Poster #047, Stanford University
Gene X Regulates Clonal Expansion and Lineage Differentiation of Human Hematopoietic Stem and Progenitor Cells
Mentors: James Chavez and Ravindra Majeti, MD, PhD
Clonal hematopoiesis (CH), the expansion of blood stem cell clones carrying somatic driver mutations, is common in older adults, and clonal expansion rate, not clone presence, predicts progression to acute myeloid leukemia (AML). Why clonal expansion rates differ widely across individuals remains unclear. To address this gap, we explored natural genetic variation as a determinant for differences in expansion rates by performing genome-wide association studies (GWAS) using passenger-approximated clonal expansion rate (PACER) across 43,000 people with CH. Through this, we linked the variant rsXXXXXXXX, near the transcription co-factor Gene X, to clonal expansion rate. Since rsXXXXXXXX sits in a putative enhancer predicted to contact the Gene X promoter, we explored whether modulating this region impacts Gene X expression and how Gene X dosage affects clonal expansion rate. We hypothesized that the enhancer regulates Gene X expression and that modulating Gene X changes how hematopoietic stem and progenitor cells (HSPCs) proliferate. To test our hypotheses, we knocked out the region around rsXXXXXXXX/Gene X with CRISPR-Cas9 and overexpressed Gene X lentivirally in cell lines and primary CD34+ HSPCs. Targeted sequencing confirmed that each edit generated insertions and deletions (INDELs) at the correct loci, and our lentivirus successfully transduced OCI-M1 cells, together providing a validated toolset for modulating Gene X expression. We next applied these tools in primary HSPCs with and without CH driver mutations and explored Gene X’s role in HSPCs by performing colony-forming and differentiation assays, revealing that loss of Gene X in HSPCs reduces erythrocyte and megakaryocyte generation while increasing myeloid differentiation. By manipulating cells’ Gene X levels, we elevate rsXXXXXXXX from statistical associations identified by GWAS to an experimentally supported regulator of HSPC differentiation. Together, our findings establish that Gene X levels influence how HSPCs differentiate and their clonal expansion rate, inaugurating Gene X as a compelling candidate for regenerative medicine approaches to restrain premalignant clonal expansion before progression to AML.