Involvement of Rho-Associated Coiled-Coil Containing Kinase (ROCK) in BCR-ABL1 Tyrosine Kinase Inhibitor Cardiovascular Toxicity

Abstract

Background: Second- and third-generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) are associated with cardiovascular adverse events (CVAEs) in patients with Philadelphia chromosome-positive (Ph+) leukemia.

Objectives: We hypothesized that second- and third-generation BCR-ABL1 TKIs may cause CVAEs through the activation of Rho-associated coiled-coil containing kinase (ROCK).

Methods: Peripheral blood mononuclear cells from 53 Ph+ patients on TKIs and 15 control patients without Ph+ leukemia were assessed for ROCK activity through capillary electrophoresis (median follow-up = 26 months [Q1-Q3: 5-37 months]). We also investigated the effects of TKIs and ROCK on endothelial dysfunction in vitro, which could contribute to CVAEs.

Results: Patients receiving second- and third-generation TKIs had 1.6-fold greater ROCK activity compared with patients receiving imatinib and control patients. Elevated ROCK activity was associated with an increased incidence of CVAEs in Ph+ leukemia patients. In endothelial cells in vitro, we found that dasatinib and ponatinib treatment led to changes in actin intensity and endothelial permeability, which can be reversed by pharmacologic inhibition of ROCK. Ponatinib led to decreased cell proliferation, but this was not accompanied by senescence. Dasatinib and ponatinib treatment led to phosphor-inhibition of endothelial nitric oxide synthase and decreased nitric oxide production. ROCK inhibition reversed endothelial permeability and endothelial nitric oxide synthase-related endothelial dysfunction. Imatinib and nilotinib induce phosphorylation of p190RhoGAP.

Conclusions: Our findings suggest ROCK activity may be a prognostic indicator of CVAEs in patients receiving BCR-ABL1 TKIs. With further study, ROCK inhibition may be a promising approach to reduce the incidence of CVAEs associated with second- and third-generation BCR-ABL1 TKIs.

Keywords: 2G/3G, second/third generation; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; CVAE, cardiovascular adverse event; PBMC, peripheral blood mononuclear cell; Ph+, Philadelphia chromosome-positive; Philadelphia chromosome; ROCK, Rho-associated coiled-coil containing kinase; Rho-associated protein kinase; TKI, tyrosine kinase inhibitor; chronic myeloid leukemia; eNOS, endothelial nitric oxide synthase; endothelial dysfunction; tyrosine kinase inhibitor.

Graphical abstract

Figure 1

ROCK Activity Association With 2G/3G BCR-ABL1 TKI Treatment and Cardiovascular Adverse Events Rho-associated coiled-coil containing kinase (ROCK) activity was assessed from patient peripheral blood mononuclear cells by capillary electrophoresis by taking the ratio of phospho-Thr⁸⁵³ myosin phosphatase target subunit 1 (MYPT1) to MYPT1. (A) ROCK activity of patients by therapy. Patients who switched between tyrosine kinase inhibitors (TKIs) were included corresponding to the most recent TKI actively received. If a patient initially not on TKI was later active on TKI, they were included in both groups (n = 7). (B) ROCK activity change for patients before and during TKI initiation (n = 7). (C) Kaplan-Meier survival curves in TKI-receiving patients comparing the incidence of cardiovascular adverse events (CVAEs) between patients with ROCK activities above/below the third quartile (1.24). The P value is from the log-rank test. Error bars represented as SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

Second-/Third-Generation BCR-ABL1 TKIs Increase ROCK Activity in Endothelial Cells Immunoblots and quantification of ROCK activity (the ratio of phosphor-Thr853 MYPT1 to total MYPT1) of human aortic endothelial cells treated with 0 to 1,000 nmol/L of (A) imatinib, (B) nilotinib, (C) dasatinib, and (D) ponatinib for 24 hours. n = 10 to 15 per treatment concentration. Error bars represented as SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. GAPDH = glyceraldehyde-3-phosphate dehydrogenase; other abbreviations as in Figure 1.

Figure 3

Ponatinib and Dasatinib Alter Actin and Permeability of Endothelial Cells (A) Human aortic endothelial cells were cultured on coverslips coated with gelatin and treated or cotreated with 250 nmol/L imatinib, nilotinib, dasatinib, or ponatinib; 20 μmol/L Rho-associated coiled-coil containing kinase inhibitor Y-27632 (Y), or control (dimethyl sulfoxide) for 24 hours. F-actin and nuclei were stained with phalloidin and 4′,6-diamidino-2-phenylindole, respectively, to visualize the effect of treatments on cellular actin organization. Images are representative of 3 to 5 fields per condition. (B) Quantification of the F-actin intensity in endothelial cells. (C) Quantification of fluorescein isothiocyanate–labeled 40 kDA dextran in transwell permeability assays after 24 hours. Scale bars in A are 50 μm. For actin fluorescence intensity, n = 5 views per treatment. For transwell permeability assays, n = 5. Error bars represented as SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

Second-/Third-Generation TKIs Lead to Endothelial Cell Dysfunction Immunoblots and quantification of Tyr⁴⁹⁵ endothelial nitric oxide synthase (eNOS) phosphorylation to total eNOS in human aortic endothelial cells treated with 0 to 1,000 nmol/L (A) imatinib (ima), (B) nilotinib (nilo), (C) dasatinib (dasa), and (D) ponatinib (pona) with/without 20 μmol/L ROCK inhibitor Y-27632 (Y) for 24 hours. (E) Visualization of nitric oxide concentration in endothelial cells with the fluorescent probe 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM). (F) Quantification of DAF-FM fluorescence. (G) Quantification of nitrite via the Griess reaction per 50,000 endothelial cells. For E and F, all treatments were performed with 250 nmol/L of each respective TKI (ima, nilo, dasa, and pona) with and without 20 μmol/L Y-27632 for 24 hours while control (con) indicates treatment with dimethyl sulfoxide for 24 hours. n = 3 to 6 per treatment. Error bars represented as SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 1.

Figure 5

Tyrosine Kinase Inhibitors Exert Differential Phosphorylation Patterns on p190RhoGAP (A) Immunoblotting and (B) quantification of phosphorylation of Tyr¹¹⁰⁵ on p190RhoGAP to total p190RhoGAP after 250 nmol/L treatment of imatinib, nilotinib, dasatinib, or ponatinib at 15 minutes, 2 hours, and 24 hours in human aortic endothelial cells. n = 6 per treatment. Error bars represent SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Central Illustration

Activation of Rho-Associated Coiled-Coil Containing Kinase by Second-Generation/Third-Generation Tyrosine Kinase Inhibitors Can Increase Cardiovascular Adverse Events Rho-associated coiled-coil containing kinase (ROCK) may play a role in the cardiotoxicity of second-/third-generation (2G/3G) tyrosine kinase inhibitors (TKIs). (Left) Our clinical results indicate that patients with Philadelphia chromosome–positive (Ph+) leukemia on 2G/3G TKIs have elevated ROCK activity compared with control patients without Ph+ leukemia and patients receiving imatinib. Patients above the third quartile of ROCK activity have a higher incidence of cardiovascular adverse events. (Right) Our in vitro results suggest that each TKI can also exert specific effects on endothelial cells. Imatinib and nilotinib lead to p190RhoGAP phosphorylation. Nilotinib, dasatinib, and ponatinib lead to ROCK activation. Dasatinib and ponatinib lead to increased actin intensity, endothelial permeability, and endothelial dysfunction. Pharmacologic ROCK inhibition can reverse TKI-induced changes in endothelial function. Increase (green up arrow), decrease (red down arrow), and no change (yellow horizontal arrow).

Figure 6

BCR-ABL1 TKIs and the Rho/ROCK Signaling Pathway ARG can directly phosphorylate p190RhoGAP at tyrosine 1105, which leads to cycling of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) on RhoA, a small GTPase, inactivating RhoA. Our study indicates that imatinib and nilotinib lead to hyperphosphorylation of p190RhoGAP, although the exact mechanisms underlying this are not well characterized. GTP-bound RhoA leads to ROCK activation, which affects diverse signaling pathways, including directly phosphorylating eNOS at threonine 495 and MYPT1 at threonine 853. Our study indicates that dasatinib and ponatinib lead to ROCK activation. However, the mechanisms underlying this are unclear and likely to include effects that are independent of p190RhoGAP phosphorylation. Abbreviations as in Figures 1 and 4.