome, with emerging evidence implicating LZTR1 in predisposition to childhood acute lymphoblastic leukemia (ALL), though its role in hematopoiesis remains poorly defined. Screening 1,587 children with ALL identified LZTR1 variants in 44 patients (2.8%). Germline variants were detected in 32 patients (2.0%), a frequency comparable to that observed in the general population (1.75%; 1,925/110,017; p=0.50). Somatic LZTR1 alterations were identified in 22 patients (1.4%) and were predominantly bi-allelic, arising through either a germline-plus-somatic or dual somatic configuration. They persisted at relapse. Despite enrichment in favorable-risk subtypes (ETV6::RUNX1, high-hyperdiploid, ERG/DUX4), bi-allelic LZTR1-mutated cases showed delayed minimal residual disease clearance and higher late relapse risk, identifying a subgroup unsuitable for treatment de-escalation. LZTR1 expression was increased in most wild-type leukemias, consistent with a compensatory response to aberrant RAS pathway activation. Bi-allelic LZTR1 inactivation abolished RAS regulation, leading to deregulated canonical RAS expression and ectopic expression of the non-canonical RIT1 protein, whose involvement in ALL has not previously been reported. These findings establish LZTR1 as a classical tumor suppressor in ALL via a two-hit model. Monoallelic alterations show insufficient signaling perturbation and low germline penetrance, whereas bi-allelic inactivation acts as a driver event linked to a high risk of late relapse despite favorable genomics.

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

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

Clonal hematopoiesis (CH) is an age-associated condiion defined by over-representation of hematopoietic stem cells (HSCs) and their progeny carrying somatic mutations or variants that confer a selective advantage. CH is associated with increased risk of hematologic malignancies (1), cardiovascular disease and inflammatory bone loss (2, 3). Chronic inflammation is increasingly recognized as a central mediator of CH-mutant hematopoietic stem and progenitor cell (HSPC) expansion underlying CH (4). DNA methyltransferase 3a ( Dnmt3a )-mutant cells produce higher levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) (5), and blocking these pathways reduces the competitive advantage of Dnmt3a -mutant HSPCs (4, 6). The upstream mediators initiating inflammatory signaling in CH are unknown. Strong candidates are S100A8 and S100A9, members of the S100 calcium-binding protein family that regulate inflammatory signaling in the hematopoietic system. These proteins form a heterodimer complex and activate innate immune signaling through receptors including Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE) (7). S100A8/A9 signaling promotes production of pro-inflammatory cytokines and inflammasome activation leading to poor prognosis in myelodysplastic syndrome and myeloproliferative neoplasms (8, 9). Across multiple myeloid malignancies, neutrophils are the primary bone marrow (BM) source of this alarmin (8, 10, 11) and pharmacologic inhibition of S100A9 with tasquinimod reduces disease severity without disrupting normal hematopoiesis (10, 12). Given the role of S100A8/A9 in establishing an inflammatory milieu, here we investigated the role of S100A8/A9 in Dnmt3a -mutant hematopoiesis. We identify neutrophils as a major source of elevated S100A8/A9 in the BM of Dnmt3a -mutant mice and this increase correlates with production of the inflammatory cytokines TNFα and IL-6. We show that tasquinimod reduces TNFα and IL-6 levels and selectively reduces the Dnmt3a -mutant HSPC compartment.

Internal-tandem-duplication of the receptor tyrosine kinase FLT3 (FLT3-ITD) generates ligand-independent signaling and is highly recurrent in acute myeloid leukemias (AMLs). One way signaling pathways can quickly influence cell fates is by phosphorylating key fate-determining proteins to trigger their proteolysis. We investigated the master transcription factor (MTF) driver of granulo-monocytic lineage-fates, CEBPA, for regulation by this mechanism because we found high CEBPA mRNA but little CEBPA protein in FLT3-ITD versus FLT3-wildtype AML cells, and inhibiting FLT3-ITD signaling with tyrosine kinase inhibitors (TKI) rapidly rescued CEBPA protein. Mass spectrometry analyses of CEBPA and its interactome demonstrated prominent interactions with major ubiquitin-proteosome pathway (UPP) components UHRF1 and USP7. TKI treatments decreased CEBPA and USP7 phosphorylations at serine 21 and serine 18 respectively alongside shifts in CEBPA interactions from degradative ubiquitin-ligase UHRF1 toward protective deubiquitinase USP7. The rescued CEBPA activated granulocytic-differentiation. Supporting that the serine-phosphorylations were phospho-degrons, UPP-inhibitors (bortezomib, MG132) increased phosphorylated and total CEBPA and USP7. The MTF regulator of apoptosis p53 is a known USP7 client, therefore, we also evaluated p53 status: TKIs and UPP-inhibitors stabilized USP7 and p53, triggering apoptosis in addition to granulocytic-differentiation specifically in FLT3-ITD but not FLT3-wildtype AML cells. UPP-inhibitors produced these consequences in TKI-resistant FLT3-ITD AML cells also. These data predicted genetic loss-of-function to CEBPA or TP53 is redundant in the FLT3-ITD context, borne out by mutual exclusivity of the mutations in clinical series. In summary, FLT3-ITD signals for CEBPA and p53 proteolysis to block lineage-maturation and apoptosis, positioning UPP-inhibitors as therapeutic candidates acting downstream of TKIs. KEY POINTSO_LIThe oncoprotein kinase FLT3-ITD signals for CEBPA and p53 proteolysis and hence suppresses lineage-differentiation and apoptosis C_LIO_LIProteosome-inhibitors are candidate remedies to restore CEBPA and p53, acting downstream of presently used FLT3-ITD kinase inhibitors C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/738455v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@6ae211org.highwire.dtl.DTLVardef@12003bforg.highwire.dtl.DTLVardef@d62eb9org.highwire.dtl.DTLVardef@1958693_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myelodysplastic syndromes (MDS) are driven by somatic mutations in hematopoietic stem and progenitor cells (HSPCs), leading to clonal expansion and ineffective hematopoiesis. Hypomethylating agents (HMAs; azacitidine or decitabine) are the standard of care for higher-risk MDS. However, their effects on the bone marrow (BM) microenvironment, and the extent to which these changes correlate with clinical response, remain poorly understood. We performed longitudinal analyses of BM aspirates, trephine biopsies, and peripheral blood samples from MDS patients treated with azacitidine in a clinical trial (NCT03493646), integrating CyTOF, 5' single-cell RNA and TCR sequencing, plasma proteomics, and multiplex immunofluorescence microscopy to characterize changes associated with azacitidine response. Clinical responders showed expansion of GzmBCD56CD8 T cells together with increased type I and type II interferon signaling within the T-cell compartment. Responders also exhibited marked alterations in circulating platelet- and myeloid-derived factors with the potential to remodel the BM niche. Spatial analyses revealed expansion of neighborhoods enriched for CXCL12-abundant reticular cells and CD8 T cells in responders, whereas HSPC-enriched neighborhoods were largely unchanged. In contrast, several HSPC-enriched neighborhoods expanded in non-responders. These microenvironmental changes were accompanied by evidence of enhanced myelopoiesis in clinical responders. Our findings support a model in which azacitidine response extends beyond direct effects on malignant hematopoietic cells to involve coordinated remodeling of the BM microenvironment which may be reinforced by platelet- and myeloid-derived signals that establish a feed-forward circuit promoting productive hematopoiesis.

Targeting oxidative phosphorylation (OXPHOS) represents an attractive therapeutic strategy in acute myeloid leukemia, which exhibits exceptional dependence on mitochondrial respiration compared to normal hematopoietic cells. However, clinical attempts to exploit this vulnerability have been limited by on-target toxicity to healthy tissue. Here, we comprehensively compare the cellular consequences of inhibiting distinct nodes of the electron transport chain in AML. We demonstrate that selective inhibition of the F1 subunit of ATP synthase with EB2023 (ammocidin A) delivers an energetic stress to AML cells without the profound redox stress that characterizes complex I inhibition, preventing NAD/NADH imbalance and allowing continued TCA cycling. Further, the duration of OXPHOS inhibition is transient in nature in vivo, a finding revealed through pharmacokinetic and serial pharmacodynamic monitoring of AMPK phosphorylation accompanied by OPA1-mediated mitochondrial structural remodeling that primes AML cells for BCL2 inhibitor synergy. EB2023 in combination with venetoclax demonstrates potent anti-AML activity across cell lines and patient-derived xenograft models at doses that spare normal hematopoietic progenitors and avoid the neuropathy and sustained detrimental systemic metabolic rewiring in healthy tissues associated with prior efforts to target OXPHOS. These findings establish F1-selective ATP synthase inhibition as a clinically actionable therapeutic strategy in AML and establish the duration of OXPHOS inhibition as a critical and previously underappreciated determinant of therapeutic index.

Background CD19-directed CAR-T cell therapy can induce durable remissions in chronic lymphocytic leukemia (CLL), but response rates are lower than in other B-cell malignancies, in part because CLL is characterized by T-cell dysfunction, defective immune synapse formation, and impaired target-cell co-stimulation. Lenalidomide is an immunomodulatory drug with the potential to act on both sides of the CAR-T/CLL interface by improving T-cell fitness and modifying malignant B-cell susceptibility to immune engagement. Methods We are conducting an open-label, non-randomized phase I/II clinical trial VTB-CLL002 (ClinicalTrials.gov identifier: NCT06762431) evaluating the safety and efficacy of CD19 CAR-T cell therapy combined with concomitant lenalidomide in patients with relapsed or refractory CLL and small lymphocytic lymphoma followed by lenalidomide maintenance. The primary endpoint was safety. The secondary endpoint included overall response rate (ORR), complete response (CR), progression-free survival (PFS) and overall survival (OS). Results Twelve patients were treated. The median age was 60 years and the median number of prior lines of therapy was 2. All patients were BTK inhibitor-naive, and all had measurable disease at the time of infusion. CAR-T manufacturing was successful in all patients. All treated patients achieved complete remission, with a median time to response of 1 month. CAR T-cells expansion was observed in all patients, with a median peak expansion of 137 cells/L and a median time to peak expansion of 14 days. CAR T-cells remained detectable at the last follow-up in all patients, with persistence documented up to 24 months. At dose levels 2-3, eight of nine patients had ongoing MRD-negative responses at the time of analysis. Toxicity was clinically meaningful. Cytokine release syndrome (CRS) occurred in all patients, with severe CRS observed in 2 of 12 patients. ICANS occurred in 5 of 12 patients, including severe ICANS in 4 of 12 patients. One patient developed late grade 4 ICANS temporally associated with lenalidomide reintroduction and secondary CAR-T expansion. Early and late immune effector cell-associated hematotoxicity were common. In mechanistic studies, lenalidomide enhanced CAR-T proliferation and cytotoxicity, shifted CAR-T cells toward effector-associated phenotypes, reduced selected exhaustion markers during repeated antigen challenge, and increased IL-2 and IFN-{gamma} secretion. Lenalidomide also increased CAR-T/CLL conjugate formation and upregulated CD54/ICAM-1 on CLL target cells without broad induction of CD80, CD86, or CD40, consistent with improved adhesive target-cell engagement rather than classical co-stimulation. Transcriptomic profiling supported enhanced Th1/cytotoxic and T-cell activation-associated programs with lower T reg -associated genes in lenalidomide-treated CAR-T cells. Conclusions Lenalidomide-augmented CD19 CAR-T therapy demonstrated strong early clinical activity in relapsed/refractory CLL, characterized by deep responses, durable CAR-T persistence, and substantial incidence of immune effector-associated toxicities. These findings support further evaluation of lenalidomide as a rational CAR-T partner in CLL and suggest that its activity may involve both improved CAR-T fitness and enhanced target-cell engagement. Future studies should optimize lenalidomide timing and dosing to preserve response depth while reducing delayed immune-effector toxicity.

Transcription factor CEBPA is mutated in 10-15% of acute myeloid leukaemia (AML), a haematopoietic malignancy with high mortality. CEBPA mutations show a distinct pattern, and most patients are biallelic, carrying both an in frame C-terminal mutation and a frameshift N-terminal mutation on opposing alleles. Rare N-terminal germline cases have 100% penetrance to AML, all with an acquired a C-terminal mutation. This suggests a selective pressure from one CEBPA mutation to develop another. Our zebrafish models faithfully recapitulate the human disease. All biallelic mutant combinations die by 4-6 weeks of age, with pre-leukaemic haematopoietic stem cell (HSC) expansion. C-terminal and N-terminal mutants show phenotypic differences in myeloid primed HSC and differences in the myeloid differentiation block. RNA-Seq identified differentially expressed genes in opposing vectors. We identified phosphatase receptor PTPRJ as a candidate driver of clonal selection, with knock-out of ptprja in our fish accelerating pre leukaemic expansion of HSC in C-terminal mutants, decelerating it in N-terminal mutants. Cebpa mutant murine cells exhibit changes in differentiation and a clonal advantage with loss of Ptprj, which perturbs key signalling pathways. Our data suggest that PTPRJ contributes to the mechanism of leukaemogenesis in CEBPA mutant AML by driving the selective pressure from each mutation to develop the other.

Background: High ambient temperature in early pregnancy has been linked to an increased risk of childhood acute lymphoblastic leukemia (ALL). To better understand biological mechanisms, the current study evaluated potential interaction between temperature and genetic characteristics. Methods: We used data from California birth records (1982-2008) and California Cancer Registry (1988-2011) to identify ALL cases (n=3,353) diagnosed <=14 years of age and non-cancer controls (n=3,530) matched 1:1 on sex, race, ethnicity, and birth year and month. Weekly ambient temperatures throughout pregnancy were assessed on a 1-km grid around the birth address, while genetic data were available from a genome-wide association study using neonatal blood spots. We evaluated the association between ambient temperature and ALL risk by quartiles of established genetic risk score for ALL. Next, we formally tested gene-temperature interactions in the association with ALL, correcting for multiple testing, for genes previously identified with epigenetic changes due to both temperature and ALL. All analyses were adjusted for potential confounders. Results: The elevated risk of ALL per 5 degrees C increase of weekly mean ambient temperature, confined to early pregnancy, was more pronounced among children with the lowest genetic susceptibility to ALL, especially among Latino children (first quartile: odds ratio [OR] = 1.50, 95% confidence interval [CI]: 1.14-1.97); fourth quartile: OR=1.03, 95% CI: 0.83-1.28). There were significant interactions (p<0.002) between ambient temperature and polymorphisms in BNC1 among non-Latino White children, and suggestive interactions (p<0.05) with TBPL2 and NRXN1 in the full population. Conclusions: Our findings suggest that there may be interactions between ambient temperature in early pregnancy and offspring genotype in the risk of childhood ALL. Impact: If replicated, these findings could help elucidate the biological mechanisms linking high ambient temperature in early pregnancy and the risk of childhood ALL.

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

Background: Clonal hematopoiesis (CH) is associated with increased risks of diverse cardiovascular diseases, hematologic malignancies and mortality, yet no preventive therapies are approved. As emerging data implicate lipid pathways in CH pathogenesis, we investigated the association of statin use and genetically proxied inhibition of HMG-CoA reductase (HMGCR) with CH risk, and validated findings using primary peripheral blood mononuclear cells (PBMCs). Methods: We performed an observational analysis of 416,118 UK Biobank participants of European ancestry using multivariable logistic regression to compare CH prevalence among statin users and nonusers. Mendelian randomization (MR) analyses evaluated the causal association of genetically proxied lowering of low-density lipoprotein cholesterol (LDL-C) with risk of CH using two instruments; (i) the lead HMGCR variant (rs12916) which proxied LDL-C lowering by statins, and, (ii) 303 genome-wide LDL-C-lowering variants representing polygenic mechanisms. Summary statistics were obtained from the Global Lipid Genetics Consortium genome-wide association study (N = 842,634). Experimentally, primary PBMCs from a DNMT3AR882 hotspot mutation carrier were cultured in methylcellulose with pravastatin or vehicle control to evaluate colony-forming dynamics. Results: Among 416,118 individuals, 20,488 had CH, including 11,550 with single DNMT3A-mutant and 4,375 with single TET2-mutant CH. Pre-recruitment statin users had reduced odds of DNMT3A-mutant CH (OR=0.93; 95% CI:0.88-0.98; P=0.009), driven primarily by associations with DNMT3AR882-mutant (OR=0.78; 95% CI:0.66-0.92; P=0.003), but not TET2-mutant CH (OR=1.05; 95% CI:0.97-1.14; P=0.20). Similarly, genetically predicted HMG-CoA-reductase inhibition equivalent to a 1 SD reduction in circulating LDL-C levels was associated with lower odds of DNMT3A-mutant CH (OR=0.66; 95% CI:0.45-0.95; P=0.03) but not TET2-mutant CH (OR=1.34; 95% CI:0.76-2.36; P = 0.31). By contrast, polygenic estimation of LDL-C lowering was not associated with DNMT3A-mutant CH (OR=1.05; 95% CI:0.97-1.14; P=0.20), suggesting protective effects were independent of LDL-C lowering per se. Genetically predicted HMG-CoA reductase inhibition had wide effects on blood cell counts and indices, suggesting effects on bone marrow cell dynamics. In vitro, pravastatin selectively suppressed colony formation of primary human DNMT3AR882-mutant relative to wild-type cells (P=0.031). Conclusions: Statin therapy and genetically predicted lifelong inhibition of HMG-CoA reductase were significantly associated with reduced risk of DNMT3A-mutant CH, likely via LDL-C-independent mechanisms, which may be specific to DNMT3A-mutant CH. This provides a strong rationale for prospective trials evaluating the effect of statins on risk of developing DNMT3A-mutant CH, subsequent clonal expansion, and associated clinical sequelae.

In human acute myeloid leukemia (AML), a sub-population of leukemia stem cells (LSCs) drive disease initiation, therapeutic resistance, and relapse. However, the lack of reliable markers to distinguish LSCs from bulk leukemia cells has impeded progress in studying LSC pathogenesis and developing meaningful LSC-specific diagnostics and therapeutics. Existing LSC gene signatures, derived from bulk populations, cannot definitively identify LSCs at single-cell resolution. To address this, we analyzed large patient cohorts with bulk gene expression data and single-cell multi-omic assays to identify a prognostic gene signature that is specifically enriched in a clinically adverse AML sub-population. Using this signature, we defined and prospectively isolated CD34+CD90-CLL1-CD69+CD53- immunophenotypic LSCs that are significantly enriched for LSC content based on limiting dilution xenotransplantation assays. Our findings demonstrate the power of single-cell multi-omics to precisely identify a clinically relevant LSC population and establish a clear framework for future translational research in AML. Key PointsO_LISingle cell multi-omics identifies human AML LSCs at high resolution. C_LIO_LIHOPX and SOCS2 co-expression (hrLSC2) defines a prognostic gene signature in de novo acute myeloid leukemia. C_LIO_LIhrLSC2 marks an AML subpopulation (iLSCs) with a distinct immunophenotype. C_LIO_LIiLSCs can be purified using flow cytometry and are significantly enriched for LSCs. C_LI

Myelofibrosis (MF) originates from the stepwise acquisition of somatic mutations in Hematopoietic Stem and Progenitor Cells (HSPCs). Alongside driver events triggering JAK-STAT pathway hyperactivation, several additional mutations, usually affecting the epigenetic machinery, contribute defining therapeutic response. Specifically, JAK-inhibition (JAKi) relieves MF symptoms but rarely eradicates the neoplastic clone. To elucidate clonal dynamics associated with JAKi, we conducted a longitudinal single-cell proteogenomic study on 6 responders and 6 non-responders MF patients. Mutational analysis revealed that the mutation acquisition order determines JAKi sensitivity. Indeed, driver-only clones are highly sensitive to JAKi, while co-mutated clones persist after treatment. JAKi response is mainly limited to the differentiated myeloid compartment, while mutant HSPCs are often maintained in JAKi-responders. Co-mutated clones may evade JAKi and outcompete other neoplastic cell populations, thus contributing to disease persistence.

Background Graft versus host disease (GVHD) remains a major determinant of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (allo HSCT). Existing GVHD prediction models demonstrate modest discrimination and limited generalizability, and calibration drift across external populations is rarely characterized despite its essential role in the clinical interpretability of predicted probabilities. Objectives To develop and externally validate an explainable machine learning framework for predicting acute and chronic GVHD and associated overall survival in patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndromes (MDS) undergoing allo HSCT, and to systematically characterize calibration across heterogeneous external validation cohorts to inform deployment requirements. Study Design The model was developed on three publicly available registry-derived datasets (N = 2,509) and externally validated across six independent cohorts (N = 14,788) comprising adult and pediatric allo HSCT recipients, including a regional Middle Eastern cohort (UAE and Jordan). A standardized preprocessing pipeline harmonized heterogeneous datasets. Gradient boosting models (CatBoost) were used for binary GVHD prediction; exploratory overall survival analysis used a Cox proportional hazards model with predicted acute GVHD risk as a covariate. Discrimination (AUROC with bootstrap 95% CI), calibration (logistic recalibration intercept and slope with analytical 95% CI), and feature importance (SHapley Additive exPlanations, SHAP) were assessed in training out-of-fold and all external cohorts. Results In internal validation, AUROC was 0.63 (95% CI 0.61-0.65) for acute GVHD and 0.72 (95% CI 0.70-0.74) for chronic GVHD. External validation demonstrated AUROC ranges of 0.51-0.57 (acute) and 0.54-0.64 (chronic), with consistent performance across disease subgroups despite substantial heterogeneity in transplant practices and feature availability. In exploratory survival analysis, the acute-GVHD-informed Cox model achieved a training-cohort C-index of 0.679 (95% CI 0.658-0.697); external C-indices ranged from 0.47-0.53. Calibration analysis identified systematic external risk overestimation (negative calibration intercept in 10 of 11 evaluable external cohort-target combinations) with heterogeneous slope drift requiring cohort-specific recalibration. Key predictors included recipient age, graft source, conditioning intensity, GVHD prophylaxis, and HLA match ratio. Conclusions An explainable, externally validated GVHD prediction framework was developed using heterogeneous registry-derived datasets, with systematic characterization of calibration drift across multiple external cohorts, an analysis rarely reported in prior GVHD prediction literature. Predictive performance was modest for acute GVHD and moderate for chronic GVHD, constrained by missing immunobiological variables and incomplete HLA characterization. Per-cohort recalibration is required before clinical deployment, with prospective validation and benchmarking against established GVHD risk scores identified as priority next steps.

While therapy resistance in acute myeloid leukemia (AML) is often attributed to leukemic stem cells (LSCs), their functional properties are not fully captured by their well-established genetic landscape and cell lineage transcriptional programs. Here, we explore AML cell states and their associations to drug response and systemic immune context. We performed integrated single-cell transcriptomics and immunophenotyping on diagnostic AML samples (n=6) to define transcriptional cell state gene signatures. These were projected onto bulk RNA-seq data from 448 AML patients to assess associations with drug sensitivity, plasma proteomics, clinical features, and established prognostic scores. Longitudinal single-cell data from external cohorts and cell-cell communication analyses were used to examine treatment dynamics and microenvironmental signaling. We defined nine AML cell states, including progenitor-like, stromal-like, antigen-presenting, and monocytic programs. Stemness features were distributed across multiple states, with lymphoid-primed and stress-adapted progenitors showing the strongest alignment with established stemness scores. Distinct drug sensitivities emerged, including cell cycle checkpoint inhibitor sensitivity in stress-adapted progenitors and kinase inhibitor sensitivity in cycling progenitors, alongside shared resistance to BH3 mimetics in monocytic states. Stress-adapted progenitors were associated with adverse clinical features and expanded following venetoclax-based therapy. Monocytic states acted as immunosuppressive hubs via TIGIT signaling, while stromal-associated states received niche-derived survival signals. Overall, we define a framework that associates AML cell states with stemness, drug response, and microenvironmental interactions. These findings highlight distributed stemness, state-specific vulnerabilities, and niche-driven resistance mechanisms, informing more precise therapeutic strategies in AML.

Transcriptional condensates anchored by chromatin readers are increasingly recognized as organizing hubs for gene expression, but how their assembly and stability are regulated remains poorly understood. Here, we identify an acetylation-dependent feed-forward circuit that controls the integrity of the Super Elongation Complex (SEC), a key driver of transcriptional elongation. We show that the SAGA histone acetyltransferase catalytic subunits KAT2A/KAT2B license acetylation of both histone H3 lysine 9 (H3K9ac) and SEC components themselves, including ENL, AFF1, and AFF3. Loss of this dual acetylation activity, achieved via a cereblon-recruiting PROTAC (GSK983/GSK699), displaces the chromatin reader ENL from target loci, dissolves ENL-anchored transcriptional condensates, and disrupts SEC-dependent transcriptional output - linking histone and non-histone acetylation to the physical integrity of a core transcriptional machine. Using genome-scale dependency data, we show that the SAGA complex is a selective chromatin dependency in acute myeloid leukemia (AML) AML and hematological malignancies and disrupting this feed-forward transcriptional circuit in AML demonstrates subtype independent antileukemia effects. KAT2A/B degradation drives potent, broad-spectrum antileukemic activity across genetically diverse AML cell lines, primary patient samples, and an isogenic KMT2A-rearranged model bearing cooperating oncogenic mutations, with H3K9ac loss concentrated asymmetrically at core AML oncogene loci such as MYC, MYB, and the HOXA cluster. Together, these findings define an acetylation-dependent circuit governing SEC integrity and establish KAT2A/B degradation as a mechanism-based, pan-AML therapeutic strategy, with implications for transcriptional condensate regulation beyond leukemia. HIGHLIGHTSO_LIThe SAGA complex is a selectively essential chromatin dependency across hematological malignancies and particularly in AML C_LIO_LIKAT2A/B degradation drives broad anti-leukemic activity across genetically diverse AML subtypes including chemo-refractory disease C_LIO_LIKAT2A/B degradation depletes H3K9ac at AML oncogene loci and dismantles ENL-anchored condensates C_LIO_LIKAT2A/B licenses regulation of super elongation complex acetylation and ENL interaction with SEC complex components C_LI

Thrombotic primary antiphospholipid syndrome (thrPAPS) outcomes are associated with thrombosis type (arterial versus venous), recurrence, and antiphospholipid antibody (aPL) profile (single versus triple-aPL). We investigated molecular signatures underlying disease status and high-risk phenotypes. We performed whole-blood transcriptomics and mass spectrometry-based plasma proteomics in patients with thrPAPS and age/sex-matched healthy controls. Analyses included differential expression, pathway enrichment, weighted gene co-expression network analysis (WGCNA) and machine learning. Multi-Omics Factor Analysis (MOFA2) and network propagation were applied to identify latent molecular programmes associated with high-risk phenotypes. Transcriptomic and WGCNA analyses revealed an interferon-associated module associated with high-risk phenotypes. Plasma proteomics distinguished thrPAPS from healthy controls through a coordinated thromboinflammatory signature encompassing complement, acute-phase, platelet, and coagulation-associated pathways. Complement factor D, a rate-limiting enzyme of the alternative complement pathway, discriminated recurrent from single-event thrPAPS (AUC = 0.79) and correlated with thrombotic event count (Spearman's correlation = 0.62, p < 0.001). Mixed arterial/venous phenotype showed the greatest degree of subgroup-specific dysregulation, including complement and coagulation/fibrinolysis-related proteins. MOFA2 identified a proteome-dominant latent factor that increased with aPL burden (Spearman's correlation = 0.33, p = 0.017) and was enriched for complement cascade proteins. Network propagation embedded this signature within immune-cell signalling (STAT-1, PI3K-AKT, MAPK8, SRC), N-linked glycosylation, and mitochondrial oxidative phosphorylation. Longitudinal profiling identified reactive oxygen species-associated proteins during active disease. ThrPAPS is characterised by a complement-, interferon- and platelet-driven thromboinflammatory programme that scales with aPL and thrombosis burden, converging on innate immune activation as a central feature of high-risk disease.