(P23-031-23) Inhibition of Choline-Phospholipid Metabolism Prevents Clonal Hematopoiesis Progression

Objectives: With age the cells that populate blood system, hematopoietic stem and progenitor cells (HSPCs), accumulate genetic mutations (e.g., asxl1, tet2, or ezh2) that can increase the competitive potential of individual HSPC clones causing clonal hematopoiesis (CH). CH predisposes individuals to blood cancers and cardiovascular disease, but no therapies exist. Our objective was to identify targetable metabolic vulnerabilities in mutant HSPCs that underlie their clonal amplification.

Methods: We combined mosaic mutagenesis of CH-driving genes and HSPC color barcoding allowing for isolation of non-dominant and dominant mutant HSPCs in the zebrafish. We then isolated non-dominant and dominant asxl1- or ezh2-mutant HSPCs via flow cytometry for untargeted metabolomics and followed up with in vivo d9-choline stable isotope tracing studies. Further experiments inhibiting the first step in the choline-phospholipid synthesis pathway, choline kinase A (Chka), pharmacologically (RSM932A) were performed in zebrafish with large asxl1-mutant clones ( >10% variant allele frequency [VAF] in blood) via intraperitoneal injection or by inclusion in expansion medium of Cas9-edited human ASXL1-mutant HSPCs.

Results: Our untargeted metabolomics revealed two catabolic products of choline were decreased in dominant clones – acetylcholine (FC -0.42, p< 0.01) and betaine (FC -0.68, p< 0.01). Dominant HSPCs had enhanced choline-betaine conversion (p < 0.01) but this was not able to maintain normal levels of intracellular betaine. We considered that levels of betaine, necessary to support one carbon metabolism, were decreased to facilitate choline-phospholipid synthesis leading to aberrant DNA or histone methylation. Chka inhibition decreased asxl1 VAF in peripheral blood by 49.95 ± 38.46% whereas vehicle control treatment increased 42.61 ± 85.80% (p < 0.01). Furthermore, CHKA inhibition for 14 days reduced the total number of cultured human ASXL1-mutant HSPCs compared to vehicle control (p < 0.0001).

Conclusions: Our data demonstrate that clonally dominant HSPCs exhibit perturbed choline metabolism and that pharmacological inhibition of Chka in vivo in zebrafish and in human HSPCs in vitro abates clonal dominance. To date this is the first identification of a targetable metabolic liability in CH-causing HSPCs.

Funding Sources: Canadian Institutes of Health Research