MOLECULAR MECHANISMS UNDERLYING CLONAL EXPANSION OF HEMATOPOIETIC STEM CELLS

项目总结

PROJECT SUMMARY Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risks of de novo and therapy-related hematological neoplasms, suggesting that mutations identified in CHIP likely drive disease development. Acquired somatic mutations in tumor suppressor gene TP53 rank in the top five among mutations identified in CHIP. Clinical studies suggest that expansion of hematopoietic stem cells (HSCs) with TP53 mutations predisposes the elderly to hematological neoplasms and other aging-related diseases. However, there is a significant gap in knowledge regarding the mechanisms by which TP53 mutations promote HSC expansion. We recently found that TP53 mutations identified in CHIP enhance HSC self-renewal in mice in vivo. We showed that several mutant p53 proteins found in CHIP, but not wild–type p53, interact with epigenetic regulator EZH2 and enhance its activity. Moreover, we discovered that mutant p53 increases the chromatin accessibility to the NLRP1 inflammasome gene and that NLRP1 expression was upregulated in mutant HSCs, leading to increased secretion of pro-inflammatory cytokine IL-1β. We hypothesize that mutant p53 promotes mutant HSC expansion through two distinct mechanisms. First, mutant p53 interacts with and enhances the activity of epigenetic regulator EZH2 to promote mutant HSC self-renewal. Second, mutant p53 suppresses wild type HSC function through NLRP1 inflammasome-mediated pro-inflammatory pathway. In this proposed research, we will utilize biochemical, genetic, molecular, immunologic, and pharmacological approaches as well as vertebrate models to investigate the cell autonomous and non-autonomous mechanisms by which mutant p53 promotes clonal expansion of HSCs. To test if mutant p53 HSC expansion is mediated through enhanced EZH2 activity and increased H3K27me3, we will determine the impact of genetic and pharmacological inhibition of EZH2 activity on mutant p53 HSC self-renewal in mice. We will then introduce hot-spot TP53 mutations identified in CHIP into human primary HSPCs using retroviruses and examine if mutant p53 increases H3K27me3 levels and promotes HSPC expansion. To elucidate the cell non- autonomous mechanisms of mutant p53-mediated NLRP1 inflammasome activation on HSC expansion, we will determine if mutant p53 HSCs are more resistant to IL-1β exposure than wild-type HSCs. We will also interrogate cytokine secretion in both mouse and human hematopoietic cells using cytokine arrays. Finally, we will perform competitive bone marrow transplantation assays to determine the impact of genetic and pharmacological inhibition of the NLRP1 inflammasome on the clonal expansion of both mouse and human HSCs with TP53 mutations. Our results will provide novel insights into developing new potential target-based therapeutics for prevention and treatment of CHIP and other aging-associated diseases.