Abstract PR001: Dissecting the role of aging and inflammation on clonal hematopoiesis

Abstract

Abstract Aging is associated with the appearance in multiple tissues of somatic mutant clones often harboring mutations observed in cancers. In the hematopoietic system this phenomenon is named clonal hematopoiesis of indeterminant potential (CHIP) or “age-related clonal hematopoiesis”. The incidence of CHIP increases exponentially with aging, and has been associated with increased risk of myeloid malignancies, cardiovascular disease, and overall mortality. While mutations are thought to be acquired at a constant rate during life, whether aging plays a role in driving the expansion of these mutant clones is of crucial importance, as it can inform on the potential to prevent or halt the expansion of CHIP clones by intervening with the specific hallmark of aging fueling such expansion. To determine the effect of aging on the expansion of CHIP clones, we have established a method that relies on in vitro manipulation of mouse hematopoietic stem and progenitor cells (HSPC) in order to introduce by CRISPR-Cas9 selected mutations, followed by transplant into young and old recipient mice. Our method has several advantages: i) it allows rapid introduction of the desired mutations from mice of any background, including aged mice, bypassing the need to create transgenic lines; ii) by expanding the cultured cells in previously developed PVA media, it allows efficient engraftment in mildly (busulfan) conditioned recipient mice, avoiding the damaging effects on the bone marrow microenvironment that result from irradiation; iii) by manipulating HSPC with gRNAs targeting either the gene of interest (GFP tagged) or the safe harbor Rosa26 locus (dTomato tagged) prior to pooling, we can assess the competitive advantage of the mutation of interest relative to the Rosa26 control cells and determine their fitness advantage at various time points. We used this technique to generate Dnmt3a and Tet2 mutant clones and tested their expansion in young and aged recipient mice. Our data reveal that Dnmt3a exhibits a relatively mild fitness advantage and no difference in the rate of expansion between young and aged mice. By contrast Tet2-KO HSPC show a significantly faster expansion in old compared to young recipient mice, indicating a greater fitness advantage in the aged background. Tet2 mutant HSPC were previously shown to preferentially expand in inflammatory conditions such as stimulation with microbial products, IL-6 and TNFa. We have preliminary data that shows that co-housing old mice with young syngeneic mice alters their gut microbiome and results in a reduction of inflammation and restoration of more youthful gene expression profiles in stem and progenitor cells in the bone marrow. We are currently testing whether altering the gut microbiome or applying genetic models of reduced inflammation prevents the expansion of Tet2-KO cells, with associated “omics” analyses to explore underlying mechanisms. In all, these studies should contribute to our understanding of the aging-dependent occurrence of CHIP and suggest potential interventions to limit its prevalence. Citation Format: Marco De Dominici, Johannes Menzel, James V. DeGregori. Dissecting the role of aging and inflammation on clonal hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR001.