Haematologica

The non-receptor protein-tyrosine phosphatases Shp1 (PTPN6) and Shp2 (PTPN11) play critical roles in hematopoietic signaling networks, yet their specific functions in human megakaryopoiesis and thrombopoiesis remain incompletely understood. While Shp2 is recognized in murine models as a positive regulator of thrombopoietin (Tpo)-mediated signaling through the Ras/MAPK and PI3K/AKT pathways, Shp1 has been implicated in RhoA-dependent cytoskeletal remodeling. However, the extent to which these roles translate to human megakaryocyte (MK) development and platelet production is not known. In this study, we systematically investigated the contributions of Shp1 and Shp2 to human MK development and function using CRISPR/Cas9-mediated gene deletion of PTPN6 and PTPN11 in CD34+ hematopoietic stem and progenitor cells (HSPCs), combined with pharmacological inhibition of Shp2 using the structurally-distinct allosteric inhibitors SHP099 and RMC-4550. Efficient gene editing of PTPN6 and PTPN11 resulted in efficient ablation of Shp1 and Shp2 in CD34+ HSPC-derived MKs. Genetic deletion or pharmacological inhibition of Shp2 markedly impaired MK proliferation, polyploidization, maturation, and proplatelet formation, whereas loss of Shp1 expression did not. Further, Shp2 inhibition significantly reduced platelet production in a 3-dimensional human bone marrow tissue model. Deletion and inhibition of Shp2 abrogated Tpo-induced ERK1/2 and AKT phosphorylation, confirming its essential role in Mpl receptor signaling. These findings demonstrate the distinct functional roles of Shp1 and Shp2 in MKs and establish Shp2 as a critical positive regulator of Mpl- mediated megakaryopoiesis and thrombopoiesis. Key PointsO_LIEfficient deletion of Shp1 and Shp2 in human CD34 progenitor cell-derived MKs using CRISPR/Cas9. C_LIO_LILoss of Shp2 expression impairs thrombopoietin-induced human MK maturation, proplatelet formation and Mpl signaling. C_LI

Wiskott-Aldrich syndrome (WAS) is an inborn error of immunity with a broad disease spectrum, classified into class I (late onset) or II (early onset) variants. Thrombocytopenia and small platelets are the most consistent findings among patients, difficult to treat and related to development of autoimmunity. To identify new treatment options for thrombocytopenia in WAS, we developed a FACS-based screening for drug repurposing. We identified parthenolide as a lead small molecule that increased WASp abundance in cells with residual WASp expression. Using the megakaryocytic MEG-01 cells, gene edited to express WAS class I and II genetic variants, parthenolide induced megakaryocyte maturation as evident by upregulation of CD61, increased cell size and complexity, increased phosphorylation of ERK1/2, and higher DNA ploidy. We generated a new mouse model harboring a WAS class I missense variant WASp-R88C, corresponding to human WASp-R86C, with reduced expression of WASp. WASp-R88C mice had lower numbers of platelets compared to WT mice. Bone marrow-derived WASp-R88C and WASp-KO megakaryocytes, differentiated in the presence of parthenolide derivative, DMAPT, showed increased ploidy and upregulation of the maturation markers CD61, CD41 and CD42d, resulting in improved platelet production in vitro. Treatment with parthenolide derivative, DMAPT, led to increased platelet numbers in vivo in WASp-R88C mice and dampened the hyperactivation of WAS patient platelets by reducing thrombin-induced CD62P exposure after activation. The identification of parthenolide offers a promising therapeutic approach for WAS patients who are unresponsive or unsuitable for definitive therapies.

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.

Type 2B von Willebrand disease (VWD) is characterized by missense variants in exon 28 of the von Willebrand factor (VWF) gene that encodes the VWF-A1 domain. These missense mutations result in single amino acid residue substitutions that cause activation of VWF-A1 and promote spontaneous interaction with platelet GPIba;. Importantly, enhanced VWF clearance has been shown to play a key pathogenic role in >90% patients with Type 2B VWD. Although this VWF clearance occurs via mechanisms that are independent of VWF-platelet complex formation, the biological mechanisms responsible for the increased clearance of type 2B VWD variants remain poorly understood. In this study, we investigated a series of different type 2B amino acid substitutions within the VWF-A1 domain (R1306W, R1308C, W1313C and R1379L). We demonstrate that these variants all exhibit significantly increased macrophage binding. In part, the enhanced macrophage interactions are mediated via increased binding of type 2B VWD variants to LRP1 extracellular cluster II and IV. Our findings further demonstrate that the K1405-K1408 lysine cluster in the VWF-A1 domain plays a key role in enabling enhanced LRP1 interactions for type 2B VWD variants. In addition, we show that type 2B VWF variants demonstrate significantly enhanced interaction with the macrophage MGL receptor. Finally, and importantly from a clinical perspective, we demonstrate that the increase in macrophage binding for type 2B variants is significantly attenuated in the presence of BT200. Collectively, our findings have direct translational relevance with respect to the clinical heterogeneity and treatment of type 2B VWD.

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.

Haplo-identical hematopoietic cell transplantation (haploHCT) is an integral treatment paradigm for patients with leukemia. While overall survival (OS) post-haploHCT has steadily improved, relapse-free survival (RFS) remains relatively stagnant. Upon the discovery of killer immunoglobulin-like receptors (KIRs) on natural killer (NK) cells and their cognate human leukocyte antigen (HLA) ligands, algorithms have been developed to enhance graft versus leukemia effects. However, these algorithms fail to yield consistent predictions in patient outcomes. We utilized a combination of in silico protein folding and interactions to determine KIR:HLA reactivity in conjunction with in vitro acoustic force microscopy to measure cell avidity (CA) as a readout for KIR signal strength. CA was determined using monoallelic HLA expressing K562 cell lines, monoallelic KIR Jurkat cells, and peripheral blood NK cells. We extended the CA results and performed standard cytotoxicity assays as well. We discovered that HLA-B*35 interacts with KIR2DS4. We applied the newly discovered interaction to predict outcomes for HCT patients. Stratifying patients based on their HLA-B*35 positivity and donor KIR2DS4 status, we delineated a correlation to survival (P=0.061) when donors only had full-length KIR2DS4. Patients who received a haploHCT and NK cell addback from donors with only full-length KIR2DS4 had a significantly improved RFS (P=0.001) and OS (P=0.016) compared to truncated (KIR1D) and full-length KIR2DS4 donors. This was independently validated in a diverse 10/10 HLA matched European cohort with RFS (P=0.0255) and OS (P=0.0388). Thus, the identified novel KIR2DS4:HLA-B*35 interaction axis predicts patient survival, in both haplo-identical and fully matched, HCT and highlights that our current understanding of the KIR:HLA interactome is incomplete and requires remapping for enhanced therapeutic applications.

Purpose: Adults with high- or very high-risk acute leukemia (AL) or myelodysplastic syndrome (MDS) face substantial relapse risk after allogeneic hematopoietic stem-cell transplantation. We evaluated single-unit cord blood (CB) transplantation after ex vivo expansion with UM171 in this population. Patients and Methods: Two prospective, single-arm phase II trials at four centers enrolled 64 adults with high- or very high-risk AL or MDS; 60 received a UM171-expanded CB transplant and comprised the analysis population. CB units were preferentially selected at a 5/8 HLA match to maximize the graft versus leukemia effect. Patients received intermediate- or high-intensity conditioning with tacrolimus/mycophenolate mofetil graft-versus-host-disease (GVHD) prophylaxis. Endpoints included safety, feasibility, non-relapse mortality (NRM), relapse-free survival (RFS), overall survival (OS), GVHD, GVHD-free relapse-free survival (GRFS), chronic GVHD-free relapse free survival (CRFS). Results: Thirty-two percent of patients had undergone previous transplantation, 17% of patients with AL were not in remission and 24% of those with AML/MDS had TP53 mutations. Of 62 patients who remained eligible for transplantation, 60 had a graft successfully manufactured and infused. Median times to neutrophil and platelet engraftment were 17 and 38 days, respectively. NRM was 5.1% at day 100 and 15.2% at 1 year. Two-year cumulative incidence of relapse was 22.3%. Two-year OS and RFS were 63.9% and 60.4%, respectively. Grade III-IV acute GVHD incidence was 20.3% at 1 year and moderate-to-severe chronic GVHD incidence was 6.8% at 2 years. Conclusion: UM171-expanded CB transplantation was feasible and provided prompt engraftment, durable disease control, and infrequent clinically significant chronic GVHD in adults with high- and very high-risk AL/MDS. Comparative studies are warranted to define its role relative to contemporary donor platforms.

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

Immune thrombocytopenia purpura (ITP) is an autoimmune disorder characterized by a reduction in circulating platelet levels, primarily due to generation of autoantibodies to platelet surface antigens followed by their spleen macrophage-mediated clearance. Emerging evidence implicates neuraminidase (sialidase) enzymes including neuraminidase 1 (NEU1) in platelet clearance and ITP severity; however, the underlying cellular mechanisms remain unknown. Using tissue-specific NEU1 knockout mouse models, we studied the contribution of platelet and macrophage NEU1 to ITP pathogenesis and evaluated whether pharmacological inhibition of NEU1 could preserve platelet counts in a murine ITP model. Constitutive and macrophage-specific, but not platelet-specific, NEU1 knockout mice showed a protection against reduction of platelet counts in the passive ITP model suggesting that macrophage, but not platelet, NEU1 promotes platelet clearance. Genetic deletion or pharmacological blockade of macrophage NEU1 also reduced platelet phagocytosis by cultured macrophages in vitro. The selective NEU1 inhibitor CG33301 protected mice against anti-CD41a antibody-induced thrombocytopenia and showed a higher efficacy compared to pan neuraminidase inhibitor oseltamivir phosphate. Our results demonstrate that the macrophage pool of NEU1 plays a central role in platelet clearance by splenocytes during ITP by activating their phagocytosis and suggest that selective NEU1 inhibition may be a promising therapeutic strategy for this disease.

ERCC6L2 disease (ED) is an inherited bone marrow failure (BMF) syndrome that progresses almost exclusively to erythroid, TP53-mutated myeloid malignancy, but the somatic evolution underlying the progression in patients is unknown. We characterized the genetic landscape of 29 ED patients with longitudinal sampling, integrating whole-genome and single-cell RNA sequencing, and compared the findings with Shwachman-Diamond syndrome (SDS), another TP53 mutation-prone disease, to define somatic evolution across disease stages. ED was defined by early, intense, and recurrent selection of TP53-mutant clones: TP53 mutations were more frequent, more often multiple, and showed steeper age-related expansion than in SDS; all TP53-negative ED patients were children. The burden of multiple TP53 mutations exceeded that of sporadic TP53-mutated myeloid neoplasia and was evident before malignancy. Missense mutations dominated the BMF phase, whereas non-missense mutations were seen in patients with malignancy, consistent with biallelic second hits, marking a stage-dependent shift in p53 inactivation. Beyond TP53, the landscape was strikingly restricted, with PPM1D as the only recurrent driver gene. Notably, despite the DNA-repair function of ERCC6L2, no ED-specific mutational signature emerged; but instead reflected age-associated processes. Malignant progression was accompanied by a complex karyotype, recurrent chromosome 5, 7, and 12p losses enriched in erythroid progenitors, and kataegis, consistent with branched, erythroid-predominant clonal evolution. Our findings establish ED as a paradigm of preleukemic dependency on p53-pathway attenuation. As progression is driven predominantly by p53 and captured longitudinally from the earliest clones, ED offers a model of how TP53-mutated leukemia arises and evolves, with relevance beyond a rare syndrome.

Acquired aplastic anaemia is caused by immune-mediated loss of haematopoietic stem and progenitor cells (HSPCs), but the regulatory states that sustain cytotoxic immunity and their relationship to inherited susceptibility remain incompletely understood. We integrated two single-cell RNA-sequencing cohorts spanning healthy, non-severe and severe aplastic anaemia with single-cell chromatin accessibility profiling, genome-wide association meta-analysis, Bayesian fine-mapping and stratified LD-score regression. Single-cell transcriptomics revealed a coordinated shift across the immune and haematopoietic compartments. Cytotoxic CD8 and {gamma}{delta} T cells converged on a shared NKG7/CCL5/PRF1 effector program, indicating that cytotoxic differentiation extends across T-cell lineages. Effector-memory T cells combined inflammatory signalling with SOCS, DUSP, TNFAIP3, RGS1 and TOX, consistent with sustained stimulation accompanied by extensive feedback regulation. With increasing disease severity, these inflammatory states were further coupled to hypoxic, oxidative and unfolded-protein-response programmes, suggesting qualitative remodeling of the immune compartment rather than uniform amplification of perforin-granzyme expression. Single-cell chromatin accessibility provided a regulatory counterpart to these transcriptional states. Naive and memory-associated cells retained TCF7/LEF1/BACH2 accessibility, whereas cytotoxic cells acquired coordinated accessibility across CCL5, NKG7, PRF1, granzymes and killer-receptor loci. Pseudotime, motif activity and integrated RNA-chromatin profiles positioned AP-1, NFAT and TBX21 along this transition, linking loss of memory-associated regulation to acquisition of cytotoxic effector competence. Genetic meta-analysis independently recovered association at the HLA-B region, reinforcing antigen presentation as the principal inherited susceptibility axis. Fine-mapping additionally prioritized a non-HLA locus without resolving its effector gene, while stratified LD-score regression found no detectable preferential enrichment of common-variant heritability within effector-memory or cytotoxic regulatory elements. Integrated with the cellular data, these findings support a mechanistic hierarchy in which HLA-linked antigen presentation establishes the selective context, persistent cytotoxic T-cell state remodeling maintains pathogenic immune pressure, and IFN{gamma}-responsive HSPC suppression translates this pressure into haematopoietic failure.

IDH inhibitors promote differentiation and hematological improvement in myeloid malignancies by reversing epigenetic dysregulation. However, molecular predictors of response remain poorly defined. We retrospectively analyzed 79 patients with IDH mutated myeloid malignancies treated with either ivosidenib (IDH1; n=36) or enasidenib (IDH2; n=43). Hematologic benefit was assessed using a composite complete hematologic response (CHR), defined by neutrophil and platelet recovery, and transfusion independence. Targeted sequencing was performed at baseline and at best hematologic response. Associations between co mutations, hematologic response, and treatment duration were evaluated using multivariable models. Overall, 43% of patients achieved CHR, with similar rates between inhibitors. Ivosidenib treated patients achieving CHR demonstrated a significant reduction in IDH1 VAF (p=0.010), which was not observed in enasidenib-treated CHR patients. Within the CHR cohort, ivosidenib patients had a greater reduction in IDH VAF relative to enasidenib patients (mean VAF change -16.74% vs -0.05%, p=0.034). Ivosidenib responders demonstrated a trend toward more durable treatment duration compared with enasidenib responders (p = 0.0908). RUNX1 and BCOR mutations were significantly enriched among patients with incomplete hematologic response and were independently associated with shorter treatment duration after adjustment for clinical factors (RUNX1: HR 2.53, p=0.0024; BCOR: HR 2.31, p=0.0058). IDH1 inhibition was associated with clonal contraction and durable hematologic benefit, a pattern not observed with IDH2 inhibition. Co mutations in RUNX1 and BCOR identified patients unlikely to achieve sustained hematologic improvement, suggesting a secondary block to differentiation. These findings support the integration of mutational and molecular profiling to refine patient selection and guide combinatorial strategies in IDH mutated myeloid malignancies.

The genetic risk factors of immune cytopenias are unclear. Immune cytopenias have been reported in various genetic contexts: 1) inherited error of immunity genes, mainly due to rare germline variants, 2) systemic lupus erythematosus, associated with common germline variants, 3) hematological malignancies, and 4) clonal hematopoiesis, the latter two due to somatic variants. However, the respective contribution and interaction of these variants remain to be investigated. Here, we used two large biobanks with whole genome sequencing data to systematically investigate the genetic contribution to immune cytopenia. We found that the four types of genetic variants independently contribute to immune cytopenia risk. We notably found that carriers of variants in some autosomal recessive genes of inherited error of immunity had an increased risk of immune cytopenia. Additionally, common variant-mediated risk of systemic lupus erythematosus also increased the risk of immune cytopenia. Overall, a third to a half of patients with immune cytopenia carried at least one of the four genetic risk variants investigated. Combining the four variants allowed stratifying the risk of immune cytopenia in both general and high-risk population. In general population, the 10-year incidence of immune cytopenia in the lowest and highest risk groups was 0.08% and 1.5%, respectively. In sum, this work identified that different genetic risk factors can lead to immune cytopenia. A large proportion of individuals with immune cytopenia carried an underlying genetic risk factor. Finally, combining these genetic risk factors enabled risk stratification.

Commitment events to specific blood lineages arise from single hematopoietic stem cells (HSCs) and are influenced by stress, inflammation and disease. However, the understanding of how such events are regulated in human haematopoiesis is limited by the lack of tractable in vitro models. In this study, we introduce a novel Early Progenitor Differentiation (EPD) assay to study the initial lineage commitment of human 49+ HSCs, in a system faithfully recapitulating cell states observed in vivo. Combining single cell -omics approaches and single cell functional assays, we show that IL-33 acts directly on human 49f+ HSCs activating the MAPK pathway to enhance their commitment towards Megakaryocytic-Erythroid-Mast cell Progenitors and subsequently megakaryopoiesis. This occurs without affecting HSC self-renewal via accelerated establishment of chromatin programmes associated with Erythroid and Megakaryocyte and mast cells lineages. Our findings demonstrate the utility of the EPD model to identify molecular regulators of human HSC differentiation and uncover a new role of IL-33 in haematopoiesis.

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.

Mechanical circulatory support essential for managing severe heart failure frequently triggers bleeding complications, driven by shear stress-induced over-proteolysis of von Willebrand factor (VWF) by ADAMTS13. Inhibiting ADAMTS13 presents a rationale to treat this condition, known as acquired von Willebrand syndrome (AVWS). However, conventional therapeutic strategies remain limited due to the risk of triggering thrombotic thrombocytopenic purpura. Here we show that HA10, a humanized anti-ADAMTS13 antibody, preserves a residual level of ADAMTS13 activity above the thrombosis-associated threshold. Multimodal structural and biophysical analyses--including NMR, SAXS, and SANS--revealed that HA10 bound to the disintegrin-like domain of ADAMTS13, dynamically competing with VWF while leaving 10- 20% residual enzymatic activity. The therapeutic efficacy and safety of HA10 were verified in non-human primate models of AVWS. Our findings establish a novel paradigm of enzymatic calibration rather than complete blockade, offering a mechanistically targeted and safe therapeutic approach for cardiovascular bleeding.

Background: Translocation t(11;14) defines a biologically distinct subset of multiple myeloma (MM) enriched for BCL2 dependency. Treatment with venetoclax, selective BCL2 inhibitor, has shown varied responses in clinical trials and retrospective cohorts. Efficacy of venetoclax-based combination therapies and optimal timing of treatment in t(11;14) MM patients remain incompletely characterized. Methods: We have compared the overall survival of t(11;14) MM patients who received Venetoclax (N = 97) at any moment in time, to those who never did (N =284), in the largest retrospective cohort (N = 381) reported to date. We used longitudinal fluorescence in situ hybridization (FISH) to assess cytogenetic evolution and genomic complexity. We performed transcriptomic profiling of CD138 enriched tumors using RNA sequencing in a subset of samples and gene expression signatures (UAMS/HALLMARKS) were used to define molecular subtypes. Ex vivo drug sensitivity assays integrated with paired RNA sequencing were used to identify subtype specific therapeutic vulnerabilities and rational venetoclax based combination strategies. Results: Venetoclax exposure was associated with an improvement of median overall survival by nearly four years compared to non VEN exposed patients (p = 0.0003). Longitudinal cytogenetic analysis demonstrated stability of the primary t(11;14) translocation over time, while secondary abnormalities tend to accumulate, including those harboring high-risk secondary cytogenetic abnormalities such as del13q, amp/gain1q21, del17p, and del1p, resulting in increasing genomic complexity with disease progression. Transcriptomic analyses identified selective enrichment of CD1/CD2 signature (associated with t(11;14) NDMM) by single sample gene set enrichment analysis as a marker of prolonged progression free survival. However, patients with more than five prior lines of therapy were enriched for transcriptionally complex biology characterized by CD1/CD2 with either proliferative/hypermetabolic or inflammatory transcriptional programming which were associated with inferior outcomes with VEN based treatment. Ex vivo drug sensitivity profiling revealed subtype specific vulnerabilities, identifying daratumumab, lenalidomide, ixazomib, and panobinostat as rational partners for venetoclax depending on transcriptional context. Conclusion: Venetoclax-based therapy significantly improved overall survival in t(11;14) MM. Clinical benefit, defined by improved progression free survival, was greatest when venetoclax was administered earlier, preceding the emergence of transcriptomic reprogramming that reduces BCL2 dependency. These findings support transcriptomic biomarker guided, subtype specific venetoclax based treatment strategies to optimize outcomes in patients with t(11;14) MM.