Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions.

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.

MicroRNA-687 Induced by Hypoxia-Inducible Factor-1 Targets Phosphatase and Tensin Homolog in Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury contributes to tissue damage and organ failure in clinical settings, but the underlying mechanism remains elusive and effective therapies are still lacking. Here, we identified microRNA 687 (miR-687) as a key regulator and therapeutic target in renal ischemia-reperfusion injury. We show that miR-687 is markedly upregulated in the kidney during renal ischemia-reperfusion in mice and in cultured kidney cells during hypoxia. MiR-687 induction under these conditions was mediated by hypoxia-inducible factor-1 (HIF-1). Upon induction in vitro, miR-687 repressed the expression of phosphatase and tensin homolog (PTEN) and facilitated cell cycle progression and apoptosis. Blockade of miR-687 preserved PTEN expression and attenuated cell cycle activation and renal apoptosis, resulting in protection against kidney injury in mice. Collectively, these results unveil a novel HIF-1/miR-687/PTEN signaling pathway in ischemia-reperfusion injury that may be targeted for therapy.

DNA damage response in cisplatin-induced nephrotoxicity.

Cisplatin and its derivatives are widely used chemotherapeutic drugs for cancer treatment. However, they have debilitating side effects in normal tissues and induce ototoxicity, neurotoxicity, and nephrotoxicity. In kidneys, cisplatin preferentially accumulates in renal tubular cells causing tubular cell injury and death, resulting in acute kidney injury (AKI). Recent studies have suggested that DNA damage and the associated DNA damage response (DDR) are an important pathogenic mechanism of AKI following cisplatin treatment. Activation of DDR may lead to cell cycle arrest and DNA repair for cell survival or, in the presence of severe injury, kidney cell death. Modulation of DDR may provide novel renoprotective strategies for cancer patients undergoing cisplatin chemotherapy.

Checkpoint kinase 1 (Chk1)-short is a splice variant and endogenous inhibitor of Chk1 that regulates cell cycle and DNA damage checkpoints.

Checkpoint kinase 1 (Chk1) is a key regulator of checkpoint signaling in both the unperturbed cell cycle and DNA damage response. Under these conditions, Chk1 becomes active to prevent premature CDK1 activation and mitotic entry until DNA is properly replicated or repaired. It is unclear how Chk1 activity is controlled in the unperturbed cell cycle. During DNA damage, Chk1 is activated by ataxia telangiectasia and Rad3 related (ATR)-mediated phosphorylation; however, it is not entirely clear how this phosphorylation results in Chk1 activation. Here we report an N-terminally truncated alternative splice variant of Chk1, Chk1-S. Importantly, we show that Chk1-S is an endogenous repressor and regulator of Chk1. In the unperturbed cell cycle, Chk1-S interacts with and antagonizes Chk1 to promote the S-to-G2/M phase transition. During DNA damage, Chk1 is phosphorylated, which disrupts the Chk1-Chk1-S interaction, resulting in free, active Chk1 to arrest the cell cycle and facilitate DNA repair. Higher levels of Chk1-S are expressed, along with Chk1, in fetal and cancer tissues than in normal tissues. However, forced overexpression of Chk1-S in cultured cells and tumor xenografts induces premature mitotic entry, mitotic catastrophe, and reduction of tumor growth. The identification of Chk1-S as a unique splice variant and key regulator of Chk1 provides insights into cell cycle regulation and DNA damage response.

Checkpoint kinase 1 in DNA damage response and cell cycle regulation.

Originally identified as a mediator of DNA damage response (DDR), checkpoint kinase 1 (Chk1) has a broader role in checkpoint activation in DDR and normal cell cycle regulation. Chk1 activation involves phosphorylation at conserved sites. However, recent work has identified a splice variant of Chk1, which may regulate Chk1 in both DDR and normal cell cycle via molecular interaction. Upon activation, Chk1 phosphorylates a variety of substrate proteins, resulting in the activation of DNA damage checkpoints, cell cycle arrest, DNA repair, and/or cell death. Chk1 and its related signaling may be an effective therapeutic target in diseases such as cancer.

hMSH2 recruits ATR to DNA damage sites for activation during DNA damage-induced apoptosis.

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by "master" regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein. Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-α, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling.

PKC-delta promotes renal tubular cell apoptosis associated with proteinuria.

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.

Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia.

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+) 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+ 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.

OCTN1 Is a High-Affinity Carrier of Nucleoside Analogues.

Resistance to xenobiotic nucleosides used to treat acute myeloid leukemia (AML) and other cancers remains a major obstacle to clinical management. One process suggested to participate in resistance is reduced uptake into tumor cells via nucleoside transporters, although precise mechanisms are not understood. Through transcriptomic profiling, we determined that low expression of the ergothioneine transporter OCTN1 (SLC22A4; ETT) strongly predicts poor event-free survival and overall survival in multiple cohorts of AML patients receiving treatment with the cytidine nucleoside analogue cytarabine. Cell biological studies confirmed OCTN1-mediated transport of cytarabine and various structurally related cytidine analogues, such as 2'deoxycytidine and gemcitabine, occurs through a saturable process that is highly sensitive to inhibition by the classic nucleoside transporter inhibitors dipyridamole and nitrobenzylmercaptopurine ribonucleoside. Our findings have immediate clinical implications given the potential of the identified transport system to help refine strategies that could improve patient survival across multiple cancer types where nucleoside analogues are used in cancer treatment. Cancer Res; 77(8); 2102-11. ©2017 AACR.

A phosphotyrosine switch regulates organic cation transporters.

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.

CCR 20th anniversary commentary: BMS-247550­microtubule stabilization as successful targeted therapy.

In a landmark article published in the May 1, 2001, issue of Clinical Cancer Research, Lee and colleagues reported the original preclinical studies demonstrating anticancer activity of BMS-247550 (ixabepilone) against taxane-sensitive and taxane-resistant cancers. Subsequent clinical trials established the clinical efficacy of ixabepilone, leading to its regulatory approval for the treatment of drug-resistant metastatic or locally advanced breast cancers.

Cisplatin-induced renal injury is independently mediated by OCT2 and p53.

PURPOSE: Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 (Oct1/2(-/-)mice), and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis is not completely absent in Oct1/2(-/-)mice, we hypothesized that alternate pathways are involved in the observed injury. EXPERIMENTAL DESIGN: Studies were done in wild-type, Oct1/2(-/-), or p53-deficient animals, all on an FVB background, receiving cisplatin intraperitoneally at 15 mg/kg. Cisplatin metabolites were analyzed using mass spectrometry, and gene expression was assessed using Affymetrix microarrays and RT-PCR arrays. RESULTS: KEGG pathway analyses on kidneys from mice exposed to cisplatin revealed that the most significantly altered genes were associated with the p53 signaling network, including Cdnk1a and Mdm2, in both wild-type (P = 2.40 × 10(-11)) and Oct1/2(-/-)mice (P = 1.92 × 10(-8)). This was confirmed by demonstrating that homozygosity for a p53-null allele partially reduced renal tubular damage, whereas loss of p53 in Oct1/2(-/-)mice (p53(-/-)/Oct1/2(-/-)) completely abolished nephrotoxicity. We found that pifithrin-α, an inhibitor of p53-dependent transcriptional activation, inhibits Oct2 and can mimic the lack of nephrotoxicity observed in p53(-/-)/Oct1/2(-/-)mice. CONCLUSIONS: These findings indicate that (i) the p53 pathway plays a crucial role in the kidney in response to cisplatin treatment and (ii) clinical exploration of OCT2 inhibitors may not lead to complete nephroprotection unless the p53 pathway is simultaneously antagonized.

Nek1 interacts with Ku80 to assist chromatin loading of replication factors and S-phase progression.

NIMA-related kinases (Neks) play divergent roles in mammalian cells. While several Neks regulate mitosis, Nek1 was reported to regulate DNA damage response, centrosome duplication and primary cilium formation. Whether Nek1 participates in cell cycle regulation is not known. Here we report that loss of Nek1 results in severe proliferation defect due to a delay in S-phase of the cell cycle. Nek1-deficient cells show replication stress and checkpoint activation under normal growth conditions. Nek1 accumulates on the chromatin during normal DNA replication. In response to replication stress, Nek1 is further activated for chromatin localization. Nek1 interacts with Ku80 and, in Nek1-deficient cells chromatin localization of Ku80 and several other DNA replication factors is significantly reduced. Thus, Nek1 may facilitate S-phase progression by interacting with Ku80 and regulating chromatin loading of replication factors.

Nek1 phosphorylates Von Hippel-Lindau tumor suppressor to promote its proteasomal degradation and ciliary destabilization.

Loss of function in either VHL or Nek1 leads to cyst formation in tissues, especially in kidneys. Whether there is a connection between pVHL and Nek1 regulation is unknown. Here, we report that the VHL protein (pVHL) may be a substrate of Nek1. While Nek1 can phosphorylate pVHL at multiple sites, the phosphorylation at serine-168 results in pVHL degradation. Nek1-mediated phosphorylation of pVHL does not significantly affect hypoxia-inducible factors (HIF), a known target of pVHL. However, non-phosphorylable pVHL reconstituted in VHL-deficient cells induces more stable cilia than wild-type VHL during serum stimulation and Nocodazole treatment. The results suggest a possible regulation of pVHL by Nek1 that may contribute to ciliary homeostasis and cystogenesis.

Curtailing side effects in chemotherapy: a tale of PKCδ in cisplatin treatment.

The efficacy of chemotherapy is often limited by side effects in normal tissues. This is exemplified by cisplatin, a widely used anti-cancer drug that may induce serious toxicity in normal tissues and organs including the kidneys. Decades of research have delineated multiple signaling pathways that lead to kidney cell injury and death during cisplatin treatment. However, the same signaling pathways may also be activated in cancer cells and be responsible for the chemotherapeutic effects of cisplatin in tumors and, as a result, blockade of these pathways is expected to reduce the side effects as well as the anti-cancer efficacy. Thus, to effectively curtail the side effects, it is imperative to elucidate and target the cell killing mechanisms that are specific to normal (and not cancer) tissues. Our recent work identified protein kinase C δ (PKCδ) as a new and critical mediator of cisplatin-induced kidney cell injury and death. Importantly, inhibition of PKCδ enhanced the chemotherapeutic effects of cisplatin in several tumor models while alleviating the side effect in kidneys, opening a new avenue for normal tissue protection during chemotherapy.

Inhibition of PKCδ reduces cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in mouse models of cancer.

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 unclear, and renoprotective approaches during cisplatin-based chemotherapy are lacking. Here we have identified PKCδ as a critical regulator of cisplatin nephrotoxicity, which can be effectively targeted for renoprotection during chemotherapy. We showed that early during cisplatin nephrotoxicity, Src interacted with, phosphorylated, and activated PKCδ in mouse kidney lysates. After activation, PKCδ regulated MAPKs, but not p53, to induce renal cell apoptosis. Thus, inhibition of PKCδ pharmacologically or genetically attenuated kidney cell apoptosis and tissue damage, preserving renal function during cisplatin treatment. Conversely, inhibition of PKCδ enhanced cisplatin-induced cell death in multiple cancer cell lines and, remarkably, enhanced the chemotherapeutic effects of cisplatin in several xenograft and syngeneic mouse tumor models while protecting kidneys from nephrotoxicity. Together these results demonstrate a role of PKCδ in cisplatin nephrotoxicity and support targeting PKCδ as an effective strategy for renoprotection during cisplatin-based cancer therapy.

The copper transporter Ctr1 contributes to cisplatin uptake by renal tubular cells during cisplatin nephrotoxicity.

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 Ctr1 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 Ctr1 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.

ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis.

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.

Effects of targeted Bcl-2 expression in mitochondria or endoplasmic reticulum on renal tubular cell apoptosis.

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.