Off target mechanisms of kinase inhibitor toxicities

摘要 蛋白激酶协调大多数正常细胞功能所必需的信号转导途径。 重要的是，蛋白激酶信号传导的失调既是几种人类疾病的原因，也是其后果。 疾病，尤其是癌症。由于它们的药物性，超过70种小分子治疗剂， 阻断激酶的ATP结合位点已被批准，主要用于肿瘤疾病。由于ATP- 结合位点是相对保守的，大多数靶向治疗剂可以同时抑制开-以及关- 靶向激酶，影响疗效以及毒性。值得注意的是，化学蛋白质组学分析现在 发现沿着脱靶激酶，蛋白激酶抑制剂也可以结合和抑制非激酶 proteins.特别地，位于大肠杆菌中的血红素生物合成酶亚铁螯合酶（FECH）具有 作为一种常见的靶点，可以被超过10%的激酶抑制剂抑制。如何FECH 然而，抑制作用影响药物反应，尤其是毒性，仍然是未知的。鉴于其 广泛的临床应用，有一个明确的未满足的需要，了解激酶抑制剂的机制基础 毒性，特别是与非激酶蛋白的抑制有关。在这方面，我们最近的研究提供了 第一个证据表明BRAF激酶抑制剂维罗非尼的脱靶FECH抑制作用有助于肾功能损害， 体外肾小管上皮细胞（RTEC）死亡和体内肾毒性。然而，在以下方面存在知识差距： 我们对激酶抑制剂如何被转运到正常细胞中的理解，以及随后的FECH如何被转运到正常细胞中的理解， 抑制驱动线粒体功能障碍，以及这些途径如何在男性与 女性为了解决这些问题，我们进行了全基因组RNAi和基于CRISPR的筛选， 这提供了两个关键的见解：（i）我们已经确定了负责维罗非尼的假定机制 在一些实施方案中，所述方法包括抑制FECH摄取、FECH抑制、线粒体功能障碍和RTEC细胞死亡。(ii)我们发现了一种独特的 毒性的性别特异性差异，其中维罗非尼治疗诱导雌性特异性FECH RTEC的上调赋予对肾毒性的抗性。在本申请中，我们提出利用 一套体外和体内化学遗传学和基因敲除方法，以进一步阐明调控 控制与维罗非尼诱导的FECH抑制相关的毒性的机制。使用这种方法， 在目标1中，我们将使用RTEC特异性条件性敲除小鼠、原代细胞和基于CRISPR的敲除小鼠。 细胞系，以检查cubilin依赖性内吞作用在维罗非尼摄取、FECH抑制和 线粒体功能障碍在目标2中，我们将利用条件性敲除小鼠和原代细胞来检查 RUNX 1在女性特异性FECH上调和对维罗非尼肾毒性的抵抗中的作用。这些 预期研究将为激酶抑制剂在正常人中的药理作用提供广泛的见解。 组织，包括细胞摄取，非激酶靶向抑制，线粒体功能障碍，和转录 毒性中性别差异的潜在机制。

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.