RNA seq analysis of calcium channel blocker, nifedipine in combination with metformin, MCC950 and anakinra treated Tet2-/- + Boy/J (1:1 ratio, mixed BM cells) transplanted Ob/Ob recipient mice

Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis (CH) of indeterminate potential (CHIP) involves expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy and cardiovascular disease (CVD). However, risk factors that contribute to CHIP-associated CH are poorly understood. A common, pro-inflammatory comorbidity responsible for enhancing the risk for developing type 2 diabetes mellitus (T2DM), CVD and cancer, obesity may exacerbate CHIP-associated diseases. We analyzed exome sequencing and clinical data from 47,466 individuals with valid CHIP in the UK Biobank. CHIP was present in 5.8% of the study population, and was associated with a significant increase in waist-to-hip ratio (WHR). The cancer genome atlas (TCGA) interrogation revealed that the mutation rate of CHIP is higher in patients with high BMI (>30 kg/m2) compared to those with low BMI (≤25 kg/m2) in six different cancer types. Utilizing mouse models of obesity bearing CHIP mutations, we show that both the compound mutant mice (Tet2-/-;Ob/Ob, Dnmt3a+/-;Ob/Ob) and CHIP mutant bone marrow (BM; Tet2-/-, Dnmt3a+/-, Asxl1+/- and Jak2+/-) transplanted into Ob/Ob mice develop rapid CH, leading to severe MPN/AML as well as CVD, which among other things, is associated with upregulation of intracellular calcium (Ca2+) levels and pro-inflammatory cytokines. Remarkably, calcium channel blocker nifedipine, in combination with metformin, MCC950 and anakinra, suppressed the growth of mutant CHIP cells, cardiovascular disease and restored normal hematopoiesis as well as serum Ca2+ and inflammatory cytokines. Thus, obesity is highly associated with the presence of CHIP and targeting CHIP mutant cells with a combination of metformin/nifedipine/MCC950/anakinra is a safe and inexpensive approach to treat CH and its associated abnormalities in obese individuals. To explain the mechanism of action of calcium channel blocker, nifedipine in combination with metformin, MCC950, anakinra treatment on Tet2-/- + Boy/J (1;1 ratio, mixed BM cells) transplanted into Ob/Ob mice, total RNA was isolated form the BM cells of 7 different drug treatment groups (Group 1: Vehicle, Group 2: Metformin, Group 3: Pioglitazone, Group 4: Nifedipine, Group 5: metformin+pioglitazone+MCC950+anakinra, Group 6: metformin+nifedipine+MCC950+anakinra and Group 7: Tet2-/-+Boy/J to WT (Un-treated) mice). RNA-sequencing (RNA-seq) was performed on samples from 3 independent mice from each group. treatment groups (Group 1: Vehicle, Group 2: Metformin, Group 3: Pioglitazone, Group 4: Nifedipine, Group 5: metformin+pioglitazone+MCC950+anakinra, Group 6: metformin+nifedipine+MCC950+anakinra and Group 7: Tet2-/-+Boy/J to WT (Un-treated) mice). RNA-sequencing (RNA-seq) was performed on samples from 3 independent mice from each group.