Nilotinib-induced vasculopathy: identification of vascular endothelial cells as a primary target site

Abstract

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

Figure 1. Nilotinib exerts pro-atherogenic and anti-angiogenic effects in vivo.

(A) ApoE-/- mice were fed with a high-caloric lipid-diet and treated either with vehicle control (Control, 9 mice), Imatinib (50 mg/kg twice daily, 9 mice) or Nilotinib (37.5 mg/kg twice daily, 8 mice) for 8 weeks. Then, mice were sacrificed and their aortic roots were evaluated for plaque-formation. Results are expressed as percent of aortic areas affected by plaque-formation per mouse and show all mice in each group as well as median values (black bars). The difference in plaque-formation between the control group and Nilotinib group was statistically significant (p<0.05) by Mann-Whitney test. Overall, however, no statistically significant differences were found when comparing all three mouse groups by Kruskal Wallis test. (B) Hind-limb ischemia was induced in C57BL/6 wild-type mice by ligation of their left femoral artery. Thereafter, mice (13 mice per group) were treated with vehicle control, Imatinib (50 mg/kg twice daily) or Nilotinib (75 mg/kg/day) for 28 days. The ratio of blood flow in the ischemic versus non-ischemic limb was assessed by Laser Doppler Perfusion Imaging (LDPI). Results represent the mean±S.D. of all mice in each group. (C) Bone marrow sections were obtained from 7 CML patients before and after Nilotinib treatment for more than 1 year. Sections were stained using an antibody against CD34 to assess microvessel density. Results show microvessels per HPF and represent the mean±S.D. of all patients. Statistical tests: (B) Kruskal Wallis test; (C) Wilcoxon matched-pairs signed ranks test. *: p<0.05.

Figure 2. Nilotinib inhibits growth and tube formation of endothelial cells in vitro.

(A) Human umbilical vein endothelial cells (HUVEC), the human endothelial cell line HMEC-1, and human coronary artery endothelial cells (HCAEC) were incubated with control medium or various concentrations of Imatinib or Nilotinib at 37°C for 48 hours. Thereafter, proliferation was examined by measuring ³H-thymidine uptake. Results are expressed as percent of control and represent the mean±S.D. of at least 3 independent experiments. (B) HUVEC cells were cultured in medium containing 0.5% bovine serum albumin in the absence (left part of image) or presence (right part of image) of VEGF (50 ng/ml) together with various concentrations of Imatinib or Nilotinib (as indicated) for 16 hours. Results show the relative BrdU-uptake compared to control and represent the mean±SD of 4 independent experiments. (C) HUVEC were cultured in matrigel in the absence or presence of VEGF (50 ng/ml) (as indicated) together with 0.1 µM Imatinib or 0.1 µM Nilotinib at 37°C for 6 hours. Then capillary tubes were counted. Results show tube formation relative to control and represent the mean±S.D. of 3 independent experiments. Statistics: (A-C) unpaired, two-way t-test. *: p<0.05.

Figure 3. TKI target expression profiles in endothelial cells.

Target expression profiles for Nilotinib and Imatinib determined by chemical proteomics profiling (CPP) and mass spectrometry (MS) performed with lysates obtained from HUVEC (A) and HMEC-1 (B). The figures show targets bound by Nilotinib (red color), Imatinib (green) or both drugs (blue). Kinase targets are indicated by colored ellipses and non-kinase targets by grey ellipses.

Figure 4. Risk factors for vascular events in TKI-treated patients.

(A) Our Nilotinib-treated patients were separated into three risk-groups according to the European Society of Cardiology (ESC) SCORE. The numbers of patients in each ESC-cohort were: 17 in ESC 0-1; 13 in ESC 2-4; and 5 in ESC ≥5. The percentage of patients without AOD (open bars) and with AOD (black and grey bars) is shown for each group. The black parts of the black and grey bars represent the percentages of severe AOD events relative to total AOD events. (B) The curves show the reduction of the cumulative fraction of individuals without AOD in our Nilotinib-treated patients and a risk-factor-matched population in high-income countries described in a meta-analysis by Fowkes et al.. The difference in reduction of the cumulative fraction between the two groups is statistically significant by log-rank test: p<0.05. (C) Overview of ARCH-related gene variants in all 36 Nilotinib-treated patients. Peripheral blood samples were analyzed for the presence of ARCH mutations at the time of best response. White numbers indicate the variant allele frequencies. The right column shows BCR-ABL1 mRNA levels (% of ABL1) at the time of sampling; levels lower than 1% are depicted in green, levels above 1% in red. The difference in the percentages of patients carrying ARCH mutations between the two groups (AOD patients versus patients without AOD) was significant (p<0.05) as assessed by the chi-squared test. AOD: Arterial occlusive disease; ARCH: age-related clonal hematopoiesis (at least one ARCH-mutation detected).