The anti-MPL antibody AMM2 prevents cardiovascular dysfunction in a mouse model of myeloproliferative neoplasms Free

Introduction Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders marked by expansion of hematopoietic stem/progenitor cells and overproduction of mature, often dysfunctional blood cells. Up to 40-50% of patients develop arterial or venous thrombosis, with cardiovascular diseases (CVDs) the leading cause of morbidity and mortality — far exceeding age-matched controls. The acquired kinase mutation JAK2V617F is central to MPN pathogenesis and is found in both blood and vascular endothelial cells (ECs). Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoiesis. MPL is also expressed on vascular ECs, and TPO/MPL signaling can influence cardiovascular function. We previously showed MPL contributes to CVD in MPN mouse models with JAK2V617F-mutant ECs. Because JAK2V617F-mutant blood cells, even in clonal hematopoiesis with normal blood counts, are associated with markedly increased cardiovascular risk, we investigated whether targeting MPL with the antibody AMM2 could prevent CVD in murine models of JAK2V617F-mutant hematopoiesis.

Methods Chimeric mice with JAK2V617F-mutant blood cells and wild-type ECs were generated by bone marrow transplantation. 8 weeks post-transplant, after full donor engraftment, mice were assigned to: (1) regular chow diet, (2) high-fat diet, or (3) high-fat diet plus the AMM2 (IBL #10401; 0.1mg/kg, i.v. every 10 days for ~40 days). Chimeric mice with wild-type blood cells on either chow diet or high-fat diet served as controls.

Results At 8 weeks post-transplant, JAK2V617F-mutant mice showed elevated neutrophiles (2 vs. 0.7 x 10³/ul), hemoglobin (15 vs 13 g/dL), and platelets (380 vs 282 x 10³/ul) compared to wild-type controls; AMM2 treatment did not affect their blood counts. On regular chow diet, mutant mice maintained normal cardiac function over 6 months by serial transthoracic echocardiography. After 5 weeks of high-fat diet, they developed reduced left ventricular (LV) ejection fraction (59 vs. 68%, p=0.001) and fractional shortening (31 vs. 37%, p=0.001), with increased LV end-systolic (21 vs. 14ul, p<0.001) and end-diastolic (51 vs. 43ul, p=0.027) volumes compared to wild-type controls (n=10-15 per group). No overt atherosclerosis, infarction, thrombosis, or coronary arteriole stenosis was observed. However, histology revealed endocardial disruption and increased perivascular collagen.

Next, we tested whether AMM2 could prevent high-fat diet-induced cardiovascular dysfunction in JAK2V617F-mutant mice. AMM2-treated mutant mice (n=14) maintained LV ejection fraction (68%), fractional shortening (37%), and LV end-systolic (15 vs. 14ul) and end-diastolic volumes (47 vs. 43ul) comparable to wild-type controls. Histology showed that AMM2 largely reversed high-fat diet-induced endocardial pathology and perivascular fibrosis in all examined mice (n=6).

To investigate the mechanisms of high-fat diet-induced CVD in JAK2V617F-mutant MPN mice and how AMM2 provides protection, we performed single-cell RNA sequencing of cardiac cells. Compared with regular diet, high-fat diet markedly increased cardiac ECs, fibroblasts, and smooth muscle cell populations in mutant mice; these changes were reversed by AMM2. Gene set enrichment analysis in cardiac ECs revealed upregulated endothelial-to-mesenchymal transition (EndoMT), TNFa/NFkB signaling, coagulation, P53 pathway, and inflammatory response pathway in high-fat diet-treated mutant mice versus diet-matched wild-type controls; most of these changes occurred in the endocardial ECs. AMM2 largely reversed these transcriptional changes.

Taken together, our data show that JAK2V617F-mutant blood cells induce endocardial disruption and EndoMT under high-fat diet challenge, leading to cardiovascular dysfunction. These effects can be prevented by the c-MPL monoclonal antibody AMM2, likely through inhibition of endocardial dysfunction and EndoMT. Given the pivatol role of endocardial ECs in postnatal coronary angiogenesis and cardiac function, targeting endocardial EC function and EndoMT may represent a promising therapeutic strategy for CVDs associated with mutant clonal hematopoiesis.