Phosphoproteomics reveals a novel mechanism underlying the proarrhythmic effects of nilotinib, vandetanib, and mobocertinib.

The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of "cellrhythmia" (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3 µM NIL, VAN, and MOB for 1.5 h. Using these identified proteins as substrates, we performed kinase-substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Fingding indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally,three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs. Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.

Phosphorylated proteome analysis of a novel germline ABL1 mutation causing an autosomal dominant syndrome with ventricular septal defect.

BACKGROUND: A gain-of-function mutation in germline ABL1 causes a syndrome including congenital heart defects. However, the molecular mechanisms of this syndrome remain unknown. In this study, we found a novel ABL1 mutation in a Japanese family with ventricular septal defect, finger contracture, skin abnormalities and failure to thrive, and the molecular mechanisms of these phenotypes were investigated. METHODS AND RESULTS: Whole-exome sequencing on several family members revealed a novel mutation (c.1522A > C, p.I508L) in the tyrosine kinase domain of ABL1, and complete co-segregation with clinical presentations was confirmed in all members. Wild-type and mutant ABL1 were transfected into human embryonic kidney 293 cells for functional analysis. Western blotting confirmed that tyrosine phosphorylation in STAT5, a substrate of ABL1, was enhanced, and the novel mutation was proved to be a gain-of-function mutation. Since this novel mutation in ABL1 enhances tyrosine kinase activity, phosphorylated proteome analysis was used to elucidate the molecular pathology. The proteome analysis showed that phosphorylation in proteins such as UFD1, AXIN1, ATRX, which may be involved in the phenotypes, was enhanced in the mutant group. CONCLUSIONS: The onset of congenital heart defects associated with this syndrome appears to involve a mechanism caused by UFD1 common to 22q.11.2 deletion syndrome. On the other hand, AXIN1 and ATRX may be important in elucidating the mechanisms of other phenotypes, such as finger contracture and failure to thrive. Verification of these hypotheses would lead to further understanding of the pathophysiology and the development of treatment methods.