U2AF1S34F mutant HSPCs have a fitness advantage in aged and inflamed bone marrow Free

Introduction. Clonal Hematopoiesis of Indeterminant Potential (CHIP) associated splicing factor mutations, such as U2AF1^S34F, are strongly associated with age and increased risk for myeloid malignancies. While aging and inflammation are known contributors to clonal expansion of other CHIP mutations, the factors governing selection of splicing factor mutations remain poorly understood. Existing studies of splicing factor mutations, including U2AF1^S34F,primarily rely on young mouse or non-physiologic overexpression models, failing to accurately reflect the clinical context. Here we used an inducible, endogenous U2af1^S34F mouse model to understand how age and inflammatory conditions drive selection of mutant hematopoietic stem and progenitor cells (HSPCs).

Methods. To model the endogenous expression of the U2AF1^S34F neomorphic aging-associated mutation, we used aged floxed conditional knock-in U2af1^S34F/+mutant mice and U2af1^+/+ wild-type (WT) control mice and activated expression of the S34F mutation ex-vivo with cell permeable TAT-Cre fusion protein. A lox-stop-lox EYFP reporter at the Rosa26 locus enables in-vivo tracking of mutant cells (Rosa26^EYFP/+). To address if aging can alter the selection of mutant cells, lineage depleted bone marrow (BM) cells from 1.5- and 21-month-old (m.o.) U2af1^S34F/+or U2af1^+/+ mice was isolated, treated with TAT-Cre, and transplanted within 2 hours into mildly conditioned 1.5-, 8-, and 21-m.o. WT congenic recipient mice. The recipient mice were pre-conditioned with low dose busulfan and CD4 and CD8 cell-depleting antibodies to clear HSPCs and remove T-lymphocytes, respectively. The expansion of the U2af1^S34F/+ mutant EYFP⁺ cells, relative to U2af1^+/+ EYFP⁺ cells, was monitored in the peripheral blood every three weeks, and upon termination, in the BM. Tamoxifen-inducible HSC specific Cre mice (hScl-CreER^T) were crossed to our Rosa26^EYFP/+; U2af1^S34F/+ or U2af1^+/+ conditional knock-in mice for in-vivo inflammation and single cell RNA sequencing (scRNA-seq) studies.

Results. We demonstrated that young U2AF1^S34F mutant donor cells were strongly selected against in young (1.5 m.o.) recipients. However, when both donor and recipient were old (21 m.o.), mutant HSPCs significantly expanded post-transplant, indicating positive selection. Intermediate phenotypes were observed when only the donor or the recipient was old, or when either donor or recipient were , suggesting both intrinsic and extrinsic influences of aging on selection. Additionally, S34F mutant cells demonstrated impaired hematopoietic differentiation in vivo compared to WTcounterparts, with age compounding this defect.

Next, we examined the impact of inflammation on selection of U2AF1^S34F mutant HSPC using IL-1β, a cytokine elevated during aging. In vitro, IL-1β reversed the negative selection of mutant HSPCs observed in basal conditions. Similarly, in vivo administration of IL-1β to young mutant mice promoted the expansion of U2AF1^S34F mutant HSPCs compared to vehicle and WT controls.

Preliminary gene set enrichment analysis from scRNA-seq revealed aberrant downregulation of inflammatory signaling in old U2AF1^S34Fmutant HSCs when compared to old WT HSCs (p.adj<0.01), suggesting an adaptation of old mutant HSCs to resist inflamed microenvironments Analyses of alternative exon usage and polyadenylation are currently ongoing.

Conclusions. Using a novel physiological model, this study reveals that aging and inflammation influence the selection of endogenously expressed U2AF1^S34Fmutant HSPCs. Importantly, both an aged stem cell and an aged bone marrow niche are required for positive selection of this high-risk CHIP mutation. Of note, IL-1β (a cytokine known to increase with age) can recapitulate this selection in young animals, directly linking inflammation to increased clonal expansion. Unlike other studied CHIP mutations (e.g., TET2 truncating mutations), the strong selection against U2AF1 CHIP mutations in youth may reflect the severity of the risk that this mutant CHIP mutation confers. These studies show how context dependent selection can contribute to malignant clonal evolution that manifests primarily in the elderly. In all, our findings support the value of implementing lifestyle changes aimed at reducing inflammation with age, along with targeted monitoring, to mitigate clonal expansion and lower the risk of associated adverse outcomes including MDS and AML.