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Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat–fed, and high-fat–fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.

Resistance to cancer chemotherapies leads to deadly consequences, yet current research focuses only on the roles of somatically acquired mutations in this resistance. The mutational status of the germline is also likely to play a role in the way cells respond to chemotherapy. The carrier status for the POLB rs3136797 germline mutation encoding P242R DNA polymerase beta (Pol β) is associated with poor prognosis for lung cancer, specifically in response to treatment with cisplatin. Here, it is revealed that the P242R mutation is sufficient to promote resistance to cisplatin in human cells and in mouse xenografts. Mechanistically, P242R Pol β acts as a translesion polymerase and prefers to insert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair (NER) pathway, removal of crosslinks, and resistance to cisplatin. In contrast, wild-type (WT) Pol β preferentially inserts the incorrect nucleotide initiating mismatch repair and cell death. Importantly, in a mouse xenograft model, tumors derived from lung cancer cells expressing WT Pol β displayed a slower rate of growth when treated with cisplatin, whereas tumors expressing P242R Pol β had no response to cisplatin. Pol β is critical for mediating crosstalk in response to cisplatin. The current data strongly suggest that the status of Pol β influences cellular responses to crosslinking agents and that Pol β is a promising biomarker to predict responses to specific chemotherapies. Finally, these results highlight that the genetic status of the germline is a critical factor in the response to cancer treatment. Implications: Pol β has prognostic biomarker potential in the treatment of cancer with cisplatin and perhaps other intrastrand crosslinking agents. Mol Cancer Res; 15(3); 269–80. ©2017 AACR.

// Molly Gale 1 , Joyce Sayegh 1, 3 , Jian Cao 1 , Michael Norcia 2 , Peter Gareiss 2 , Denton Hoyer 2 , Jane S. Merkel 2 , Qin Yan 1 1 Department of Pathology, Yale School of Medicine, New Haven, CT, USA 2 Yale Center for Molecular Discovery, Yale University, West Haven, CT, USA 3 Current address: Department of Biology and Chemistry, Azusa Pacific University, Azusa, CA, USA Correspondence to: Qin Yan, email: qin.yan@yale.edu Keywords: histone demethylase inhibitor, KDM5A, anti-cancer drug, drug resistance, epigenetics Received: February 23, 2016 Accepted: May 05, 2016 Published: May 21, 2016 ABSTRACT Lysine demethylase 5A (KDM5A/RBP2/JARID1A) is a histone lysine demethylase that is overexpressed in several human cancers including lung, gastric, breast and liver cancers. It plays key roles in important cancer processes including tumorigenesis, metastasis, and drug tolerance, making it a potential cancer therapeutic target. Chemical tools to analyze KDM5A demethylase activity are extremely limited as available inhibitors are not specific for KDM5A. Here, we characterized KDM5A using a homogeneous luminescence-based assay and conducted a screen of about 9,000 small molecules for inhibitors. From this screen, we identified several 3-thio-1,2,4-triazole compounds that inhibited KDM5A with low μM in vitro IC 50 values. Importantly, these compounds showed great specificity and did not inhibit its close homologue KDM5B (PLU1/JARID1B) or the related H3K27 demethylases KDM6A (UTX) and KDM6B (JMJD3). One compound, named YUKA1, was able to increase H3K4me3 levels in human cells and selectively inhibit the proliferation of cancer cells whose growth depends on KDM5A. As KDM5A was shown to mediate drug tolerance, we investigated the ability of YUKA1 to prevent drug tolerance in EGFR-mutant lung cancer cells treated with gefitinib and HER2+ breast cancer cells treated with trastuzumab. Remarkably, this compound hindered the emergence of drug-tolerant cells, highlighting the critical role of KDM5A demethylase activity in drug resistance. The small molecules presented here are excellent tool compounds for further study of KDM5A’s demethylase activity and its contributions to cancer.

Through a combination of cell culture studies and in vitro experiments we have begun to investigate whether MT plays a role in modulating the toxicity and genotoxicity of Ni(II), Cr(VI) and As(III). Our data suggest that MT participates in oxidant-generating redox reactions with Cr(VI) and As(III) that increase the toxicity of these metals and create potentially DNA-damaging species. In the case of Ni(II), MT appears to play a protective role, scavenging radical(s) generated by Ni(II) and H2O2, and preventing Ni(II) from replacing Zn(II) in zinc fingers of transcription factors.

Loss of functional DNA repair pathways is a common occurrence in cancer cells and discovery of novel drugs that selectively kill repair deficient tumors cells could identify new cancer therapies. In this work we describe the use of high-throughput screening to determine chemicals that sensitize cells deficient in the fanconi anemia protein, FANCD2. Cell growth of human fibroblasts proficient (PD20+D2) and deficient (PD20) for FANCD2 were screened using two libraries containing over 1000 bioactive compounds and kinase inhibitors. The cells were treated with compounds continuously for 3 days and cell growth was assessed using a luciferase-based assay measuring ATP levels. The initial screen identified numerous compounds inducing differential survival in cells with and without FANCD2 and follow-up testing will be required to confirm these potential hits. However, it is promising that, in addition to novel compounds, several crosslinking agents, including mitomycinC, which are known to sensitize fanconi anemia cells to cell death are among the positive hits. Our results validate chemical screening as a tool to discover potential new cancer therapies targeting repair deficient cells lines. In addition, other than their role in crosslink repair, there is still not much known about the fanconi anemia protein family and the discovery of novel compounds involved in FANCD2 dependent cell survival could lead to the discovery of new functions of this protein family. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2185.