Selection Against U2AF1^S34F Mutant HSPCs Is Rescued Via IL-1B Treatment

Clonal Hematopoiesis of Indeterminant Potential (CHIP) is associated with adverse outcomes including progression to hematological malignancies such as MDS and AML, which have poor prognosis in older (over 60 years old) patients. Human studies reveal that heterozygous mutations in the splicing factor gene U2AF1 are primarily detected in aged CHIP patients and are strongly associated with increased malignancy risk. It is not well understood whether these mutations are gradually selected for across life or whether the mutation shows differential selection, with negative selection in youth converting to positive selection in old age.

It has been previously shown that chronic infection and elevated levels of inflammatory interferon (IFN)-γ were sufficient to drive expansion of hematopoietic stem and progenitor cells (HSPCs) with other common CHIP mutations. Aged individuals experience chronic inflammation, and administration of inflammatory cytokines (e.g., IL-1β) to young mice mimics an aging phenotype, suggesting that this could be a potential driver of U2AF1 mutant HSPC expansion in old age. Understanding how aging and inflammation influence selection for malignancy-associated U2AF1 mutations is critical for the development of interventions to limit the progression of associated malignancies.

To address if aging can alter the selection of U2AF1 S34F mutant cells, young (1.5 months) and middle-aged (8 months) donor cells were transplanted into young and middle-aged recipients. Because non-specific Cre activation can occur with time, Floxed (FL) conditional knock-in U2AF1 S34F mutant mice (heterozygous for the mutation) that did not contain an internal Cre driver were used as donors. Upon the exogenous addition of recombinant TAT-Cre fusion protein ex vivo, a wild-type (WT) U2af1 cDNA is recombined out and the S34F mutation is expressed at the endogenous U2af1 locus. To facilitate tracking of mutant cells in vivo, these mice also express a lox-stop-lox EYFP reporter at the Rosa26 locus.

Bone marrow (BM) from 1.5- and 8-month-old FL EYFP/U2af1 S34F mice or FL EYFP control mice (WT for U2af1) was isolated and recombined HSPCs were transplanted into mildly conditioned 1.5- and 8-month-old WT congenic recipient mice (C57BL/6J). The recipient mice were pre-conditioned with busulfan and CD4 and CD8 cell-depleting antibodies to clear HSPCs and remove T-Lymphocytes, respectively. The expansion of the U2AF1 mutant EYFP⁺ cells, relative to U2AF1 WT EYFP⁺ cells, was monitored in the peripheral blood every three weeks, and upon termination, in the BM.

In contrast to U2AF1 WT EYFP⁺ donor cells, our peripheral blood data suggest that U2AF1 S34F mutant donor cells are negatively selected against in recipient mice of similar ages, particularly the 1.5-month cohort. The same number of WT or S34F EYFP+ HSPCs were transplanted into recipients, however, the significant difference seen in the contribution to hematopoiesis is a strong indicator of negative selection against HSPCs with the S34F mutation. Interestingly, only in the context of 8-month-old S34F donors into 8-month-old recipients did we fail to observe significant continued loss of mutant HSPC from 3 to 9 weeks post-transplant. This suggests that older age (even if middle aged) may reduce negative selection of the S34F mutation.BM data from these mice suggest that while there is strong selection against the S34F mutation (5 to 10 fold), particularly in the context of young age, the small percentage of remaining EYFP+ U2AF1 S34F mutant cells appear to be skewed toward a stem-like phenotype. Furthermore, preliminary results in vitro and in vivo also suggest that the selection against U2AF1 S34F mutant LSKs can be rescued by the addition of the inflammatory cytokine IL-1β. Ongoing experiments will determine whether old age (>20 months) can

In conclusion, our studies indicate that a known malignant driver mutation is strongly selected against in youth, congruent with the idea that natural selection eliminates detrimental mutations to preserve the individual's fitness during youth. Furthermore, the context in which these mutations arise influences their expansion, and thus, their contribution to a malignant phenotype.

No relevant conflicts of interest to declare.