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Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-030871) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further supports the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for chemotherapeutic discovery.

We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5’-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK.

The orphan nuclear receptor estrogen-related receptor α (ERRα) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRα, bound to a co-activator protein (e.g., peroxisome proliferator receptor γ co-activator-1α, PGC-1α), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRα is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRα, but not to ERRβ or ERRγ, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRα's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1α and ERRα from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30 mg/kg given once daily and 100 mg/kg given once weekly. In a combination study, SR16388 (10 mg/kg, once daily) and paclitaxel (7.5 mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRα biology.

The peroxisome proliferator-activated receptor delta (PPARdelta) is a ligand-activated, nuclear receptor transcription factor that has a documented role in glucose and lipid homeostasis. Recent studies have implicated this nuclear receptor in numerous aspects of oncogenesis. We report herein the characterization of a novel small-molecule (SR13904) that inhibits PPARdelta agonist-induced transactivation and functions as a PPARdelta antagonist. SR13904 also antagonizes PPARgamma transactivation, albeit with much weaker potency. SR13904 displays inhibitory effects on cellular proliferation and survival in several human carcinoma lines, including lung, breast and liver. These inhibitory effects of SR13904 on tumor cells were linked to a G(1)/S cell cycle block and increased apoptosis. Molecular studies show that SR13904 treatment of a lung cancer cell line, A549, results in markedly reduced levels of a number of cell cycle proteins including cyclin A and D, and cyclin dependent kinase (CDK) 2 and 4. The inhibitory effects on CDK2 appear to be transcriptional. Several of these cell cycle-related genes are known to be upregulated by PPARdelta. The antitumor activities of SR13904 suggest that antagonism of PPARdelta-mediated transactivation may inhibit tumorigenesis and that pharmacological inhibition of PPARdelta may be a potential strategy for treatment or prevention of cancer.

We have been studying the relationship between the activity of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the response of mammalian cells to oxygen deprivation (e.g., see references 21, 43, and 50) and the regulation of c-Jun/AP-1 transcription factors (31, 32). We determined that c-Jun N-terminal phosphorylation is induced by low-oxygen conditions (hypoxia or anoxia; called hypoxia hereafter) in an HIF-1-dependent manner (31) and showed that this HIF-1-dependent c-Jun phosphorylation absolutely requires extracellular glucose utilization (32). Together, these findings suggest that enhanced glucose absorption and/or glycolytic activity mediated by HIF-1 in response to hypoxia activates c-Jun/AP-1, as well as other targets of c-Jun N-terminal kinases. To further investigate this potential mechanism, we focused on determining the contribution of bioenergetics—ATP depletion—to hypoxia-inducible c-Jun phosphorylation in wild-type (WT) and HIF-1-null mouse embryo fibroblasts (MEFs). While exploring cellular mechanisms of ATP regulation, we observed that 5′-AMP-activated protein kinase (AMPK) activity was induced in both cell types, particularly under conditions of hypoxia and glucose deprivation. This observation suggested the hypothesis that AMPK is important for the adaptive responses of energetically stressed cells in the hypoxic and glucose-deprived microenvironments present in solid tumors (e.g., reviewed in references 35 and 59). AMPK activity is defined by a class of evolutionarily conserved serine/threonine kinases that are sensitive to various environmental stresses, especially those that perturb cellular energy status (reviewed in references 9, 19, and 47). Different members of the AMPK catalytic subunit subfamily have been characterized; the α subunits (collectively, AMPKα1 and -α2) are the most widely expressed in mammalian cells (36). AMPK is a heterotrimeric complex consisting of an α subunit and β and γ regulatory subunits, each of which is encoded by distinct genes (α1 and α2; β1 and β2; γ1, γ2, and γ3) (19). In terms of a role in ATP regulation, decreased cellular ATP levels promote AMPK activation through the allosteric binding of AMP, which in effect enables AMPK to sense increases in the cellular [AMP]/[ATP] ratio. Full activation of AMPK also requires specific phosphorylation within the activation loop of the catalytic domain of the α subunit (at Thr172 in humans and mice) by LKB1, a serine/threonine protein kinase and tumor suppressor (36, 37, 52). LKB1 is thus an AMPK kinase. Recently, mammalian Ca2+/calmodulin-dependent kinase kinases have also been identified as AMPK kinases (reviewed in reference 6). Activated AMPK phosphorylates diverse targets, including many that are directly involved in controlling cellular energy metabolism (22, 34). In cells exposed to an energy-depleting stress, AMPK is believed to function as an energy sensor that inhibits ATP-consuming processes and stimulates ATP-producing processes to optimize total cellular ATP levels for maintaining critical physiological functions (or for survival in response to extreme stress) (19). For example, in cells exposed to hypoxic or ischemic conditions that significantly deplete total ATP, activated AMPK can stimulate ATP generation by increasing both glucose absorption and glycolysis (e.g., see references 2, 19, and 22). AMPK can also generate ATP by phosphorylating and inhibiting the metabolic enzymes acetyl coenzyme A (acetyl-CoA) carboxylases 1 and 2 (ACC1/2), which synthesize malonyl-CoA (19). Malonyl-CoA synthesized by ACC1 is necessary for de novo fatty acid synthesis, whereas that synthesized by ACC2 inhibits fatty acid transport into the mitochondrion, the site of ATP production by the process of fatty acid β oxidation (18). Thus, AMPK-dependent inhibition of ACC1/2 can divert cellular metabolism from consuming ATP during fatty acid biosynthesis to producing ATP by oxidizing fatty acid stores. In the present study, we found that the combination of hypoxia and glucose deprivation decreased total cellular ATP levels to the same extent in both WT and HIF-1α-null cells. This finding supports our previous conclusion that increased intracellular glucose, rather than decreased ATP levels, is responsible for the stimulation of c-Jun N-terminal kinase activity in WT cells exposed to hypoxia. AMPK activity, conventionally defined by phosphorylation of AMPK target sites on the metabolic enzymes ACC1/2 (19), was strongly activated in both WT and HIF-1α-null cells under the same conditions of hypoxia and glucose deprivation, which is consistent with its function as a sensor of ATP depletion. However, AMPK activity was also rapidly induced in both cell types following exposure to hypoxia in the presence of glucose, even though total cellular ATP was not significantly depleted. By using genetically manipulated MEFs nullizygous for AMPK expression, we directly demonstrated that AMPK activity is sensitive to a wide range of low-oxygen conditions, at least in mesenchymal cells. To determine whether these hypoxia-inducible responses of AMPK also occur in vivo, we prepared tumor xenografts from transformed derivatives of the same WT and HIF-1α-null cells, and exposed tumor-bearing mice to the hypoxia probe pimonidazole (3, 10, 44, 45). Immunohistochemical analysis of these tumors indicated that AMPK activity was prevalent in hypoxic regions of both tumor types, especially in viable areas near necrosis. By using tumor xenografts prepared from identically transformed WT and AMPKα-null cells, we determined that the absence of AMPK activity greatly inhibited the growth of this experimental tumor type. We propose that activation of AMPK in hypoxic or ischemic microenvironments may be critical for cell survival and thus would represent a novel protective mechanism for metabolically depressed or ATP-deficient cells.

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5'-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [(13)C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.

Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment.

We found that adenosine 5'-monophosphate-activated protein kinase (AMPK), which is considered the "fuel sensor" of mammalian cells because it directly responds to the depletion of the fuel molecule ATP, is strongly activated by tumor-like hypoxia and glucose deprivation. We also observed abundant AMPK activity in tumor cells in vivo, using subcutaneous tumor xenografts prepared from cells transformed with oncogenic H-Ras. Such rapidly growing transplants of tumor cells, however, represent fully developed tumors that naturally contain energetically stressed microenvironments that can activate AMPK. Therefore, to investigate the induction of AMPK activity during experimental tumorigenesis, we used an established model of brain tumor (glioma) development in the offspring of rats exposed prenatally to the mutagen N-ethyl-N-nitrosourea. We observed that immunostaining for a specific readout of AMPK activity (AMPK-dependent phosphorylation of acetyl-CoA carboxylase) was prominent during N-ethyl-N-nitrosourea-initiated neurocarcinogenesis, from the occurrence of early hyperplasia (microtumors) to the emergence of large gliomas. Moreover, we observed that immunostaining for activating phosphorylation of AMPK correlated with the same stages of glioma development, notably in mitotic tumor cells in which the signal showed punctate as well as cytoplasmic patterns associated with spindle formation. Based on these observations, we propose that neurocarcinogenesis requires AMPK-dependent regulation of cellular energy metabolism.

AMPK has been termed the fuel sensor of mammalian cells because it directly responds to the depletion of the fuel molecule ATP. In previous work, we found that AMPK is strongly activated by tumor-like hypoxia and glucose deprivation, independently of the oxygen response system associated with HIF-1. We also observed high levels of AMPK activity in tumor cells in vivo, using different model tumors. These findings suggested the hypothesis that modulation of AMPK activity could have therapeutic value for the treatment of solid tumors. To investigate this hypothesis, we have been conducting a SAR study of potential small-molecule modulators of AMPK activity. Here we report that the chemotherapeutic drug SU11248 (sunitinib) is at least as potent an inhibitor of AMPK as compound C, which is a commonly used experimental direct inhibitor of the enzyme. We also provide a computational model of the binding pose of SU11248 to an AMPKα subunit, which suggests a structural basis for the affinity of the drug for the ATP site of the catalytic domain. The ability of SU11248 to inhibit AMPK has potential clinical significance--there may be populations of SU11248-treated patients in which AMPK activity is inhibited in normal as well as in tumor tissue.

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalpha's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

Hypoxia and anoxia are important microenvironmental stresses that contribute to pathological events such as solid-tumor development. We have been investigating the effects of hypoxia and anoxia on expression of the proto-oncogene c-jun and the regulation of c-Jun/AP-1 transcription factors. In earlier work using genetically manipulated mouse embryo fibroblasts (mEFs), we found a functional relationship among c-jun expression, c-Jun N-terminal phosphorylation, and the presence of hypoxia-inducible factor 1 alpha (HIF-1 alpha), the oxygen-regulated subunit of the HIF-1 transcription factor. Both the induction of c-jun mRNA expression and c-Jun N-terminal phosphorylation in cells exposed to hypoxia or anoxia were found to be dependent on the presence of HIF-1 alpha, but this was not the case in cells exposed to less-severe hypoxia. Here we describe new findings concerning HIF-1-dependent c-Jun N-terminal phosphorylation in cells exposed to hypoxia or anoxia. Specifically, we report that hypoxia-inducible c-Jun N-terminal kinase (JNK) activity, which involves JNKs or stress-activated protein kinases (SAPKs), is dependent on enhanced glucose utilization mediated by HIF-1. These results suggest a model in which hypoxia-inducible JNK activity is connected to oxygen sensing through increased glucose absorption and/or glycolytic activity regulated by the HIF-1 system. We also found that basal threonine and tyrosine phosphorylation (within the TEY motif) of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the corresponding ERK1/2 activity were defective in hypoxic HIF-1 alpha-null mEFs but not in wild-type mEFs, independently of glucose uptake. Therefore, the activities of both JNKs/SAPKs and ERK1/2 are sensitive to HIF-1-dependent processes in cells exposed to hypoxia or anoxia.

The c-jun gene is a major regulator of proliferative and stress responses of both normal and transformed cells. In general, during immortalization/transformation c-jun cooperates with oncogenic signals rather than acting as an oncogene itself. Here we report a novel example of this cooperation, the requirement for c-jun to sustain expression of the matrix metalloproteinase-2 (MMP-2) gene in cells immortalized by SV40 large T-antigen (TAg). MMP-2 encodes a type IV collagenase that is secreted by cells within normal and tumor microenvironments. We used wild-type and c-jun null primary and TAg-immortalized mouse embryonic fibroblasts (mEFs) to investigate the importance of c-jun for the regulation of this activity, and observed that c-jun is essential for MMP-2 expression in immortalized but not primary mEFs. This finding directly demonstrates a cooperative interaction of c-jun with an oncogene, and suggests that TAg dependent immortalization/transformation may require other c-Jun/AP-1-dependent genes.

Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.

p97 (also called VCP in metazoans and CDC48 in yeast) is a highly conserved, ubiquitously expressed, and essential AAA ATPase. p97 plays a key role in endoplasmic reticulum-associated degradation of misfolded secretory and membrane proteins as well as ubiquitin-dependent turnover of a subset of cytoplasmic substrates of the ubiquitin-proteasome system. p97 also plays a critical role in specific cellular processes including Golgi membrane reassembly, membrane fusion, autophagy and DNA repair mechanisms. p97 expression is elevated in a number of cancer types including prostate cancer, non-small-cell lung carcinoma and leukemia. Given its cellular functions in protein homeostasis and the successful clinical development of proteasome inhibitors, p97 has emerged as an exciting new target for cancer therapy. We used p97 siRNA and a recently identified small molecule p97 inhibitor, N2,N4-dibenzylquinazoline- 2,4-diamine (DBeQ) as tools to develop functional assays suitable for further evaluating the activity of potential p97 inhibitors. A panel of unfolded protein response marker genes that are consistently modulated by p97 siRNA or DBeQ treatment has been identified. In addition, methods to monitor autophagic activity have been developed, including high content analysis of LC3B-GFP puncta formation. Cell viability assays were used to determine the susceptibility of various cancer cell lines to p97 inhibition. These assays will be used to evaluate the effects of potential p97 inhibitors on p97-dependent pathways and anti-proliferative activity. Acknowledgement: Funded by NExT Chemical Biology Consortium, NCI Contract No. HHSN261200800001E. Citation Format: Yihui Shi, Xiaohe Liu, Joy Calaoagan, Steven Samuelsson, Tsui-Fen Chou, Raymond J. Deshaies, Lidia C. Sambucetti. Development of functional assays for p97/VCP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2146. doi:10.1158/1538-7445.AM2013-2146

SR16388 is a novel amino steroid that targets estrogen-binding proteins including genomic estrogen receptors (ERs). Preclinical studies have demonstrated that SR16388 is a well-tolerated, orally active compound with compelling properties as an experimental antitumor agent: SR16388 has been demonstrated to 1) inhibit the proliferation and viability of diverse human cancer cell lines in vitro; 2) have antiangiogenic activity in vivo; and 3) reduce the growth of human tumor xenografts in mice. SR16388, which binds to and potently inhibits ERα and ERβ in breast cancer cells, also inhibits the widely expressed orphan nuclear receptor ERRα that has been implicated in the development of various human malignancies. ERRα is thought to regulate tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. We have been exploring potential mechanisms that could explain SR16388’s broad anti-proliferative effects toward human tumor cell lines with different genotypes and tissues of origin. In these studies, we observed that SR16388 can initiate a cell-cycle arrest at G1 or G2 and ultimately cause cytotoxicity (e.g., by apoptosis) even in cells that do not express ERs or ERRα. We recently observed that SR16388 can induce persistent macroautophagy (autophagy) in diverse tumor cells, including cells from hematopoietic malignancies (e.g., multiple myeloma/MM cells) in addition to carcinomas such as prostate and breast cancers. We demonstrated induction of autophagy by using established assays (e.g., LC3I/II processing; fluorescent reporters for acidic vacuole formation; long-lived protein degradation). Here we present results demonstrating that SR16388 can rapidly and potently induce autophagy in cultures of MM cells (U266, RPMI-8226) and PC3 prostate cancer cells. Induction of autophagy in PC3 cells, in particular, was detected in tumor xenografts as early as 3 hours after dosing of mice with a therapeutic dose level of SR16388. We will present data supporting the hypothesis that SR16388 exerts its anti-proliferative and anti-tumor effects in part through the induction of stress pathways modulated by perturbations of protein homeostasis. These effects could yield therapeutic benefits that derive from targets within solid tumor microenvironments. Citation Format: Lidia C. Sambucetti, Wei Zhou, Joy Calaoagan, Laurie Kara, Xiaohe Liu, Barbara Sato, Steve Samuelson, Nathan Collins, Keith Laderoute. SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1692. doi:10.1158/1538-7445.AM2013-1692