Off target mechanisms of kinase inhibitor toxicities

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ABSTRACT Protein kinases orchestrate signal transduction pathways that are essential for most normal cellular functions. Importantly, dysregulation of protein kinase signaling is both a cause and consequence of several human diseases, especially cancer. Due to their druggability, more than seventy small-molecule therapeutics that block the ATP-binding site of kinases have been approved, mostly for oncological diseases. Since the ATP- binding site is relatively conserved, most targeted therapeutics can simultaneously inhibit on- as well as off- target kinases, impacting efficacy as well as toxicity. Remarkably, chemical proteomic profiling has now revealed that along with off-target kinases, protein kinase inhibitors can also bind and inhibit non-kinase proteins. Particularly, the mitochondria-localized heme biosynthesis enzyme, ferrochelatase (FECH) has emerged as a common target which can be inhibited by more than 10% of kinase inhibitors. How FECH inhibition influences drug responses, especially toxicities, nevertheless remains unknown. Given their widespread clinical use, there is a clear unmet need to understand the mechanistic basis of kinase inhibitor toxicities, especially related to inhibition of non-kinase proteins. In this regard, our recent study has provided the first evidence that off-target FECH inhibition by BRAF-kinase inhibitor vemurafenib contributes to renal tubular epithelial cell (RTEC) death in vitro and nephrotoxicity in vivo. However, there is a knowledge gap in our understanding of how kinase inhibitors are transported into normal cells, how the subsequent FECH inhibition drives mitochondrial dysfunction, and how these pathways are differentially regulated in males versus females. To address these questions, we have performed genome-wide RNAi and CRISPR based screens, which have provided two key insights: (i) we have identified putative mechanisms responsible for vemurafenib uptake, FECH inhibition, mitochondrial dysfunction, and RTEC cell death. (ii) We have uncovered a unique gender-specific difference in toxicity, wherein vemurafenib treatment induces a female specific FECH upregulation in RTECs imparting resistance to nephrotoxicity. In the current application we propose to utilize a suite of in vitro and in vivo chemical genetic and gene knockout approaches to further illuminate the regulatory mechanisms that govern toxicities associated with vemurafenib-induced FECH inhibition. Using this approach, in Aim 1 we will employ RTEC-specific conditional knockout mice, primary cells, and CRISPR-based knockout cell lines to examine the role of cubilin-dependent endocytosis in vemurafenib uptake, FECH inhibition, and mitochondrial dysfunction. In Aim 2 we will utilize conditional knockout mice and primary cells to examine the role of RUNX1 in female specific FECH upregulation and resistance to vemurafenib nephrotoxicity. These studies are expected to provide broad insights into the pharmacological action of kinase inhibitors in normal tissues including cellular uptake, non-kinase target inhibition, mitochondrial dysfunction, and transcriptional mechanisms underlying gender differences in toxicities.