Mutation-Specific Differences in the Relationship between Obesity and Clonal Hematopoiesis, with a Focus on JAK2^V617F and MPN Prevention

Introduction:

Although the inferred “fitness” of clonal hematopoesis (CH) driver mutations differs depending on the gene mutation, the impact of environmental factors that may promote or impair CH expansion remain largely unknown. We hypothesized that obesity influences the clonal expansion rate of common CH driver mutations.

Methods:

To test this hypothesis, we first interrogated data from the UKBiobank (UKBB) (n=425,573 exomes) to evaluate the relationship between body mass index (BMI) (kg/m2) and four common CH mutations, namely DNMT3A, TET2, ASXL1, and JAK2. For functional studies we focused on JAK2^V617F, using a novel Fgd5-CreER-Jak2^V617F mouse model we generated. In this model, Jak2^V617F expression is induced in a small percentage of long-term (LT)-HSC which are tracked sequentially in primary mice in an unirradiated bone marrow niche. To quantify Jak2^V617F expression in the model, we developed a digital droplet PCR assay. We also generated a chimeric bone marrow transplant Jak2^V617F model and performed bulk RNA-sequencing (RNAseq) on purified lineage^low Sca-1⁺ c-kit^high (LSK) cells expressing Jak2^V617F or wild-type Jak2, isolated from the same mouse. Finally, we performed single-cell RNAseq on Jak2^V617F-expressing c-kit^high cells purified from chimeric transplant mice. Mice fed an obesity diet received 60 kcal% fat while mice fed a control diet received 10 kcal% fat.

Results:

In UKBB analyses, we identified genotype-specific patterns of association between BMI and the presence of CH mutations. JAK2 (OR 0.51; 95% CI 0.27-0.96; p=0.036) and DNMT3A mutations (OR 0.92; 95% CI 0.87-0.97; p=0.0028) were negatively associated with a BMI >30 relative to BMI <25, whereas mutations in ASXL1(OR 1.40; 95% CI 1.23-1.60; p=8.8x10^-7) and TET2 (OR 1.16; 95% CI 1.04-1.31; p=0.0095) were positively associated with BMI >30.

To further explore the negative association between JAK2^V617F and obesity, we generated a cohort of Fgd5-CreER-Jak2^V617F mice and fed half the mice an obesity diet and the other half a control diet. After 24 weeks, Jak2-mutant mice fed the control diet developed a significantly higher hematocrit (HCT) as compared to Jak2-mutant mice fed the obesity diet (p=0.0043). The HCT remained significantly higher in control mice as compared to obese mice over a 46-week period (p<0.0001). Strikingly at 46 weeks, 5/7 mice (71%) fed the control diet showed signs of MPN as compared with 0/6 mice (0%) fed the obesity diet. Furthermore, overall survival was significantly improved in Jak2-mutant obese mice as compared to Jak2-mutant mice fed a control diet (p=0.03). Subsequently, after the death of most control mice, one Jak2-mutant obese mouse developed an elevated HCT, indicating that obesity prolonged the latency to MPN development in Jak2^V617F mice.

In the chimeric transplant model, gene set enrichment analysis (GSEA) showed significantly increased interferon alpha (NES=2.55, FDR≈0), interferon gamma (NES=2.26, FDR≈0), and interleukin-6 (NES=2.15, FDR=5.46x10^-05) signaling in Jak2-mutant LSK cells isolated from obese animals as compared to wild-type LSK cells, a finding not observed in control mice. Single-cell RNAseq experiments further validated these findings, where we identified interferon induced transmembrane protein 1 (IFITM1) as one of the most differentially upregulated genes in Jak2-mutant LT-HSCs from obese mice (adjusted p-value=1.08x10^-54) as compared to wild-type LT-HSCs, and unlike Jak2-mutant LT-HSCs from control mice.

Conclusions:

To our knowledge, our study is the largest to date to evaluate the relationship between obesity and CH and the first to investigate the negative association between JAK2^V617F CH and obesity, using functional studies. We developed a novel Fgd5-CreER-Jak2^V617F mouse model, which allowed us to study the transition from CH to overt MPN in an unperturbed, non-irradiated bone marrow niche. Transcriptomic profiling points to activation of the interferon signaling pathway in Jak2-mutant HSC as a possible mechanism by which JAK2-mutant LT-HSC may preferentially exhaust during obesity. These findings support testing pegylated interferon as a potential treatment for individuals with JAK2-mutant CH and a clinical protocol is currently under development at our institution. Ongoing functional studies are investigating additional metabolic targets in LT-HSC with the goal of uncovering novel treatment approaches for JAK2-mutant CH.

Bick:TenSixteen Bio: Consultancy. Mullally:Incyte: Consultancy; Protagonist: Consultancy; Biomarin: Consultancy; Morphic: Consultancy, Research Funding; Cellarity: Consultancy; PharmaEssentia: Consultancy; Relay Therapeutics: Research Funding.