Phosphatidylcholine synthesis inhibition diminishes ASXL1 and DNMT3a mutation driven clonal hematopoiesis Free

The acquisition of somatic mutations that increase the competitive potential of hematopoietic stem and progenitor cell (HSPC) clones causes Clonal Hematopoiesis (CH). CH, where one HSPC clone becomes dominant, occurs in >10% of individuals over 65 and predisposes these individuals to hematological malignancy and cardiovascular disease. No approved therapies currently exist. We posited that metabolically profiling dominant, mutant HSPCs could reveal targetable metabolic vulnerabilities.

We combined mosaic mutagenesis of CH-driving genes and HSPC color barcoding to isolate dominant HSPC clones in zebrafish. Untargeted metabolomics was performed on dominant asxl1- or ezh2-mutant or wildtype (WT) HSPCs. Only 7 of 90 detected metabolites differed significantly. Two choline-related metabolites were decreased in dominant HSPC clones: betaine (FC -0.68, p<0.01) and acetylcholine (FC -0.42, p<0.01). This metabolic signature was confirmed in an independent asxl1-mutant CH cohort using a standard containing choline metabolite profiling assay. We hypothesized that decreased betaine and acetylcholine levels reflected increased partitioning of choline into phospholipid biosynthesis (e.g., phosphatidylcholines). Lipid profiling of asxl1-mutant and WT zebrafish HSPCs revealed a global decrease in phosphatidylcholine levels; species 16:1/18:1 (FC 1.33, p<0.05), 16:0/16:0 (FC 1.44, p<0.05), and 16:0/18:2 (FC 1.50, p<0.05) were the most affected. Primary human CD34⁺ HSPCs from two donors were edited via CRISPR/Cas9 in exon 12 of ASXL1 and showed increased levels of the same phosphatidylcholine species observed in zebrafish after 7 days of culture (16:1/18:1, FC 2.73; 16:0/16:0, FC 2.15; 16:0/18:2, FC 2.76). To determine if phosphatidylcholine biosynthesis was required to maintain clonal dominance zebrafish with large asxl1-mutant clones (>10% VAF) were treated with RSM-932A, an inhibitor of the first enzyme in phosphatidylcholine synthesis, for 5 days followed by monthly blood collection. After 2 months of treatment, RSM-932A decreased asxl1 VAF by 49.95% compared to a 42.61% increase in controls (p<0.01). RSM-932A did not affect lineage or clonal output in WT zebrafish. We next used a genetic barcoding system (GESTALT) in zebrafish to track dominant clones during monthly RSM-932A dosing. In clones contributing <60% of blood (3/5), RSM-932A reduced the clone below detection after 6 months. To validate these findings in a human model, primary human CD34⁺ HSPCs edited in ASXL1 or AAVS1 (control) were treated with RSM-932A. ASXL1-mutant HSPCs increased in number after 14 days and in EdU positivity after 4 days compared to AAVS1 control but these effects were completely abrogated by RSM-932A (total HSPC number, p<0.0001; Edu positivity, p<0.01). To test whether this treatment may be generalizable to other CH mutations, HSPCs (Lin^-Sca⁺cKit⁺CD34^-CD150⁺⁾ were isolated from mice with inducible, mutant DNMT3a^R878H and ROSA26-tdTomato alleles. RSM-932A reduced the number of DNMT3a^R878H-tdTomato⁺ cells by 49.12% (p<0.01) after 21 days of culture. To understand what underlies the need for more phosphatidylcholine synthesis transcriptomic analysis of dominant asxl1-mutant and WT zebrafish HSPCs was performed and lpcat2 (p<0.05), known to catalyze the last step of phosphatidylcholine into platelet activating factor (PAF), was upregulated. When ASXL1-mutant human HSPCs were cultured with RSM-932A and PAF, RSM-932A's suppressive effect on HSPC expansion (p<0.01, day 14) and EdU incorporation (p<0.05, day 4) was overcome. To address the potential of RSM-932A as a therapeutic approach primary, CD34⁺ HSPCs from a patient with myelodysplastic syndrome (ASXL1 VAF 17%) were cultured for 14 days with RSM-932A with and without PAF. RSM-932A reduced ASXL1 VAF by 37.27% (p<0.05), but RSM-932A with PAF was not different from control.

In sum, inhibiting phosphatidylcholine biosynthesis in ASXL1 or DNMT3a mutant HSPCs suppresses their clonal expansion in a PAF dependent manner. This is the first identification of choline metabolism as a targetable metabolic liability in CH-causing HSPCs.