Poisoning of Healthy Hematopoiesis Is an Unanticipated Mechanism Driving Clonal Dominance in Vexas Syndrome

VEXAS syndrome is a recently discovered adult-onset autoinflammatory disease burdened by a high mortality rate and caused by dominant hematopoietic clones bearing somatic mutations in the UBA1 gene. However, pathogenic mechanisms fueling clonal dominance are unknown. Moreover, the lack of disease models hampers the development of disease-modifying therapies. Here, we develop humanized models of VEXAS syndrome through base editing technology and capitalize on granular omic datasets from patients and the model itself to unravel cell state perturbations and biologic mechanisms driving clonal dominance in VEXAS syndrome. Immunophenotypic dissection of hematopoiesis in VEXAS patients (n=9) compared to controls highlighted myeloid bias, impaired lympho/erythro/megakaryo-poiesis, increased mobilization to the bloodstream, and lower primitiveness of HSPCs as major phenotypic markers in patients. Moreover, patients' HSPCs had impaired reconstitution capacity in immunodeficient mice, hampering their use for further studies. We thus leveraged base editing to develop in vitro and in vivo models of VEXAS syndrome by genetically converting human wildtype HSPCs (UBA1^wt) into UBA1 mutant (UBA1^mut) with >90% efficiency. Clonogenic and multilineage differentiation assays revealed an exclusive myeloid and NK output of UBA1^mut HSPCs. Concordantly, xenotransplantation of >80% UBA1^mut HSPCs resulted in a 10-50-fold lower human cell output in vivo compared to controls (100% UBA1^wt) due to dramatic shrinkage of the lymphoid compartment. Conversely, NK and myeloid ones were more preserved. HSPCs from bone marrow of UBA1^mut mice were lower in number and mostly myeloid-biased. Of note, myeloid cells and HSPCs were mostly UBA1^mut, while the few differentiated lymphoid cells were UBA1^wt. Single-cell RNA-sequencing (scRNA-seq) on human UBA1^mut grafts showed pervasive upregulation of inflammatory and apoptotic transcriptional responses, and lower propensity to engage cell cycle, across all hematopoietic subpopulations. Primitive HSCs from UBA1^mut mice early activate inflammatory responses, are prematurely aged, and transcriptionally imprinted toward myelopoiesis. Notably, the immunophenotype, lineage repopulation patterns, and transcriptomic programs across all human hematopoietic subpopulations strikingly mirrored those observed in VEXAS patients from our cohort, validating the reliability of the model. We then leveraged our humanized model to study how different degrees of mosaicism affect hematopoiesis, and the mechanisms underlying clonal dominance. Competitive transplants mixing human UBA1^mut and UBA1^wt HSPCs at different ratios showed that the absolute number of human cells progressively decreased at increasing input of UBA1^mut HSPCs, while the myeloid-lymphoid ratio increased. The proportion, but not the absolute number, of UBA1^mut cells was enriched from the input within HSPCs and myeloid cells at >25% UBA1^mut HSPCs infused. Conversely, B cells were only UBA1^wt but dramatically reduced in numbers considering the input of transplanted UBA1^wt HSPCs. These data suggested a threshold effect above which the pathogenic clone dominates and subverts human hematopoiesis through a “poisonous” cell-extrinsic effect, rather than cell-autonomous proliferative advantage. To uncover the factors contributing to the establishment of a poisonous microenvironment, we measured the abundance of proinflammatory cytokines within the bone marrow of UBA1^mut mice and found substantial elevation of human IL-1β, IL-18 and IL-1RA compared to UBA1^wt controls. To confirm that the bone marrow microenvironment reshaped toward an inflammatory milieu can poison bystander cells, including hematopoietic progenitors, we performed scRNA-seq on lineage-negative murine cells in UBA1^mut mice. Intriguingly, we found potent activation of NFkB-mediated inflammatory signatures, promoting cell proliferation and apoptosis. These data indicate that while mutant cells are resilient to the inflammatory milieu, wild-type ones are poisoned and progressively overwhelmed by VEXAS clones, becoming unable to support functional multilineage hematopoiesis. In summary, our study unveils an unanticipated mechanism of clonal dominance, provides new models for preclinical investigation of therapeutic strategies, and has relevant implications for clinical management of VEXAS patients.

Ciceri:ExCellThera: Membership on an entity's Board of Directors or advisory committees. Cavalli:Novartis: Current Employment. Naldini:GeneSpire;Chroma Medicine: Consultancy, Current equity holder in private company, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company.