Poster #047, Stanford University
SSBP3 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 rs77628744, near the transcription co-factor SSBP3, to clonal expansion rate. Since rs77628744 sits in a putative enhancer predicted to contact the SSBP3 promoter, we explored whether modulating this region impacts SSBP3 expression and how SSBP3 dosage affects clonal expansion rate. We hypothesized that the enhancer regulates SSBP3 expression and that modulating SSBP3 changes how hematopoietic stem and progenitor cells (HSPCs) proliferate. To test our hypotheses, we knocked out the region around rs77628744/SSBP3 with CRISPR-Cas9 and overexpressed SSBP3 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 SSBP3 expression. We next applied these tools in primary HSPCs with and without CH driver mutations and explored SSBP3’s role in HSPCs by performing colony-forming and differentiation assays, revealing that loss of SSBP3 in HSPCs reduces erythrocyte and megakaryocyte generation while increasing myeloid differentiation. By manipulating cells’ SSBP3 levels, we elevate rs77628744 from statistical associations identified by GWAS to an experimentally supported regulator of HSPC differentiation. Together, our findings establish that SSBP3 levels influence how HSPCs differentiate and their clonal expansion rate, inaugurating SSBP3 as a compelling candidate for regenerative medicine approaches to restrain premalignant clonal expansion before progression to AML.