A Murine Model with JAK2V617F Expression in Both Hematopoietic Cells and Vascular Endothelial Cells Recapitulates the Key Features of Human Myeloproliferative Neoplasm

Introduction Although murine models have provided unequivocal evidence that the JAK2V617F mutation is able to cause myeloproliferative neoplasms (MPNs), there is significant heterogeneity in disease phenotypes between different murine models and none has been able to recapitulate both the myeloproliferative phenotype and cardiovascular pathology seen in patients with MPNs. In addition, these murine models were mostly followed for less than 3-9 months and how aging affects MPN disease progression has not been studied. Endothelial cells (ECs) are an essential component of the hematopoietic niche, and they have been shown to express the JAK2V617F mutation in patients with MPNs. In this study, we investigated how MPN progresses in the JAK2V617F-bearing vascular niche during aging. Methods JAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) were crossed with Tie2-cre mice to express JAK2V617F specifically in all hematopoietic cells (including HSPCs) and vascular ECs (Tie2FF1). Results The Tie2FF1 mice developed essential thrombocythemia to primary myelofibrosisdisease transformation with extramedullary splenic hematopoiesis during 18-month follow up. No evidence of leukemia transformation was observed in the Tie2FF1 mice. (Figure 1) Hematopoietic colony formation assays, flow cytometry analysis, and in vitro culture experiments revealed that there was a loss of both HSPC number and HSPC function in the marrow of old Tie2FF1 mice during aging, mimicking the advanced phases of myelofibrosis. In contrast, the spleen of old Tie2FF1 mice was able to maintain the expansion of JAK2V617F mutant hematopoiesis during aging and MPN disease progression. (Figure 2) These differences between marrow and spleen hematopoiesis in the old Tie2FF1 mice prompted us to investigate how aging affects the JAK2V617F mutant hematopoiesis differently in the marrow and spleen. We found that, although the JAK2V617F mutant HSCs (Lin -cKit +Sca1 +CD150 +CD48 -) from old Tie2FF1 mice were more proliferative than wild-type HSCs in both the marrow and spleen, mutant marrow HSCs were more apoptotic and senescent than wild-type HSCs in the marrow while mutant spleen HSCs were relatively protected in the spleen. Examination of the hematopoietic vascular niche revealed that marrow ECs (CD45 -CD31 +) were significantly decreased in old Tie2FF1 mice compared to age-matched control mice; in contrast, spleen ECs were significantly expanded and less senescent in old Tie2FF1 mice compared to control mice. Therefore, the different vascular niche function of the marrow and spleen could contribute to the decreased marrow hematopoiesis and expanded splenic hematopoiesis we have observed in the Tie2FF1 mice during aging. (Figure 3) Previously, we reported that the Tie2FF1 mice developed spontaneous heart failure with thrombosis, vasculopathy, and cardiomyopathy at 20wk of age. Here, we followed the cardiovascular function of Tie2FF1 mice during aging. At 18mo of age, the Tie2FF1 mice continued to demonstrate a phenotype of dilated cardiomyopathy with a moderate but significant decrease in left ventricular ejection fraction and an increase in left ventricular volume and mass compared to age-matched control mice. Histology examination revealed spontaneous thrombosis in the right ventricle, pulmonary arteries, both main (epicardial) coronary arteries and scattered coronary arterioles (microvessels) in the old Tie2FF1 mice, while age-matched Tie2-cre control mice had no evidence of spontaneous thrombosis in their heart or lungs. Despite these cardiovascular dysfunctions, there was no difference in body weight nor was there any increased incidence of sudden death between the old Tie2FF1 mice and control mice. These findings suggested that there was a persistent but compensated cardiomyopathy and heart failure in the Tie2FF1 mice during aging. (Figure 4) Conclusion Compared to other MPN murine models reported so far, the Tie2FF1 mice is the first MPN murine model that faithfully recapitulated almost all the key features of the human MPN diseases. Considering the presence of the JAK2V617F mutation in microvascular ECs isolated from patients with MPNs and the recapitulation of all the key features of human MPN diseasesby the Tie2FF1 mice, the roles of endothelial dysfunction in the hematologic and cardiovascular pathogenesis of MPN shall be further investigated. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Inflammatory risk factors are not associated with coronary artery calcification in patients with myeloproliferative philadelphia-negative neoplasms

Background Myeloproliferative Philadelphia-negative Neoplasms (MPNs) are hematological cancers associated with chronic inflammation and endothelial dysfunction, conditions that may lead to development of premature atherosclerosis. Purpose To investigate whether biomarkers of inflammation and endothelial dysfunction are associated with the degree of atherosclerotic burden, measured by Coronary Artery Calcium Score (CACS), in patients with MPNs. Methods Patients with a validated MPN diagnosis; essential thrombocythemia (ET), polycythemia vera (PV) or myelofibrosis (MF), were recruited between 2016 and 2018 from one single specialized hematologic center. Patients filled out a standardized questionnaire on medical history, current medication, alcohol and smoking habits and family medical history. They were examined by cardiac computed tomography (CT), Endothelial Peripheral Arterial Tone (EndoPAT), and a range of blood analyses. The atherosclerotic burden was evaluated by CACS. High sensitivity C-reactive protein (hs-CRP) and Neutrophil:Lymphocyt Ratio (NLR) were used as indicators of chronic inflammation. EndoPAT was applied to evaluate endothelial dysfunction, which is linked to development of atherosclerosis. The JAK2V617F-mutation is a common gene mutation in MPN-patients. It affects the gene coding the Janus kinase 2 (JAK2) protein, and can be detected by qPCR of peripheral blood or bone marrow. The JAK2V617F-mutation is a risk factor associated with both chronic inflammation and endothelial dysfunction. Multivariable logistic regression analyses were used to identify associations between potential risk factors and a higher CACS value. Results Among 170 included patients, 161 patients completed cardiac CT (mean age 65.5 (SD 10.5), 52% men). Baseline data is presented in Table 1. JAK2V617F-mutation was found in 137 (85%) patients, 53 patients (35%, n=152) had hs-CRP>2.0 mg/L, 107 (67%, n=160) had NLR>2.15 and 32 (21%, n=154) had an abnormal EndoPAT. Overall, 66 patients (41%) had a CACS>100, with no significant difference between ET (41%), PV (42%) and MF (50%) (p=0.3). In patients with a history of ischemic heart disease (IHD), 92% had CACS>100, compared to 37% in patients without prior IHD (p=0.0003). Five independent factors associated with a CACS>100 were identified; age (OR: 1.3 [95% CI 1.1–1.4]), male sex (OR: 15.2 [95% CI 4.0–57.7]), prior IHD (OR: 15.9 [95% CI 1.2–202.4]), smoking (OR: 3.3 [95% CI 1.1–10.1]), and abnormal EndoPAT (OR: 4.8 [95% CI 1.1–20.0]) (Figure 1). Hs-CRP, NLR and JAK2V617F-mutation status were not significantly associated with CACS >100. Conclusion In this cohort of patients with MPNs, markers of chronic inflammation like hs-CRP and NLR were not associated with higher CACS, nor was the JAK2V617F-mutation. Traditional risk factors of cardiovascular disease seem to be sufficient to identify MPN patients with increased atherosclerotic burden, but measuring endothelial dysfunction provides additional information. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Department of Cardiology, Zealand University Hospital, Region Zealand, Denmark Table 1. Baseline characteristics Figure 1. Odds ratio for CACS >100