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The maintenance of redox and metabolic homeostasis is integral to embryonic development. Nuclear factor erythroid 2-related factor 2 (NRF2) is a stress-induced transcription factor that plays a central role in the regulation of redox balance and cellular metabolism. Under homeostatic conditions, NRF2 is repressed by Kelch-like ECH-associated protein 1 (KEAP1). Here, we demonstrate that Keap1 deficiency induces Nrf2 activation and postdevelopmental lethality. Loss of viability is preceded by severe liver abnormalities characterized by an accumulation of lysosomes. Mechanistically, we demonstrate that loss of Keap1 promotes aberrant activation of transcription factor EB (TFEB)/transcription factor binding to IGHM Enhancer 3 (TFE3)-dependent lysosomal biogenesis. Importantly, we find that NRF2-dependent regulation of lysosomal biogenesis is cell autonomous and evolutionarily conserved. These studies identify a role for the KEAP1–NRF2 pathway in the regulation of lysosomal biogenesis and suggest that maintenance of lysosomal homeostasis is required during embryonic development.

Supplementary table from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples.Significance:Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia.This article is highlighted in the In This Issue feature, p. 587

Supplementary movie from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary Data from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary Figure from Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death

Supplementary Figure S1. Chemotherapy exposure stimulates an increase in pyrimidine nucleotides in TNBC cells.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. Significance: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587

Glutamine is a conditionally essential nutrient for many cancer cells, but it remains unclear how consuming glutamine in excess of growth requirements confers greater fitness to glutamine-addicted cancers. By contrasting two breast cancer subtypes with distinct glutamine dependencies, we show that glutamine-indispensable triple-negative breast cancer (TNBC) cells rely on a non-canonical glutamine-to-glutamate overflow, with glutamine carbon routed once through the TCA cycle. Importantly, this single-pass glutaminolysis increases TCA cycle fluxes and replenishes TCA cycle intermediates in TNBC cells, a process that achieves net oxidation of glucose but not glutamine. The coupling of glucose and glutamine catabolism appears hard-wired via a distinct TNBC gene expression profile biased to strip and then sequester glutamine nitrogen, but hampers the ability of TNBC cells to oxidise glucose when glutamine is limiting. Our results provide a new understanding of how metabolically rigid TNBC cells are sensitive to glutamine deprivation and a way to select vulnerable TNBC subtypes that may be responsive to metabolic-targeted therapies.

Adenosine 5'‐monophosphate (AMP)‐activated protein kinase (AMPK) is an important cellular metabolite‐sensing enzyme that can directly sense changes not only in ATP but also in metabolites associated with carbohydrates and fatty acids. However, less is known about whether and how AMPK senses variations in cellular amino acids. Here, we show that cysteine deficiency significantly triggers calcium/calmodulin‐dependent protein kinase kinase 2 (CaMKK2)‐mediated activation of AMPK. In addition, we found that CaMKK2 directly associates with cysteinyl‐tRNA synthetase (CARS), which then binds to AMPKγ2 under cysteine deficiency to activate AMPK. Interestingly, we discovered that cysteine inhibits the binding of CARS to AMPKγ2, and thus, under cysteine deficiency conditions wherein the inhibitory effect of cysteine is abrogated, CARS mediates the binding of AMPK to CaMKK2, resulting in the phosphorylation and activation of AMPK by CaMKK2. Importantly, we demonstrate that blocking AMPK activation leads to cell death under cysteine‐deficient conditions. In summary, our study is the first to show that CARS senses the absence of cysteine and activates AMPK through the cysteine–CARS–CaMKK2–AMPKγ2 axis, a novel adaptation strategy for cell survival under nutrient deprivation conditions.

The coordinated regulation of growth control and metabolic pathways is required to meet the energetic and biosynthetic demands associated with proliferation. Emerging evidence suggests that the Hippo pathway effector Yes-associated protein 1 (YAP) reprograms cellular metabolism to meet the anabolic demands of growth, although the mechanisms involved are poorly understood. Here, we demonstrate that YAP co-opts the sterol regulatory element-binding protein (SREBP)-dependent lipogenic program to facilitate proliferation and tissue growth. Mechanistically, YAP stimulates de novo lipogenesis via mechanistic target of rapamcyin (mTOR) complex 1 (mTORC1) signaling and subsequent activation of SREBP. Importantly, YAP-dependent regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1) is required to activate mTORC1/SREBP and stimulate de novo lipogenesis. We also find that the SREBP target genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are conditionally required to support YAP-dependent proliferation and tissue growth. These studies reveal that de novo lipogenesis is a metabolic vulnerability that can be targeted to disrupt YAP-dependent proliferation and tissue growth.

Activating FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of all acute myeloid leukaemias (AMLs) and are associated with poor prognosis. The limited clinical efficacy of FLT3 inhibitor monotherapy has highlighted the need for alternative therapeutic targets and treatments for FLT3-mutant AML. Using human and murine models of MLL-rearranged AML harbouring FLT3 internal tandem duplication (FLT3-ITD) and primary patient samples, we have demonstrated that FLT3-ITD promotes serine uptake and serine synthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine synthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism in FLT3-mutant AML is dependent on the mTORC1-ATF4 axis, that drives RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5. Genetic or pharmacological inhibition of the de novo serine synthesis pathway selectively inhibited the proliferation of FLT3-ITD AML cells, and this was potentiated by withdrawal of exogenous serine. Purine supplementation effectively rescued the antiproliferative effect of inhibiting de novo serine synthesis, consistent with the idea that serine fuels purine nucleotide synthesis in FLT3-mutant AML. Pharmacological inhibition of the de novo serine synthesis pathway, using the PHGDH inhibitor WQ-2101, sensitises FLT3-mutant AML cells to the standard of care chemotherapy agent cytarabine via exacerbation of DNA damage. Collectively, these data reveal new insights as to how FLT3 mutations reprogram metabolism in AML, and reveal a combination therapy strategy to improve the treatment of FLT3-mutant AML.Statement of SignificanceFLT3 mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine metabolism, thereby rendering FLT3-ITD leukemias dependent on serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-mutant AML to chemotherapy.

Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD–driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo. Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD–induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. Significance: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD–driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD–driven AMLs to chemotherapy. This article is highlighted in the In This Issue feature, p. 1307

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an example, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies., The systemic understanding of oncogenic kinase signalling is still limited. Here, the authors combine chemical proteomics with functional screens to assess the impact of oncogenic Src on the expressed kinome and identify SGK1 as a critical mediator of Src-induced cell transformation.

N-functionalized amino acids are important building blocks for the preparation of diverse bioactive molecules including peptides. The development of sustainable manufacturing routes to chiral N-alkylated amino acids remains a significant challenge in the pharmaceutical and fine chemical industries. Herein we report the discovery of a structurally diverse panel of biocatalysts which catalyze the asymmetric synthesis of N-alkyl amino acids via the reductive coupling of ketones and amines. Reactions have been performed on a gram scale to yield optically pure N-alkyl functionalized products in high yields. Postprint

Several biocatalytic approaches for the preparation of optically pure methyl 3-oxocyclohexanecarboxylates (S)-, (R)-1 and 3-oxocyclohexanecarbonitriles (S)-, (R)-2 have been successfully demonstrated. Screening of reaction-focused enzyme collections was used to identify initial hits using three enzymatic strategies. Reaction optimization and scale-up enabled the production of chiral intermediates for route scouting efforts on scales of up to 100 g. The enzymes applied in these processes (lipases, enoate reductases, and nitrilases) have been shown to be robust catalysts for drug manufacturing and represent a green alternative to conventional methods to access these chiral cyclohexanone building blocks.

Although the BCL2 inhibitor venetoclax have been transformative in the management of AML, therapeutic resistance and relapse are frequently observed. In light of the urgent need to uncover novel therapeutic options in AML, we sought to study the potential role of ironomycin (AM5), a recently described small molecule that induces cell death through the sequestration of lysosomal iron. To evaluate the effects of ironomycin in AML, we chose a diverse panel of AML cell lines. These data showed a potent and dose-dependent effect, on proliferation, cell cycle progression and survival at a nanomolar range. In contrast to venetoclax, the cell death induced by ironomycin did not result in potent caspase activation or PARP1 cleavage. Neither the caspase inhibitor Z-VAD-fmk nor the necroptosis inhibitor necrostatin-1 did prevent cell death. Consistent with previous observations, we found that ironomycin accumulates in the lysosomes of AML cells leading to a sequestration of iron in this organelle but inhibitors of canonical ferroptosis, including ferrostatin-1 and liproxstatin-1 failed to prevent the activity of ironomycin. To gain greater insight into the molecular mechanism of ironomycin in AML cells, we performed a genome-wide positive-selection resistance screen under ironomycin selection pressure and collected several samples for sequencing. We found nine genes whose knock out conferred resistance to the drug. Interestingly, these data implicated key components of mitochondrial metabolic pathways, including phosphoglycolate phosphatase (PGP), a central phosphatase involved in glycolysis and pentose phosphate pathway (PPP) regulation and Hexokinase 2 (HK2), the first enzyme of glycolysis. Mass-spectrometry metabolomics analyses highlighted that ironomycin treatment significantly reduced key components of the TCA cycle and consequently the reducing agent nicotinamide adenine dinucleotide (NADH) and increased the intracellular concentration of amino acids. These data were corroborated with RNAseq showing a mitochondrial stress response mediated through the Activating Transcription Factor 4 (ATF4) and its paralog Activating Transcription Factor 5 (ATF5). As mitochondria are major hubs of iron utilization for oxidative respiration, we used Mass-spectrometry to measure mitochondrial iron load. We observed a rapid and dose-dependent decrease in mitochondrial iron after treatment mirroring the iron sequestration into the lysosomes and inducing the mitochondrial dysfunction. We next examined the ultrastructural appearance of mitochondria after ironomycin using transmission electron microscopy and observed a dramatic alteration of the structural integrity of mitochondria resulting in abnormal cristae, matrix density changes and mitochondrial membrane blebbing. In cells lacking BAX and BAK, the two main effectors of mitochondrial membrane permeabilization, structural changes and cell death were almost completely rescued but cell proliferation was still markedly affected, consistent with a BAX/BAK dependent cell death following mitochondrial iron deprivation. In vivo imaging confirmed that BAX activation occurred after 30 hours of treatment and preceded cell death, but we observed some major differences with canonical apoptosis induced by venetoclax. First, the structural alterations were clearly distinct. Next, delay between MOMP and cell death was significantly longer and caspase inhibitors weakly delayed cell death. Finally, BCL2 overexpression and P53 deletion did not rescue ironomycin cell death. These non-canonical features prompted us to assess the efficacy of the combination between ironomycin and venetoclax. In vitro experiment on AML cell lines found a high synergy between the two drugs. In vivo experiments on xenotransplanted mice confirmed the efficacy of the combination, which was associated with a significant increase in mice survival in comparison with the controls (Figure). Finally, primary AML samples from patients clinically resistant or refractory to venetoclax were sensitive to ironomycin in monotherapy and even more in combination with venetoclax. These results demonstrate that the novel mechanism of ironomycin action can be leveraged to resensitize AML cells to venetoclax and substitute for cytotoxic drugs as a more effective therapeutic combination in the salvage setting. Figure 1 Figure 1. Disclosures Huang: The Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Employee of the Walter and Eliza Hall Institute and eligilble for payments in relation to venetoclax. Wei: Novartis, Celgene, AbbVie, Servier, AstraZeneca, and Amgen: Research Funding; Novartis, Janssen, Amgen, Roche, Pfizer, Abbvie, Servier, BMS, Macrogenics, Agios, Gilead: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria.

Chiral secondary and tertiary amines are ubiquitous in pharmaceutical, fine, and specialty chemicals, but their synthesis typically suffers from significant sustainability and selectivity challenges. Biocatalytic alternatives, such as enzyme-catalyzed reductive amination, offer several advantages over traditional chemistry, but industrial applicability has not yet been demonstrated. Herein, we report the use of cell lysates expressing imine reductases operating at 1:1 stoichiometry for a variety of amines and carbonyls. A collection of biocatalysts with diversity in coverage of small molecules and direct industrial applicability is presented.

An epoxide resolution process was rapidly developed that allowed access to multigram scale quantities of (R)-2-butyl-2-ethyloxirane 2 at greater than 300 g/L reaction concentration using an easy-to-handle and store lyophilized powder of epoxide hydrolase from Agromyces mediolanus. The enzyme was successfully fermented on a 35 L scale and stability increased by downstream processing. Halohydrin dehalogenases also gave highly enantioselective resolution but were shown to favor hydrolysis of the (R)-2 epoxide, whereas epoxide hydrolase from Aspergillus niger instead provided (R)-7 via an unoptimized, enantioconvergent process.

Chemotherapy resistance is a major barrier to the treatment of triple-negative breast cancer (TNBC), and strategies to circumvent resistance are required. Using in vitro and in vivo metabolic profiling of TNBC cells, we show that an increase in the abundance of pyrimidine nucleotides occurs in response to chemotherapy exposure. Mechanistically, elevation of pyrimidine nucleotides induced by chemotherapy is dependent on increased activity of the de novo pyrimidine synthesis pathway. Pharmacologic inhibition of de novo pyrimidine synthesis sensitizes TNBC cells to genotoxic chemotherapy agents by exacerbating DNA damage. Moreover, combined treatment with doxorubicin and leflunomide, a clinically approved inhibitor of the de novo pyrimidine synthesis pathway, induces regression of TNBC xenografts. Thus, the increase in pyrimidine nucleotide levels observed following chemotherapy exposure represents a metabolic vulnerability that can be exploited to enhance the efficacy of chemotherapy for the treatment of TNBC. Significance: The prognosis for patients with TNBC with residual disease after chemotherapy is poor. We find that chemotherapy agents induce adaptive reprogramming of de novo pyrimidine synthesis and show that this response can be exploited pharmacologically, using clinically approved inhibitors of de novo pyrimidine synthesis, to sensitize TNBC cells to chemotherapy. Cancer Discov; 7(4); 391–9. ©2017 AACR. See related article by Mathur et al., p. 380. This article is highlighted in the In This Issue feature, p. 339

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of all acute myeloid leukaemias (AMLs) and are associated with poor prognosis. The clinical utility of FLT3 inhibitor monotherapy has been limited by the rapid development of resistance, highlighting the need for identification of alternative therapeutic targets for the treatment of FLT3-mutant AML. Using a syngeneic murine model of AML harbouring FLT3 internal tandem duplication (FLT3-ITD), we demonstrate that FLT3-ITD promotes serine uptake and serine synthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine synthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism was dependent on the mTORC1-ATF4 axis, which promoted RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5 in AML cells harbouring FLT3-ITD mutations. Genetic or pharmacological inhibition of the de novo serine synthesis pathway selectively inhibited the proliferation and viability of FLT3-ITD AML cells, and purine supplementation effectively rescued the apoptotic effects of inhibiting FLT3-ITD, consistent with the hypothesis that serine fuels purine nucleotide synthesis in FLT3-ITD AML cells. To exploit these findings in the context of standard-of-care therapy in AML, we show that pharmacological inhibition of the de novo serine synthesis pathway, using the PHGDH inhibitor WQ-2101, sensitises FLT3-ITD AML cells to the chemotherapy agent cytarabine by greatly exacerbating the DNA damage response in AML cell lines harbouring FLT3-ITD mutations, primary AML patient samples, and an aggressive PDX mouse model of FLT3-ITD-driven AML. Collectively, these data reveal new insights into FLT3-ITD-induced metabolic reprogramming in AML, and suggest a novel combinatorial therapeutic strategy for the treatment of FLT3-mutant AML.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, originating from liver cells known as hepatocytes. At the molecular level, HCC is driven by transcription factors which are able to reprogram metabolism to support tumorigenesis that are still poorly understood. The Yes-associated protein (Yap) is the nuclear effector of the Hippo pathway, responsible in regulating organ size control and metabolism. Metabolic reprogramming has recently emerged as a fundamental hallmark of cancer. In our previous studies, we found that Yap integrates the anabolic demands of tumour growth by reprogramming glutamine and glucose metabolism to support nucleotide biosynthesis. The central aim of this study was to determine the role that Yap plays in regulating lipid metabolism in the context of liver cancer. We took advantage of a larval zebrafish model in which a hyperactivated form of Yap is specifically expressed in hepatocytes (lf:YapS87A;lf:NLS-mcherry). We found that the expression of Yap was sufficient to stimulate de novo lipogenesis (DNL) and induce lipid droplet formation in hepatocytes (steatosis). To determine whether the stimulation of DNL was required for oncogenic growth, we exposed larvae to pharmacological inhibitors of DNL and examined the impact on growth at the cellular level by multiphoton microscopy. Strikingly, we identified that fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) inhibitors suppressed Yap-driven growth. To complement these studies, we took a genetic approach using CRISPR to generate FAS and SCD KO zebrafish larvae and we found that Yap-driven growth required both FAS and SCD. Importantly both chemical and genetic suppression of FAS and SCD activity had no effect on normal liver growth. Together, these findings suggest that oncogenic Yap-driven growth is conditionally dependent upon the stimulation of DNL. Consequently, these studies provide a rationale for examining the clinical efficacy of DNL inhibitors to combat liver cancer. Citation Format: Talhah M. Salmi, Srimayee Vaidyanathan, Malcolm J. McConville, Kristin K. Brown, Andrew G. Cox. Yap reprograms de novo lipogenesis to fuel liver cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-015.

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap−/− mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap−/− mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis., National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant P30DK034854), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK090311), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant R24OD017870), National Institutes of Health (U.S.) (Grant P30DK034854), National Institutes of Health (U.S.) (Grant 1R01DK090311), National Institutes of Health (U.S.) (Grant 1R01DK105198), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (Grant 1R01DK105198), National Institutes of Health (U.S.) (Grant R24OD017870), National Institute of General Medical Sciences (NIGMS) (Grant T32GM007753), NHMRC (Grant 1146558), National Cancer Institute (U.S.) (Grant 5P01CA120964), National Cancer Institute (U.S.) (Grant 5P30CA006516)

Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8+ T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8+ T cell-mediated killing and blunted antitumor immune responses in vivo. Deletion of a number of genes in the TNF pathway also emerged as the key mechanism of immune evasion from primary NK cells. Our screens also identified that the metabolic protein 2-aminoethanethiol dioxygenase (Ado) modulates sensitivity to TNF-mediated killing by cytotoxic lymphocytes and is required for optimal control of tumors in vivo. Remarkably, we found that tumors delete the same genes when exposed to perforin-deficient CD8+ T cells, demonstrating that the dominant immune evasion strategy used by tumor cells is acquired resistance to T cell-derived cytokine-mediated antitumor effects. We demonstrate that TNF-mediated bystander killing is a potent T cell effector mechanism capable of killing antigen-negative tumor cells. In addition to highlighting the importance of TNF in CD8+ T cell- and NK cell-mediated killing of tumor cells, our study also provides a comprehensive picture of the roles of the TNF, IFN, and antigen presentation pathways in immune-mediated tumor surveillance.

The assessment of the suitability of novel targets to intervention by different modalities, e.g. small molecules or antibodies, is increasingly seen as important in helping to select the most progressable targets at the outset of a drug discovery project. This perspective considers differing aspects of tractability and how it can be assessed using in silico and experimental approaches. We also share some of our experiences in using these approaches.

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

© 2016, National Academy of Sciences. All rights reserved. Selenium, an essential micronutrient known for its cancer prevention properties, is incorporated into a class of selenocysteine-containing proteins (selenoproteins). Selenoprotein H (SepH) is a recently identified nucleolar oxidoreductase whose function is not well understood. Here we report that seph is an essential gene regulating organ development in zebrafish. Metabolite profiling by targeted LC-MS/MS demonstrated that SepH deficiency impairs redox balance by reducing the levels of ascorbate and methionine, while increasing methionine sulfoxide. Transcriptome analysis revealed that SepH deficiency induces an inflammatory response and activates the p53 pathway. Consequently, loss of seph renders larvae susceptible to oxidative stress and DNA damage. Finally, we demonstrate that seph interacts with p53 deficiency in adulthood to accelerate gastrointestinal tumor development. Overall, our findings establish that seph regulates redox homeostasis and suppresses DNA damage. We hypothesize that SepH deficiency may contribute to the increased cancer risk observed in cohorts with low selenium levels.

Chemotherapy resistance is a major barrier to the treatment of triple-negative breast cancer (TNBC), and strategies to circumvent resistance are required. Using

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone1. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis, and responses to available therapy2–4. Recurrent somatic alterations in breast cancer have been described including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration5. Prior DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements 6–10. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA11, TP536, AKT112, GATA313, and MAP3K110, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking estrogen and progesterone receptors and ERBB2 expression. The Magi3-Akt3 fusion leads to constitutive activation of Akt kinase, which is abolished by treatment with an ATP-competitive Akt small-molecule inhibitor.

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.

A novel series of AKT inhibitors containing 2,3,5-trisubstituted pyridines with novel azaindazoles as hinge binding elements are described. Among these, the 4,7-diazaindazole compound 2c has improved drug-like properties and kinase selectivity than those of indazole 1, and displays greater than 80% inhibition of GSK3beta phosphorylation in a BT474 tumor xenograft model in mice.

The synthesis and evaluation of tetrasubstituted aminopyridines, bearing novel azaindazole hinge binders, as potent AKT inhibitors are described. Compound 14c was identified as a potent AKT inhibitor that demonstrated reduced CYP450 inhibition and an improved developability profile compared to those of previously described trisubstituted pyridines. It also displayed dose-dependent inhibition of both phosphorylation of GSK3beta and tumor growth in a BT474 tumor xenograft model in mice.

Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.

The phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling network is a master regulator of processes that contribute to tumorigenesis and tumor maintenance. The PI3K pathway also plays a critical role in driving resistance to diverse anti-cancer therapies. This review article focuses on mechanisms by which the PI3K pathway contributes to therapy resistance in cancer, and highlights potential combination therapy strategies to circumvent resistance driven by PI3K signaling. In addition, resistance mechanisms that limit the clinical efficacy of small molecule inhibitors of the PI3K pathway are discussed.

We have discovered that the Akt pathway modulates breast cancer cell survival in response to genotoxic agents, and discovered a new substrate of Akt, MERIT40, that is phosphorylated upon exposure of cells to chemotherapeutic drugs. We propose that this represents a major mechanism by which cells exposed to these drugs evade cell death by apoptosis and thus become resistant to the damaging effects of clinically-relevant chemotherapy agents. These findings have important ramifications for the use of chemotherapy drugs in breast cancer patients, and many also suggest that MERIT40 may be used as a clinically relevant biomarker for resistance to doxorubicin.

Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.

SummaryIntegrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

Background Isothiocyanates are phytochemicals with a broad array of effects in biological systems. Bioactivity includes the stimulation of cellular antioxidant systems, induction of apoptosis and interference with cytokine production and activity. Epidemiological evidence and experimental studies indicate that naturally occurring isothiocyanates and synthetic derivatives have anti-cancer and anti-inflammatory properties. Scope of review This review focuses on the molecular targets of isothiocyanates, and how target modification translates into a biological response. Major conclusions Isothiocyanates may mediate their effects via direct protein modification or indirectly by disruption of redox homeostasis and increased thiol oxidation. Some target proteins have been identified, but in-depth searches with new techniques are needed to reveal novel targets. Site-directed mutagenesis and isothiocyanate structure-activity relationships will assist in determining the biological significance of specific modifications. General significance Target identification is important for rational drug design and exploiting the therapeutic potential of isothiocyanates. It also provides insight into the diverse pathways that these compounds regulate.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

Enthalpies of reaction for reactions involving diatomic sulfides and oxides have been calculated at the MP n /6–31G ∗∗ ( n = 2–4) and MP4SDTQ/6–311G(2df,2pd) levels. Enthalpies of formation and singlet-triplet and doublet-quartet energy splittings have also been obtained. The species investigated include ArS + , ArO + , NeS + , NeO + , ArCl + , NeCl + , HS and HS + . The reactions investigated include proton transfer, halogen atom transfer and charge transfer. In order to facilitate the study of reaction enthalpies, the reactions have been divided into four categories: (1) isogyric and the reactant and product pairs are isoelectronic; (2) isogyric and the reactant and product pairs are valence isoelectronic; (3) isogyric; (4) not even isogyric. The results from this study are compared to those from our earlier study. It is found that the MP4SDTQ/6–311G(2df,2pd) enthalpies of reaction reproduce the experimental enthalpies within 3.5 kcal mol −1 for all but nonisogyric reactions. MP n /6–31G ∗* results reproduce the higher level computational results and the experimental results consistently well only for reactions in category (1). There is also disagreement in the energy splittings obtained with the two basis sets. Finally, some suggestions are made to experimentalists about ion-molecule reactions that may produce the above ions.

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.

Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-alpha-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.

Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity.

Isothiocyanates are a class of phytochemicals able to induce apoptosis in numerous cells including Jurkat T-lymphoma cells overexpressing the oncoprotein Bcl-2. To test if isothiocyanates are also effective against other anti-apoptotic members of the Bcl-2 family we generated Jurkat cells stably overexpressing Bcl-X(L). Phenethyl isothiocyanate (PEITC) was cytotoxic to these cells, with an LD(50) ranging from 9 to 18 microM depending on the level of Bcl-X(L) expression. Apoptosis induction in response to PEITC was confirmed by caspase activation and phosphatidylserine exposure. Isothiocyanates specifically target cysteine residues, therefore we tested the hypothesis that PEITC directly impairs Bcl-2 and Bcl-X(L) activity by interacting with their conserved cysteine residues. Jurkat cells overexpressing double cysteine mutants of Bcl-2 were generated, but they remained sensitive to PEITC. We conclude that PEITC antagonizes the action of anti-apoptotic Bcl-2 family members via an indirect mechanism.