Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase

Abstract

Background: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood.

Methods: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF.

Results: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001).

Conclusions: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

Keywords: BTK protein, human; CSK tyrosine-protein kinase; atrial fibrillation; electrophysiology; ibrutinib; protein kinase inhibitors.

Figure 1.. Long-term ibrutinib treatment predisposes mice to AF.

Evaluation of ibrutinib effects on C57BL/6J wild-type mice. A, Experimental outline of ibrutinib administration in C57BL/6J mice. Mice received intraperitoneal injection of ibrutinib (IBR) or vehicle (CTL) at 25 mg·kg⁻¹·d⁻¹ for 4 weeks (28 days), followed by in vivo electrophysiology studies. B, Representative surface lead II ECG, intracardiac atrial and ventricular electrogram recordings showing AF after atrial burst pacing in ibrutinib-treated mice (IBR), but not in CTL. C, AF inducibility in CTL (n = 9) and IBR (n = 8). Data are mean±SEM percentage of successfully induced atrial arrhythmia episodes during invasive EP studies. ***P<0.0001, Fisher exact test. D, Histological images of Masson trichrome stain of LA fibrosis in CTL and IBR. E, Quantification of tissue fibrosis levels (%) in LA sections from CTL and IBR mice. Data are mean±SEM, n = 17 LA regions of interest from 5 CTL mice, n=22 LA regions of interest from 5 IBR mice. ***P<0.0001, unpaired Student t test. F, Mean±SEM of Fn1, Col1a1, and Col3a1 mRNA levels in LA tissues from CTL and IBR mice. n=5 mice per group. *P<0.05, **P<0.01, unpaired Student t test. G, Representative cardiac magnetic resonance images of 4-chamber views (left) and ventricular short axis views (right) from CTL and IBR mice. LA is highlighted in the 4-chamber view. H and I, Quantification of LA sizes (H) and left ventricular ejection fraction (I) measured by MRI after 4 weeks of ibrutinib treatment. Data are mean±SEM, n=6 mice in CTL and n=8 mice in IBR. *P<0.05, unpaired Student t test. J and K, Quantification of plasma levels of TNFα (J) and IL-6 (K) after 4 weeks of ibrutinib treatment. Data are mean±SEM, n=13 mice in CTL and n=11 mice in IBR. *P<0.05, unpaired Student t test (J) or Mann–Whitney test (K). L, Heatmap of expression levels of 40 differentially expressed genes from RNAseq data of 3 CTL and 3 IBR LA tissues. M, Gene set enrichment analysis shows 18 differentially regulated gene categories/pathways in LA after 4 weeks of ibrutinib treatment. Barplot of gene set enrichment analysis NES with false discovery rate on the right. AF indicates atrial fibrillation; CPM, counts per million; ECG, electrocardiography; LA, left atrial; and NES, normalized enrichment score.

Figure 2.. Ibrutinib causes atrial fibrillation through off-target effects.

Evaluation of ibrutinib effects on mice lacking BTK. A, Experimental outline of ibrutinib administration in BTK mutant CBA/CaHN-BTK^xid/J mice lacking active BTK (Btk^xid) and strain-matched wild-type CBA/CaJ (WT). Mice received intraperitoneal injection of ibrutinib (IBR) or vehicle (CTL) at 25 mg·kg⁻¹·d⁻¹ for 4 weeks (28 days), followed by in vivo electrophysiology studies. B, AF inducibility in WT and Btk^xid after 4-week ibrutinib or vehicle treatment. Data are mean±SEM percentage of successfully induced atrial arrhythmia episodes during invasive EP studies in WT CTL, n=5, WT IBR, n=7, Btk^xid CTL, n=6, and Btk^xid IBR n=8 mice. *P<0.05, Fisher exact test. C, Data are LA diameters measured by echocardiography before (D0) and after 4 weeks (D28) of ibrutinib (IBR) or vehicle (CTL) injections in WT and Btk^xid mice (n=7 mice in WT CTL; n=7 in WT IBR; n=5 in Btk^xid CTL; n=8 in Btk^xid IBR). *P<0.05, D0 vs D28, paired Student t test. D, Representative sections of Masson trichrome stain of LA fibrosis in CTL and IBR of WT and Btk^xid mice. E, Mean±SEM LA fibrosis levels (%) in LA sections of WT CTL (n=5 regions of interest from 2 mice), WT IBR (n=7 from 3 mice), Btk^xid CTL (n=6 from 3 mice), and Btk^xid IBR (n=9 from 4 mice). *P<0.05, ** P<0.01, 1-way ANOVA with Sidak posttest. F, Workflow of chemoproteomic kinase profiling of ibrutinib and acalabrutinib targets in mouse heart. Five kinases and 2 kinases detected to be inhibited by >90% were identified as ibrutinib or acalabrutinib targets in the heart. A Venn diagram shows the overlap of 2 kinases inhibited by both ibrutinib and acalabrutinib. G, Mean±SEM CSK protein levels normalized to GAPDH levels in paired LA and LV tissues from adult wild-type C57BL/6J mice (n=5 mice; bottom). **P<0.01, paired Student t test. CSK protein is detected at ≈50 kDa on Western blot (top). H, The expression level of CSK in human heart was evaluated from MS-based protein intensity measurements performed on heart biopsies collected from LA and LV from 6 living humans. Normalized raw intensities measured for CSK are shown. Individual measurements from LA or LV are depicted dots. The relative protein expression levels of CSK in atrial and ventricular biopsies were compared by a paired Student t test. P=0.009. The box in the box-and-whisker plot shows the quartiles of the dataset while the whiskers represent the CI (95%) by bootstrapping. AF indicates atrial fibrillation; BTK, Bruton tyrosine kinase; CSK, c-Src terminal kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; i.p., intraperitoneal; LA, left atria; LC, liquid chromatography; LV, left ventricle; and MS, mass spectrometry.

Figure 3.. Cardiac-specific knockout of CSK causes AF and increases LA interstitial fibrosis, LA size, CD68+ macrophages, and plasma TNFα and IL-6 levels.

A, Experimental outline. Cardiac specific deletion of Csk was induced in αMHC^mercremer Csk^fl/fl mice after 5 daily doses of tamoxifen injection (αMHC Csk^−/−). Littermate Csk^fl/fl mice receiving the 5-dose tamoxifen treatment were used as control (CTL). In vivo electrophysiology studies were performed on day 32, 4 weeks after the last dose of tamoxifen. B, Polymerase chain reaction analyses of Csk^fl/fl (CTL) and αMHC^mercremerCsk^fl/fl (αMHC Csk^−/−) heart and spleen tissues 4 weeks posttamoxifen show cardiac specific deletion of Csk allele (Csk^Δ) in αMHC Csk^−/− mice. C, Atrial fibrillation inducibility in CTL and αMHC Csk^−/− mice 4 weeks after tamoxifen treatment. Data are mean±SEM percentage of successfully induced atrial arrhythmia episodes during invasive EP studies. n=6 mice per group, ***P<0.0001, Fisher exact test. D, Images of Masson trichrome stain of LA fibrosis in CTL and αMHC Csk^−/− mice. E, Mean±SEM LA fibrosis levels (%) in LA sections of CTL (n=8 ROIs from 3 mice) and αMHC Csk^−/− (n=13 from 3 mice) mice. ***P<0.0001, unpaired Student t test. F, Immunofluorescence images of LA sections stained for CD68 (green) and DAPI(blue) in CTL and αMHC Csk^−/− mice. G, Quantification of CD68⁺ cells in LA sections of CTL (n=16 HPFs from 3 mice) and αMHC Csk^−/− (n=13 from 3 mice). Data are mean±SEM; ***P<0.0001, unpaired Student t test. H, Representative cardiac magnetic resonance images of CTL and αMHC Csk^−/− mice. I, Data are LA volumes obtained by cardiac MRI on CTL and αMHC Csk^−/− mice before and 4 weeks posttamoxifen treatment (n=3 mice for CTL and n=4 mice for αMHC Csk^−/−). Data are mean±SEM, *P<0.05, paired Student t test. J and K, Quantification of plasma TNFα (J) and IL-6 (K) levels in CTL (n=7) and αMHC Csk^−/− (n=7) mice. Data are mean±SEM, *P<0.05, ** P<0.01, nonparametric Mann–Whitney test. L, Heatmap of expression levels of 896 differentially expressed genes from RNAseq data of 2 CTL and 2 αMHC Csk^−/− LA tissues. M, Gene set enrichment analysis shows 18 differentially regulated categories/pathways in LA after cardiac Csk knockout. Barplot of gene set enrichment analysis NES with false discovery rate on the right. N, A Venn diagram shows the overlap of 13 differentially regulated gene categories/pathways between ibrutinib-treated mice and cardiac Csk knockout mice. AF indicates atrial fibrillation; CPM, counts per million; CSK, C-terminal Src kinase; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high power fields under 40× objective; LA, left atria; NES, normalized enrichment score; and ROI, region of interest.

Figure 4.. CSK inhibition is highly associated with atrial fibrillation in individual case safety reports gathered in the international pharmacovigilance database, VigiBase.

A, Data are C_max/CSK IC₅₀ against atrial fibrillation information component (a Bayesian disproportionality estimator) for 16 kinase inhibitors that are reported to inhibit CSK with various potency (IC₅₀s range from 2.2 nmol/L to 66 μmol/L; Table V in the Data Supplement) out of all kinase inhibitors approved by U.S. Food and Drug Administration as anticancer drugs. CSK inhibitors are defined as C_max/CSK IC₅₀>1, and mild or not CSK inhibitors have C_max/IC₅₀<1. B, ROR for AF cases over total number of ICSRs on CSK inhibitors compared with the non-CSK inhibitors (mild or not CSK inhibitors). AF indicates atrial fibrillation; CSK, C-terminal Src kinase; ICSR, individual case safety reports; and ROR, reporting odds ratio.