Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultra-pure human RBCs, we generated the deepest contamination-free proteome to date (3,775 prote...Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultra-pure human RBCs, we generated the deepest contamination-free proteome to date (3,775 proteins) and mapped the oxygen-dependent interactome. These datasets reveal an oxygen-responsive metabolon centered on the Band 3 (SLC4A1) N-terminus. We identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that dissociates under hypoxia, coincident with increased Band 3-deoxyhemoglobin contacts. This reversible assembly functions as an oxygen-sensitive switch coordinating redox and glycolytic remodeling. Humanized mice lacking Band 3 N-terminal segments exhibit impaired oxygen-dependent regulation of BLVRB binding to band 3, impaired hypoxic activation of glycolysis, reduced 2,3-bisphosphoglycerate synthesis, and diminished exercise tolerance, demonstrating physiological relevance. Population-scale cis-pQTLs for SLC4A1 and BLVRB suggest functions beyond canonical heme catabolism. Mechanistically, biochemical analyses in vitro suggest that hemoglobin β (HBB), Band 3, and BLVRB can undergo S-nitrosation and may participate in trans-nitrosation reactions with the glycolytic enzyme GAPDH, whose modification at C152 inhibits enzymatic activity in vitro. Collectively, these findings define a Band 3-BLVRB axis that integrates oxygen-dependent protein interactions with thiol-based redox chemistry, providing a framework for understanding how an anucleate cell achieves metabolic adaptability through reversible protein-protein interactions and post-translational modification. These findings suggest that perturbation of the Band 3-BLVRB axis may influence oxygen delivery and metabolic flexibility during hypoxic stress, with potential relevance to high-altitude adaptation, exercise physiology, and cardiopulmonary disease.
Renin-angiotensin-aldosterone system inhibitors (RAASi) are suggested for treating albuminuria in patients with sickle cell disease (SCD). RAASi may exacerbate anemia in the general and diabetic population through unclea...Renin-angiotensin-aldosterone system inhibitors (RAASi) are suggested for treating albuminuria in patients with sickle cell disease (SCD). RAASi may exacerbate anemia in the general and diabetic population through unclear mechanisms. The impact of RAASi on anemia in chronic hemolytic disorders, such as SCD, is unknown. In a cross-sectional analysis of 658 Walk-PHaSST participants, RAASi use was independently associated with lower hemoglobin concentrations adjusting for age, sex, SCD genotype, estimated glomerular filtration rate, and erythroid-stimulating agent and hydroxyurea use (β -0.46±0.21; P=0.032). In two longitudinal cohorts (UIC, n=24; multicenter losartan clinical trial, n=32), RAASi therapy led to a reduction in hemoglobin concentrations compared with pre-treatment values by -0.44±0.14g/dL (P=0.006) and -0.53±0.17g/dL (P=0.005), respectively. SCD mice treated with losartan demonstrated lower hemoglobin concentrations after 6- and 14-weeks (P<0.001) and lower absolute reticulocyte counts by 14-weeks (P=0.03) versus control mice without changes in circulating erythropoietin, IL-12p70, IL-3, or IGF-1 levels. Bone marrow cells from losartan-treated SCD mice had lower colony forming units (P≤0.09) with rescue of erythroid colony formation after exogenous erythropoietin supplementation (P=0.02). Bone marrow histopathology demonstrated reduced erythroid relative to myeloid ratios in losartan-treated versus untreated SCD mice. Hemoglobin levels should be closely monitored when using RAASi in this high-risk population.
Successful transplantation of autologous gene-modified hematopoietic stem/progenitor cells (HSPCs) requires efficient ablation of resident hematopoietic stem cells. Since conventional myeloablative conditioning regimens...Successful transplantation of autologous gene-modified hematopoietic stem/progenitor cells (HSPCs) requires efficient ablation of resident hematopoietic stem cells. Since conventional myeloablative conditioning regimens are associated with non-hematologic toxicities, we evaluated CD45-directed radioimmunotherapy (RIT) using the a-emitter astatine-211 (211At) before transplantation of ex vivo gene-edited autologous HSPCs as an alternative in nonhuman primates. We humanized the CD45 antibody, BC8 (HuBC8), and labeled it with 211At. As a model, mobilized CD34+ HSPCs were multiplex gene-edited using an adenine base editor, modifying the HBG promoter to reactivate fetal hemoglobin (HbF) and deleting CD33. Two animals each received 300 or 400 µCi/kg of 211At. In contrast to historic controls conditioned with total body irradiation, CD45-RIT animals did not show any noticeable non-hematopoietic toxicities and were almost entirely transfusion independent with rapid recovery of neutrophils and platelets. 211At enabled dose-dependent engraftment of gene-edited cells. A new single cell sequencing assay revealed up to 70% combined mono- and bi-allelic gene-editing efficiency in the blood, consistent with complete replacement of the bone marrow stem cell compartment. Assessment by bulk analysis underestimated the frequency of gene-edited cells, highlighting the importance of a single cell readout. Single cell sequencing further confirmed stable and unbiased contribution of multiplex-edited HSPCs to all mature lineages in the blood, providing high-resolution data assuring successful replacement upon autologous HSPC gene therapy. Levels of edited cells remained stable for the entire follow-up of >18 months. Together, these studies identify 211At-CD45 RIT as a targeted alternative for myeloablative conditioning for autologous gene therapy.
Polycomb repressive complexes PRC1 and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin production, a process whose reversal is a goal for the treatment of sickle cell dise...Polycomb repressive complexes PRC1 and PRC2 regulate diverse developmental processes, including the fetal-to-adult switch in hemoglobin production, a process whose reversal is a goal for the treatment of sickle cell disease and b-thalassemia. PRC inhibitors show promise for various disorders, but use is limited because of pleiotropic PRC activities. We explored whether fetal hemoglobin (HbF) can be reactivated in adult erythroid cells by selective perturbations of PRC1 or PRC2 components without complete loss of PRC function. A high-density CRISPR-Cas9 mutagenesis screen identified a region in the EZH2 subunit where Cas9 induced exon 14 skipping (EZH2D14). EZH2D14, which lacks a portion of the CXC domain, relieves HbF repression while largely maintaining cellular fitness. EZH2D14 retains H3K27 methylation and repression of a PRC target gene subset. Experiments in cells derived from mice bearing human b-globin genes confirm that pathways mediating EZH2 control of HbF expression can function in a mouse model of HBG switching. These findings demonstrate that partial disruption of PRC can yield selective phenotypes, highlighting the therapeutic potential of targeting non-enzymatic domains within chromatin-modifying complexes.
Göbel C, Niccolai R, Gregoricchio S
… +12 more, de Groot MHP, Kneefel SFK, Kreft M, Kuiken HJ, Lieftink C, Eickhoff N, Hoekman L, Bleijerveld O, Zwart W, Beijersbergen RL, van Leeuwen F, Jacobs H
Germinal center (GC) B cell-like diffuse large B cell lymphoma (GCB-DLBCL) depends on the cooperative activity of the histone methyltransferases DOT1L and EZH2 to maintain its pro-proliferative GCB cell identity while re...Germinal center (GC) B cell-like diffuse large B cell lymphoma (GCB-DLBCL) depends on the cooperative activity of the histone methyltransferases DOT1L and EZH2 to maintain its pro-proliferative GCB cell identity while repressing plasma cell (PC) differentiation. To explore the mechanisms underlying the co-dependency between DOT1L and EZH2 in GCB-DLBCL, we performed an EZH2 inhibition (EZH2i)-anchored genome-wide CRISPR interference screen and identified multiple candidate genes encoding components of non-canonical (nc) PRC1 complexes, including USP7, KDM2B, RING1, and PCGF1. We identified USP7 as potential direct target of DOT1L, whose downregulation was associated with increased EZH2i sensitivity in multiple GCB-DLBCL cell lines. Furthermore, we observed that DOT1L influences the composition of chromatin-bound ncPRC1 complexes and regulates, in part, the deposition of H2AK119 monoubiquitination (H2AK119ub1) at gene promoters co-occupied by H3K27me3, here defined as PRC1/2 targets. These PRC1/2 targets were specifically enriched in PC signature genes, whose derepression was associated with DOT1L inhibition (DOT1Li)-mediated loss of H2AK119ub1. This study reveals novel insights into the role of DOT1L and its functional co-dependence with EZH2 in maintaining GCB identity in DLBCL, supporting a model in which concurrent reduction of H2AK119ub1 and H3K27me3 promotes differentiation toward an anti-proliferative, plasma cell-like state.
Arribas AJ, Cannas E, Sartori G
… +29 more, Guidetti F, Cascione L, Napoli S, Forestieri G, Fuzio F, Pesenti EDS, Tarantelli C, Spriano F, Zucchetto A, Rossi FM, Bruscaggin A, Rinaldi A, Castro de Moura M, Jovic S, Raimondi A, Bordone Pittau R, Terzi di Bergamo L, Ye X, Stathis A, Ben-David Y, Pan-Hammarström Q, Simonetta F, Stussi G, Zucca E, Gattei V, Brown JR, Esteller M, Rossi D, Bertoni F
Resistance to Bruton tyrosine kinase (BTK) inhibitors remains a major clinical challenge in B-cell lymphomas and often occurs in the absence of BTK or PLCG2 mutations. Here, we investigated non-genetic mechanisms of ibru...Resistance to Bruton tyrosine kinase (BTK) inhibitors remains a major clinical challenge in B-cell lymphomas and often occurs in the absence of BTK or PLCG2 mutations. Here, we investigated non-genetic mechanisms of ibrutinib resistance in marginal zone lymphoma (MZL) and their broader therapeutic implications. Chronic ibrutinib exposure generated a resistant MZL model that showed cross-resistance to BTK inhibitors and degraders, without evidence of multidrug resistance or genetic alterations. Integrated transcriptomic, epigenetic, and proteomic analyses revealed extensive reprogramming, including activation of PI3K/AKT, MAPK, and MYC pathways, repression of apoptosis and oxidative phosphorylation, and a prominent cytokine-secretory phenotype. Interleukin-16 (IL-16) emerged as a central mediator of resistance. IL-16 was transcriptionally upregulated, actively secreted, and sufficient to induce ibrutinib resistance across multiple CD9-positive models of MZL, mantle cell lymphoma, chronic lymphocytic leukemia (CLL), and activated B-cell-like diffuse large B-cell lymphoma. Serum IL-16 levels were elevated in ibrutinib-refractory CLL patients without BTK/PLCG2 mutations. Mechanistically, IL-16 engaged CD9-enriched membrane microdomains to activate PI3Kδ, thereby sustaining AKT and ERK signaling, stabilizing MYC, inducing NF-κB-dependent programs, and upregulating antiapoptotic effectors, including BFL1 (BCL2A1). Pharmacological or genetic disruption of the IL-16/CD9/PI3K axis restored sensitivity to BTK inhibitors and R-CHOP and abrogated IL-16-induced signaling in primary CLL samples. In conclusion, an IL-16/CD9-driven, epigenetically regulated survival pathway represents one possible mechanism of resistance to BTK inhibitors and chemoimmunotherapy, supporting therapeutic targeting of this axis in refractory B-cell lymphomas.
Ramaekers K, Tran M, Lunghi M
… +13 more, Hermans A, Thys C, De Wispelaere K, Turro E, Van Geet CVG, Van Thillo Q, Peerlinck K, Eto K, Nurden AT, Balduini A, Di Buduo CA, Labarque V, Freson K
Glanzmann Thrombasthenia (GT) is characterized by absent platelet aggregation in response to all agonists except ristocetin and is caused by recessive inactivating variants in ITGA2B or ITGB3. While GT patients typically...Glanzmann Thrombasthenia (GT) is characterized by absent platelet aggregation in response to all agonists except ristocetin and is caused by recessive inactivating variants in ITGA2B or ITGB3. While GT patients typically are described as having normal platelet counts, autosomal dominant activating variants in ITGA2B or ITGB3 cause macrothrombocytopenia. Interestingly, in our cohort of 16 GT patients, eight consistently exhibited platelet counts at the lower end of the normal range. We studied the role of integrin αIIbβ3 in platelet formation using megakaryocytes (MKs) derived from genetically modified immortalized megakaryocyte cell lines (imMKCLs), focusing on two modifications of ITGB3: ITGB3-/- (inactivating) and ITGB3WT/D673_E713del (activating). In static differentiation cultures, ITGB3-/- and ITGB3WT/D673_E713del imMKCLs exhibited normal MK differentiation but reduced proplatelet formation. Platelet production was also impaired in a 3D silk-based bone marrow system and in shaking cultures, confirming a quantitative role for ITGB3 in platelet production, independent of the type of variant. While TRAP-activated in vitro-generated platelets lacking αIIbβ3 failed to bind the activation-dependent PAC-1 antibody, ITGB3WT/D673_E713del platelets bound PAC-1 prior to activation mimicking the patient's phenotype. Transcriptome profiling and metabolomic analyses of integrin αIIbβ3 deficient MKs revealed impaired serine metabolism and downregulation of SLC3A2 (CD98hc), an amino acid transporter chaperon known to interact with the β3 subunit. Flow cytometry confirmed decreased CD98hc in mutant MKs, while re-expression of wild-type ITGB3 in ITGB3-/- MKs restored αIIbβ3 and CD98hc expression, normalized proplatelet formation, and enhanced serine uptake. These results uncover a previously unrecognized role of integrin αIIbβ3 in coupling serine metabolism to platelet biogenesis.
Small AM, Patel SM, Giugliano RP
… +13 more, Morrow DA, Goodrich EL, Hug B, Parkar S, Chen SA, Goodman SG, Joung B, Kiss RG, Wojakowski W, Weitz JI, Bloomfield D, Sabatine MS, Ruff CT
This prespecified analysis from AZALEA-TIMI 71 demonstrated that prior oral anticoagulation (OAC) experience modifies bleeding risk in atrial fibrillation. The factor XI inhibitor abelacimab consistently reduced bleeding...This prespecified analysis from AZALEA-TIMI 71 demonstrated that prior oral anticoagulation (OAC) experience modifies bleeding risk in atrial fibrillation. The factor XI inhibitor abelacimab consistently reduced bleeding irrespective of prior OAC experience, with potentially greater absolute benefit among OAC-naïve patients. CT# NCT04755283.
Ling RE, Welsh AM, Hamer L
… +24 more, Jackson T, Neil E, Elliott N, Cross JW, Wuppalapati AS, Smith AL, Chahrour C, Sevim O, Palmer DA, Bozhilov Y, Brown EJ, Olender L, Iskander D, Wang G, Rice S, O'Byrne S, Harman J, Ancliff P, Psaila B, Wilkinson AC, Morgan RG, Roberts IA, Milne TA, Roy A
Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we...Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we aimed to identify specific properties of human fetal hematopoietic stem/progenitor cells (HSPC) that promote leukemic transformation in KMT2Ar iALL using molecular, functional and in vivo assays. Through comparison of human fetal HSPC with adult HSPC transcriptomes we derived a fetal-specific gene signature and identified the fetal oncogene LIN28B and its downstream effectors among the top hits. These genes were also expressed in iALL. Functional assays revealed that LIN28B was essential in human fetal liver (FL) CD34+ cells to maintain proliferation and stem-like properties, and support B- and NK-lymphopoiesis. To interrogate the role of LIN28B in iALL, we utilized a human FL-derived CRISPR-Cas9 KMT2A::AFF1 model. In this CRISPRKMT2A::AFF1 model, human FL CD34+ cells fail to transform upon induction of KMT2A::AFF1 translocation in the absence of LIN28B. Furthermore, LIN28B-expressing CRISPRKMT2A::AFF1 leukemias were more proliferative in vitro and in vivo, with this advantage being lost upon LIN28B knockdown. Mechanistic studies showed that LIN28B acts by stabilizing key early B-lymphoid genes, epigenetic regulators, and cell cycle and anti-apoptotic genes. Thus, LIN28B has an essential role in normal human fetal B-lymphopoiesis, and is necessary for the initiation of KMT2A::AFF1 iALL in human fetal cells in the absence of co-operating mutations. LIN28B activity may help explain why KMT2A::AFF1 leukemias are so aggressive, making it a potential target in LIN28B-expressing leukemias.
Lymphodepleting pre-conditioning (LD) is essential for the efficacy of CAR T therapy in hematologic malignancies. However, in the setting of autoimmune diseases (ADs), the contribution of LD for efficacy is unclear. Here...Lymphodepleting pre-conditioning (LD) is essential for the efficacy of CAR T therapy in hematologic malignancies. However, in the setting of autoimmune diseases (ADs), the contribution of LD for efficacy is unclear. Here, we report on the early safety, efficacy, and correlative data of the first four pemphigus vulgaris (PV) subjects who received resecabtagene autoleucel (rese-cel) a fully human CD19-CAR T cell therapy, without LD in the RESET-PV® trial (NCT04422912), a sub-study of the DesCAARTes trial. Following infusion, pemphigus disease area index (PDAI) scores improved significantly in all subjects. A favorable safety profile was observed with a single episode of grade 1 cytokine release syndrome. Immune-effector-cell-associated neurotoxicity was not observed. Rese-cel expansion was similar in PV subjects as compared to other rese-cel treated AD subjects who received LD. B cell depletion was observed in all PV subjects, with 3 of 4 subjects achieving B cell aplasia. Elevations in serum BAFF were observed, with 3 of 4 subjects achieving levels within the lowest end of the range exhibited in rese-cel treated AD subjects who received LD. Pathogenic PV autoantibodies decreased in 2 of 4 subjects. Together, these preliminary data suggest that LD may not be required for humanized CAR T efficacy in patients with AD.
Hakobyan M, Langstein J, Ramos Medina MJ
… +25 more, Kleinert E, Schönung M, Hartmann M, Rohdjess H, Wojtarowicz J, Staeble S, Türe M, Pobiedonoscew Y, Claus R, Bullinger L, Oakes CC, Zoldan K, Cross M, Platzbecker U, Kneisel N, Raffel S, Germing U, Hoermann G, Haas S, Rippe K, Fröhling S, Pusch S, Plass C, Milsom MD, Lipka DB
IDH1 and IDH2 are frequently mutated in various cancers, including acute leukemias. However, the distinct mechanisms by which mutant IDH1 or IDH2 drive hematopoietic neoplasms remain poorly understood. Here, we analyzed...IDH1 and IDH2 are frequently mutated in various cancers, including acute leukemias. However, the distinct mechanisms by which mutant IDH1 or IDH2 drive hematopoietic neoplasms remain poorly understood. Here, we analyzed DNA methylation in IDH1- and IDH2-mutant AML and found neutrophil lineage-specific epigenetic alterations in IDH1-mutant cases that went along with severely impaired neutrophil differentiation. Transcriptional analysis of normal hematopoiesis in humans and mice revealed a strong physiological upregulation of IDH1/Idh1 in myeloid progenitors. To study the functional effects of Idh1 mutations on hematopoiesis in a pre-leukemic setting, we used a genetically engineered inducible mouse model expressing a heterozygous Idh1 mutation under control of the endogenous promotor. Our study revealed a cell-intrinsic block in neutrophil differentiation caused by repression of myeloid transcription programs in neutrophil progenitors. This included impaired expression of Cebpe, which encodes a key transcription factor regulating neutrophil differentiation. Reactivation of Cebpe expression, by overexpression of its upstream regulator Cebpa or following treatment with hypomethylating agents restored differentiation, indicating that the differentiation block is reversible.In summary, we found a reversible, pre-leukemic impairment of neutrophil differentiation in IDH1-mutant hematopoiesis that correlates with elevated IDH1 expression in myeloid progenitors and likely explains the strong association of IDH1 mutations with myeloid neoplasms.
Duan H, Wang B, Zheng Y
… +19 more, Lv Y, Lu T, Li H, Li P, Li R, Xie X, Yang Z, Sun G, Zhao X, Yang M, He Y, Xu C, Pu S, Zhang L, Shi J, Jiang E, Cheng T, Hu Z, Cheng H
Understanding how metabolism governs human hematopoietic stem cells (HSCs) function is essential for advancing regenerative therapies, yet direct metabolic profiling of human HSCs has been limited by their extreme scarci...Understanding how metabolism governs human hematopoietic stem cells (HSCs) function is essential for advancing regenerative therapies, yet direct metabolic profiling of human HSCs has been limited by their extreme scarcity and the technical limitations of conventional methods. Here, we apply a low-input mass spectrometry-based metabolomics platform, optimized for rare cell populations, to generate metabolic profiles of 13 immunophenotypically defined hematopoietic cell types from adult human bone marrow. Using as few as ~10,000 cells per sample, we detect over 80 metabolites and uncover both conserved metabolic programs in primitive hematopoietic stem and progenitor cells (HSPCs) and lineage-specific metabolic specializations. Notably, we identify L-carnitine-driven fatty acid oxidation (FAO) as a key metabolic feature supporting HSPC function. Mechanistically, L-carnitine activates the PPARA-TFEB signalling axis, promoting mitochondrial metabolism and autophagy to preserve regenerative capacity. Functional assays in primary CD34+ HSPCs derived from healthy donors or patients with aplastic anemia confirm that L-carnitine supplementation improves stem cell function ex vivo and in vivo. Together, this work provides a foundation for human hematopoietic metabolism and reveals a targetable metabolic circuit governing HSPC regenerative fitness with therapeutic potential for improving stem cell-based interventions.