Further Evaluation of Pro-Atherogenic and Anti-Angiogenic Effects of Nilotinib in Mice and in Patients with Ph-Chromosome+ CML
Authors: Hadzijusufovic Emir , Albrecht-Schgoer Karin , Huber Kilian , Grebien Florian , Eisenwort Gregor , Schgoer Wilfried , Kaun Christoph , Herndlhofer Susanne , Theurl Markus , Cerny-Reiterer Sabine , Hoermann Gregor , Sperr Wolfgang R , Rix Uwe , Sadovnik Irina , Jilma Bernd , Schernthaner Gerit Holger , Wojta Johann , Wolf Dominik , Superti-Furga Giulio , Kirchmair Rudolf , Valent Peter 
Published: October 2019

Abstract
Nilotinib, an inhibitor of BCR/ABL1 is increasingly used to treat patients suffering from chronic myeloid leukemia (CML). However, treatment with nilotinib is associated with the occurrence of vascular adverse events, including progressive atherosclerosis resulting in arterial occlusive disease (AOD). We have recently shown that these events are recurrent and severe and accumulate over time in our patients (n=36). The percentage of patients developing i) one or more clinically overt (symptomatic) AOD-related events and ii) one or more clinically severe AOD events requiring surgical intervention and/or prolonged hospitalization increased from 26.5% and 17.6% at a median observation time (MOT) of 24 months to 44.4% and 19.4% after a MOT of 44 months. The frequency of AOD was lower in all control cohorts examined, including risk factor- and age-matched CML patients treated with imatinib, patients suffering from myelodysplastic syndromes, patients with JAK2-mutated myeloproliferative neoplasms and patients with lymphoid neoplasms. In order to explore the potential pro-atherogenic potential of nilotinib, we employed ApoE knock-out mice. In these mice, treatment with nilotinib (75 mg/kg/day p.o. for 8 weeks) was found to promote plaque formation and thus atherosclerosis when compared to control-mice (percent aortic plaque-area: vehicle-control: 16±3.2%; imatinib: 19.4±8.2%; nilotinib: 22.4±2.9%; p<0.005 for nilotinib vs control). To evaluate the effects of nilotinib on vascular repair processes following stenosis, we also employed a mouse model of hindlimb ischemia. Here, nilotinib (75 mg/kg/day p.o. for 28 days) was found to decrease reperfusion after induction of ischemia whereas imatinib (100 mg/kg/day p.o. for 28 days) showed no comparable effect as determined by laser Doppler perfusion imaging. The decreased perfusion seen in the nilotinib-treated mice was accompanied by an increased rate of limb necrosis as well as a decrease in microvessel density when compared to imatinib-treated mice or control-mice (p<0.05). In addition, we found that the pro-atherogenic cytoadhesion molecule VCAM-1 is expressed at higher levels in vascular cells in nilotinib-treated mice compared to imatinib-treated mice or control animals (cells/high power field: control: 13.9±11.9; imatinib: 14.9±11; nilotinib: 22.6±9.3). In a next step, we examined the in vitro effects of nilotinib on cultured human umbilical vein endothelial cells (HUVEC). In these experiments, we found that nilotinib (between 1-10 µM), but not imatinib (1-10 µM) promotes the expression of the cytoadhesion molecules ICAM-1, VCAM-1 and E-Selectin on HUVEC, confirming our data obtained in mice. In addition, we were able to confirm the anti-angiogenic effects of nilotinib seen in mice by our in vitro experiments. In particular, nilotinib was found to inhibit the migration of HUVEC in a wound-scratch assay as well as angiogenesis in a tube-formation assay whereas imatinib showed no comparable effect. Moreover, nilotinib was found to inhibit the proliferation of HUVEC in a dose-dependent manner (IC50: 1.0 µM) whereas imatinib showed no substantial effect up to 5 µM. Finally, we examined bone marrow (BM) microvessel density in 8 CML patients before and during treatment with nilotinib (800 mg/day for at least 1 year). In these experiments, we found that the numbers of CD34+ endothelial cells per high power field decreases substantially during treatment with nilotinib (before nilotinib: 12.3±2 versus post-nilotinib: 5.5±2.6, p<0.05). In summary, nilotinib exerts direct proatherogenic and growth-inhibitory and thus anti-angiogenic effects on vascular endothelial cells. Whether these effects contribute to nilotinib-associated vasculopathy in patients with CML is currently under investigation.

Disclosures
Wolf: BMS: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Valent:Pfizer: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Consultancy, Honoraria, Research Funding.