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Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug transporters and tyrosine kinase inhibitors (TKIs), which has uncovered widespread phosphotyrosine-mediated regulation of drug transporters. We initially found that organic cation transporters (OCTs), uptake carriers of metformin and oxaliplatin, were inhibited by several clinically used TKIs. Mechanistic studies showed that these TKIs inhibit the Src family kinase Yes1, which was found to be essential for OCT2 tyrosine phosphorylation and function. Yes1 inhibition in vivo diminished OCT2 activity, significantly mitigating oxaliplatin-induced acute sensory neuropathy. Along with OCT2, other SLC-family drug transporters are potentially part of an extensive 'transporter-phosphoproteome' with unique susceptibility to TKIs. On the basis of these findings we propose that TKIs, an important and rapidly expanding class of therapeutics, can functionally modulate pharmacologically important proteins by inhibiting protein kinases essential for their post-translational regulation.

Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3-internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq-based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD-positive ([FLT3-ITD.sup.+]) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human [FLT3-ITD.sup.+] cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects., Introduction Advances in genomics, as well as the development of molecularly targeted drugs, have transformed cancer therapy (1). Identification of genomic alterations and oncogenic addictions in specific cancer subtypes has [...]

Cisplatin is a widely used cancer therapy drug that unfortunately has major side effects in normal tissues, notably nephrotoxicity in kidneys. Despite intensive research, the mechanism of cisplatin-induced nephrotoxicity remains [...]

The usefulness and efficacy of cisplatin, a chemotherapeutic drug, are limited by its toxicity to normal tissues and organs, including the kidneys. The uptake of cisplatin in renal tubular cells is high, leading to cisplatin accumulation and tubular cell injury and death, culminating in acute renal failure. While extensive investigations have been focused on the signaling pathways of cisplatin nephrotoxicity, much less is known about the mechanism of cisplatin uptake by renal cells and tissues. In this regard, evidence has been shown for the involvement of organic cation transporters (OCT), specifically OCT2. The copper transporter Ctrl is highly expressed in the renal tubular cells; however, its role in cisplatin nephrotoxicity is not known. In this study, we demonstrate that Ctr1 is mainly expressed in both proximal and distal tubular cells in mouse kidneys. We further show that Ctrl is mainly localized on the basolateral side of these cells, a proposed site for cisplatin uptake. Importantly, downregulation of Ctr1 by small interfering RNA or copper pretreatment results in decreased cisplatin uptake. Consistently, downregulation of Ctr1 suppresses cisplatin toxicity, including cell death by both apoptosis and necrosis. Cimetidine, a pharmacological inhibitor of OCT2, can also partially attenuate cisplatin uptake. Notably, cimetidine can further reduce cisplatin uptake and cisplatin toxicity in Ctr1-downregulated cells. The results have demonstrated the first evidence for a role of Ctr1 in cisplatin uptake and nephrotoxicity. apoptosis; necrosis; acute kidney injury

Bcl-2 family proteins are central regulators of apoptosis. As the prototypic member, Bcl-2 protects various types of cells against apoptotic insults. In mammalian cells, Bcl-2 has a dual subcellular localization, in mitochondria and endoplasmic reticulum (ER). The respective roles played by mitochondrial and ER-localized Bcl-2 in apoptotic inhibition are unclear. Using Bcl-2 constructs for targeted subcellular expression, we have now determined the contributions of mitochondrial and ER-localized Bcl-2 to the antiapoptotic effects of Bcl-2 in renal tubular cells. Wild-type Bcl-2, when expressed in renal proximal tubular cells, showed partial colocalizations with both cytochrome c and disulfide isomerase, indicating dual localizations of Bcl-2 in mitochondria and ER. In contrast, Bcl-2 constructs with mitochondria-targeting or ER-targeting sequences led to relatively restricted Bcl-2 expression in mitochondria and ER, respectively. Expression of wild-type and mitochondrial Bcl-2 showed significant inhibitory effects on tubular cell apoptosis that was induced by cisplatin or ATP depletion; however, ER-Bcl-2 was much less effective. During ATP depletion, cytochrome c was released from mitochondria into the cytosol. This release was suppressed by wild-type and mitochondrial Bcl-2, but not by ER-Bcl-2. Consistently, wild-type and mitochondrial Bcl-2, but not ER-Bcl-2, blocked Bax activation during ATP depletion, a critical event for mitochondrial outer membrane permeabilization and cytochrome c release. In contrast, ER-Bcl-2 protected against apoptosis during tunicamycin-induced ER stress. Collectively, the results suggest that the cytoprotective effects of Bcl-2 in different renal injury models are largely determined by its subcellular localizations. Bcl-2; cisplatin; adenosine 5'-triphosphate depletion; apoptosis; mitochondria; endoplasmic reticulum

Cisplatin induces renal cell injury and death, resulting in nephrotoxicity that limits its use in cancer therapy. Using cell culture models, recent work has suggested the involvement of p53 in renal cell apoptosis during cisplatin treatment. However, the signals upstream of p53 remain elusive. ATM and ATR are critical regulators of p53 under various conditions of DNA damage. Here, we show that ATM, and not ATR, was proteolytically cleaved into specific fragments of ~210 and 150 kDa during cisplatin-induced tubular cell apoptosis. ATM cleavage was paralleled by the development of apoptosis. VAD, a broad-spectrum inhibitor of caspases, attenuated the cleavage of ATM, whereas the inhibitors of specific caspases were less effective. In caspase-3-deficient MCF-7 cells, ATM was cleaved, releasing the 210- but not the 150-kDa fragment. Recombinant caspase-3 was much more effective than caspase-7 in cleaving ATM that was immunoprecipitated from cell lysates. During cisplatin incubation, VAD protected ATM and enhanced p53 phosphorylation. In vitro assay of protein kinase activity further showed that ATM immunoprecipitated from cisplatin-treated cells had significantly lower kinase activity toward p53 than that from control ceils. Importantly, the protein kinase activity was restored in ATM that was protected by VAD during cisplatin incubation. ATM deficiency sensitized the cells to cisplatin-induced apoptosis, suggesting a cytoprotective role of ATM in this experimental model. Thus proteolysis of ATM by caspases may inactivate this regulatory molecule to facilitate the progression of apoptosis. ATR; proteolysis; DNA damage; kidney; nephrotoxicity

Although the primary elimination pathway for most tyrosine kinase inhibitors (TKI) involves CYP3A4-mediated metabolism, the mechanism by which these agents are brought into hepatocytes remains unclear. In this study, we optimized and validated a competitive counterflow (CCF) assay to examine TKIs as substrates of the hepatic uptake transporter OATP1B1. The CCF method was based on the stimulated efflux of radiolabeled estradiol-17β-glucuronide under steady-state conditions in HEK293 cells engineered to overexpress OATP1B1. Of the 62 approved TKIs examined, 13 agents were identified as putative substrates of OATP1B1, and pazopanib was selected as a representative hit for further validation studies. The transport of pazopanib by OATP1B1 was confirmed by decreased activity of its target VEGFR2 in OATP1B1-overexpressing cells, but not cells lacking OATP1B1, consistent with molecular docking analyses indicating an overlapping binding orientation on OATP1B1 with the known substrate estrone-3-sulfate. In addition, the liver-to-plasma ratio of pazopanib in vivo was decreased in mice with a deficiency of the orthologous transporters, and this was accompanied by diminished pazopanib-induced hepatotoxicity, as determined by changes in the levels of liver transaminases. Our study supports the utility of CCF assays to assess substrate affinity for OATP1B1 within a large set of agents in the class of TKIs and sheds light on the mechanism by which these agents are taken up into hepatocytes in advance of metabolism., Significance: Despite the established exposure-pharmacodynamic relationships for many TKIs, the mechanisms underlying the agents' unpredictable pharmacokinetic profiles remain poorly understood. We report here that the disposition of many TKIs depends on hepatic transport by OATP1B1, a process that has toxicologic ramifications for agents that are associated with hepatotoxicity., (©2024 The Authors; Published by the American Association for Cancer Research.)

Proteinuria may contribute to progressive renal damage by inducing tubulointerstitial inflammation, fibrosis, and tubular cell injury and death, but the mechanisms underlying these pathologic changes remain largely unknown. Here, in a rat kidney proximal tubular cell line (RPTC), albumin induced apoptosis in a time- and dose-dependent manner. Caspase activation accompanied albumin-induced apoptosis, and general caspase inhibitors could suppress this activation. In addition, Bcl-2 transfection inhibited apoptosis and attenuated albumin-induced Bax translocation to mitochondria and cytochrome c release from the organelles, further confirming a role for the intrinsic pathway of apoptosis in albuminuria-associated tubular apoptosis. We observed phosphorylation and activation of PKC-delta early during treatment of RPTC cells with albumin. Rottlerin, a pharmacologic inhibitor of PKC-delta, suppressed albumin-induced Bax translocation, cytochrome c release, and apoptosis. Moreover, a dominant-negative mutant of PKC-delta blocked albumin-induced apoptosis in RPTC cells. In vivo, we observed activated PKC-delta in proteinuric kidneys of streptozotocin-induced diabetic mice and in kidneys after direct albumin overload. Notably, albumin overload induced apoptosis in renal tubules, which was less severe in PKC-delta-knockout mice. Taken together, these results suggest that activation of PKC-delta promotes tubular cell injury and death during albuminuria, broadening our understanding of the pathogenesis of progressive proteinuric kidney diseases.