Obesity-Induced Inflammation Co-Operates with Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mutants to Promote Leukemia Development and Cardiovascular Disease
Authors: Pasupuleti Santhosh Kumar , Ramdas Baskar , Burns Sarah S. , Kumar Ramesh , Pandhiri Taruni Reddy , Sandusky George , Yu Zhi , Honigberg Michael , Bick Alexander G. , Griffin Gabriel K. , Niroula Abhishek , Ebert Benjamin L. , Natarajan Pradeep , Kapur Reuben 
Published: November 2021

Abstract

Obesity is an increasing epidemic world-wide responsible for enhancing the risk for developing Type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and cancer. However, it is unclear if and how obesity contributes to the transformation of pre-leukemic stem and progenitors (pre-LHSC/Ps) into full-blown leukemia such as acute myeloid leukemia (AML) or severe form of myeloproliferative neoplasm (MPN) or CVD. We hypothesized that obesity induced chronic inflammation might be responsible for clonal selection of pre-LHSC/Ps bearing pre-leukemic clonal hematopoiesis of indeterminate potential (CHIP) mutations such as DNMT3A, TET2, ASXL1, and JAK2 and for promoting the progression of early-onset MPN, AML/leukemia and CVD. To study the linkage between obesity and CHIP in humans, we first examined the UK biobank. After exclusions, the final study cohort included 47,466 unrelated participants free of T2DM at baseline and having valid CHIP measurements. The mean (SD) age at enrollment was 56.5 (8.0), 45.0% were male, 43.9% never smoked, and 82.6% self-reported as European decedents. At baseline, the mean (SD) body mass index (BMI) was 27.3 (4.7) kg/m 2, with 43.0% overweight and 23.6% obese, and the overall mean (SD) waist-to-hip ratio (WHR) was 0.87 (0.09). CHIP was present among 5.8% of the study population the most common mutations on the DNMT3A (3.7%) and TET2 (1.0%) genes; large CHIP clone defined as CHIP mutation with variant allele fraction &gt;10% was present among 2.4% of the study population. Individuals with CHIP mutations on average had higher WHR. The presence of CHIP mutation was associated with a 0.0028 increase of WHR (p=0.03). Furthermore, CHIP prevalence increased with higher WHR: the percentage of participants with CHIP was 4.93%, 5.75%, 6.56% in the lowest, middle, and highest WHR quintiles respectively, signifying that dysfunctional metabolism may accelerate expansion of clonal hematopoiesis (CH). To better define the mechanism of obesity driven CH, we utilized several novel mouse models bearing Tet2 -/-, Dnmt3a +/-, Asxl1 +/- and Jak2 +/- mutations to mimic the human pre-LHSC/Ps condition and obesity, in the form of leptin deficient Lep Ob/Ob (Ob/Ob) mutation, which induces obesity and T2DM. We show that both the compound mutant (Tet2 -/-;Ob/Ob, Dnmt3a +/-;Ob/Ob, Asxl1 +/-;Ob/Ob and Jak2 +/-;Ob/Ob) and CHIP mutant bone marrow (BM; Tet2 -/-, Dnmt3a +/-, Asxl1 +/- and Jak2 +/-) transplanted into Ob/Ob mice develop rapid growth of mature myeloid cells and HSC/Ps leading to severe form of MPN/AML as well as CVD. This was associated with upregulation of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. Flow cytometry analysis of LSK and progenitor cells isolated from Tet2 -/-;Ob/Ob mice revealed an up-regulation of intracellular Ca2+ levels. We hypothesized that up-regulated Ca2+ signaling in Tet2 -/-; Ob/Ob HSC/Ps promotes aberrant signaling leading to an early-onset of severe MPN/AML. We performed a competitive transplantation experiment using, Tet2 -/-: Boy/J BM cells (1:1 ratio) into Ob/Ob and WT recipients. After 8 weeks post transplantation, we investigated the role of Ca2+ blockers in driving CH in Ob/Ob recipients using pharmacological inhibitors, either individually, or in combination, of metformin (100 mg/kg, orally), nifedipine (100 mg/kg, orally), MCC950 (30 mg/kg, orally) and anakinra (10 mg/kg, i.p). The combination treatment markedly reduced monocytes, neutrophils, WBC counts, and improved RBCs, hematocrits and platelets. The spleen and liver, heart, body weights and blood glucose levels were significantly reduced, along with a greater re-emergence of normal CD45.1 wild-type cells in the PB, BM, and spleen and a significant reduction in Tet2 mutant CD45.2 pre-LHSC/Ps and myeloid cells in the PB, BM, and spleen. Importantly, the frequency of leukemic blasts, LSK cells, ST-HSCs, LT-HSCs and granulocyte macrophage progenitors (GMP) were significantly reduced. Furthermore, the combination of drug treatment showed greater heart protective activity by reducing the atherosclerotic lesions in Ob/Ob recipients bearing CHIP by suppressing Ca2+ signaling. Taken together, these data suggest that obesity is highly associated with the presence of CHIP in humans and that targeting CHIP mutant cells with a combination of metformin/nifedipine/MCC950/anakinra is a safe and inexpensive way to rescue CH and its associated leukemic and cardiovascular defects.

Disclosures
Natarajan: Amgen: Research Funding; Apple: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Boston Scientific: Research Funding; Blackstone Life Sciences: Consultancy; Genentech: Consultancy; Foresite Labs: Consultancy.