Systematic functional dissection of germline noncoding risk variants impacting clonal hematopoiesis

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor condition characterized by the expansion of blood cell clones harboring somatic mutations originating in hematopoietic stem cells (HSCs). Since individuals with CHIP face a high risk of developing myeloid malignancies, targeting CHIP clones could provide a viable strategy for leukemia prevention. Despite its clinical significance, the mechanisms underlying CHIP predisposition and progression remain poorly understood. Recent genome wide association studies (GWAS) have identified several non-coding genetic loci that are strongly associated with CHIP; however, their underlying mechanisms still remain unknown. We hypothesize that risk variants in these non-coding loci modulate enhancer elements active in HSCs. To test this, we selected 1,374 non-coding variants from 51 loci associated for CHIP risk in the UK Biobank and screened them for regulatory activity using a Massively Parallel Reporter Assay (MPRA). We performed our lentiviral MPRA screen in MUTZ-3 cells, a human hematopoietic cell line relevant to HSCs, which express CD34 surface marker and are dependent on HSC-specific transcription factors. Using a MPRA library of ∼73,000 constructs in CD34+ fraction of MUTZ-3 cells, we identified 87 variants representing 32 GWAS loci. We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MPRA variants that affect the transcription of NKD2, FLT3, and MSI2. Our functional studies on MSI2 indicate that presence of higher levels of MSI2 mediated by CHIP risk allele enhances the clonal expansion of TET2 knockout hematopoietic stem and progenitor cells, providing a mechanistic link whereby non-coding genetic variants can influence the expansion of mutant CHIP clones. Competing Interest Statement RT hold patents related to the application of MPRA.