Defining Germline Predisposition to Dnmt3a-Mutant Clonal Hematopoiesis (CH)

Clonal hematopoiesis (CH) is the result of somatic mutations that confer a selective advantage to hematopoietic stem cells (HSCs), driving them to outcompete other HSCs and dominate mature hematopoietic cell production. While it is estimated that by middle age nearly all healthy individuals will have low but detectable variant allele frequency (VAF) indicating CH, only a small fraction will progress to hematologic malignancy. No methods currently exist to predict who will or will not progress to hematologic malignancy and preventative therapeutic strategies are lacking, largely due to poor understanding of the risk factors that determine whether individuals are susceptible to, or protected from, CH.

Several recent population sequencing studies have found that inherited genetic variants increase the likelihood of developing CH and that there are population differences in clonal advantages gained by specific mutations in particular genetic and environmental contexts. Limitations of these human studies are the insufficient ability to control for and distinguish genetic from environmental contributions to CH, and to directly interrogate mechanisms causing genetic predisposition to CH and progression to malignancy.

To overcome these barriers, here we have utilized the eight inbred and wild-derived founder strains of the Collaborative Cross (CC) and Diversity Outbred (DO) mouse populations (Saul MC et al., Trends in Genetics 2019). These feature well-characterized, segregated genetic variation that accurately reflects the genetic structure of human populations in a controlled environment. We crossed our recently described mouse model of CH (Dnmt3a^R878H/+Mx1-Cre on a C57BL/6 background) (Loberg MA et al., Leukemia 2019) with each of the eight founder strains. We observed that genetic background conferred sensitivity or resistance to Dnmt3a^R878H/+ HSC expansion based on phenotypic cell surface markers (Lin^- c-Kit⁺ Sca-1⁺ CD150⁺ CD48^-), relative to Dnmt3a^+/+Mx1-Cre strain littermate controls. Genetic contribution from the C57BL/6 and CAST strains permitted robust Dnmt3a^R878H/+ HSC expansion compared to Dnmt3a^+/+ controls over a period of 6 months. In this same time frame, genetic contribution from 129, NOD, NZO, A/J, PWK, WSB strains demonstrated a spectrum of resistance to Dnmt3a^R878H/+ HSC expansion.

In vitro studies using colony-forming assays of bone marrow cells isolated from selected strains show a positive correlation between serial replating efficiency and phenotypic HSC frequency, supporting that phenotypic cell surface markers reliably define cells with functional potential across these strains. Ongoing studies to interrogate in vivo functional potential by competitive transplantation of HSCs isolated from each of these strains will be discussed.

Together, our work demonstrates that HSC expansion as a consequence of Dnmt3a mutation is influenced by heritable genetic background in a controlled environmental setting. Understanding the mechanisms, at the systemic, intracellular and/or molecular levels, by which genetic background modifies HSC expansion caused by Dnmt3a or other CH mutations will be critical to develop improved prognostic tools and therapeutics to diagnose and treat high-risk versus low-risk CH. For this, utilization of genetically diverse mouse populations provides a critically needed platform to determine causative mechanisms driving clonal expansion of HSCs.