Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non-small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. Mol Cancer Ther; 9(3); 706-17. ©2010 AACR.

A new approach is described for delivering small interfering RNA (siRNA) into cancer cells by noncovalently complexing unmodifi ed siRNA with pristine single-walled carbon nanotubes (SWCNTs). The complexes were prepared by simple sonication of pristine SWCNTs in a solution of siRNA, which then served both as the cargo and as the suspending agent for the SWCNTs. When complexes containing siRNA targeted to hypoxiainducible factor 1 alpha (HIF-1 ) were added to cells growing in serum containing culture media, there was strong specific inhibition of cellular HIF-1 activity. The ability to obtain a biological response to SWCNT / siRNA complexes was seen in a wide variety of cancer cell types. Moreover, intratumoral administration of SWCNT-HIF-1 siRNA complexes in mice bearing MiaPaCa-2 / HRE tumors signifi cantly inhibited the activity of tumor HIF-1 . As elevated levels of HIF-1 are found in many human cancers and are associated with resistance to therapy and decreased patient survival, these results imply that SWCNT / siRNA complexes may have value as therapeutic agents.

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional co-activator p300. Notably, of the HIF1α activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cell, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors.

KRAS is the predominant form of mutated RAS (mut-KRAS) and is found in 25% of patient tumors across many cancer types. Mut-KRAS is well known to play a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. However, despite extensive effort, there is no effective treatment for mut-KRAS. The effects of mut-KRAS are mediated through multiple downstream signaling pathways which have been independently associated with tumorigenesis, including RAF1, RALGDS, and PI3K. Current efforts to treat mut-KRAS tumors employ concurrent treatment with inhibitors of the RAF and PI3K pathways but this does not address the potential contribution of other pathways for which there are currently no inhibitors. Using a global siRNA screen we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth using an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, and validated these hits in a similar isogenic HCT-116 colon cell line. From the screen we identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) as a top hit. CNKSR1 is found associated with KRAS in the RAS membrane associated signaling nanocluster that KRAS has to be associated with to provide growth signals. Knockdown of CNKSR1 with siRNA inhibited the growth of a panel of 10 mut-KRAS non small cell lung cancer (NSCLC) cell lines but not of 4 NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein that has a potentially druggable plekstrin homology (PH) domain responsible for binding to membrane phosphatidylinositols-3-phosphates. In order to demonstrate whether the PH domain of CNKSR1 is necessary for mut-KRAS activity we over expressed the PH domain in H1373 mut-KRAS NSCLC cells and found that it acted as a dominant negative and inhibited cell growth. We suggest that the PH domain fragment competes with the full length CNKSR1 in the cell. We also showed that knockdown of CNKSR1 inhibited KRAS dependent phosphorylation of RAF1 in multiple non small cell lung cancer cell lines. Together our results suggest that the CNKSR1 protein, acting through it's PH domain, is necessary for cell growth and down stream signaling by the KRAS oncogene. The PH domain can be targeted by inhibitors thus potentially providing agents that will selectively block mut-KRAS signaling and cell growth, creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A197.

The ability to survive hypoxic stress is an essential component of tumor development. Many rapidly proliferating solid tumors outgrow their vasculature while the abnormal tumor vasculature leads to small, transient pockets of hypoxic cells. In addition to being deprived of oxygen, these cells are also deprived of nutrients and unable to export harmful byproducts of cellular metabolism out of their immediate environment. One mechanism cancer cells employ to survive hypoxic stress is the stabilization and activation of the transcription factor hypoxia inducible factor 1α (HIF-1α), which translocates to the nucleus and induces pro-survival genes such as VEGF, Glut-1, and glycolytic enzymes. MiaPaCa-2 pancreatic carcinoma cells stably transfected with a 5X hypoxia response element (HRE) promoter regulating the firefly luciferase gene were generated, and shown to express luciferase only when HIF-1α was stabilized in hypoxia(1% O2). Using these cells, a genome wide siRNA screen was performed using the Dharmacon 22,000 gene siRNA library under the conditions of normoxia, 16hr and 72 hr hypoxia, to discover novel effectors of the HIF-1α pathway. Luciferase expression was corrected for cell number using a WST-1 cell proliferation assay prior to luciferase detection. Several hits were identified including genes which, when silenced, both increased and decreased HIF-1α transcriptional activity. Genes whose inhibition increased HIF-1α transcriptional activity in normoxia, in some cases up to 19 fold, include members of the E3 ubiquitin ligase complex which bind to and degrade HIF-1α under normal oxygen tensions, and also a number of other genes with uncharacterized function. There were also genes whose inhibition decreased hypoxia induced HIF-1α activity. Many were not well characterized, but there were a number of genes belonging to the MAP kinase pathway; the DNA repair pathway; the NF-κB pathway; and glycolysis genes, that were identified as necessary for HIF-1α transcriptional activity. Hits in these pathways were validated by secondary siRNAs and Western blots. It was discovered that silencing key genes involved in glycolysis inhibited HIF-1α transcriptional activity due to a decrease in HIF-1α protein levels. They include hexokinase-1, pyruvate dependent kinases-1,2 and 3, and 6-phosphofructo-1-kinase(M). These results suggest that increased glycolysis found in many tumors under aerobic conditions (the Warburg effect), which has been suggested to be caused by increased Myc expression, mutant Ras, mutant p53 and PI-3-kinase signaling, has an important role in increasing HIF-1α expression and activity. This stabilization and increase in activity then further induces the expression of target glycolytic genes and glycolytic activity in a feed-forward loop. 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 5505.

Cancer cells satisfy their increased need for energy under conditions of normal oxygen tension by undergoing a high rate of glycolysis followed by lactic acid fermentation, a process known as the Warburg Effect, rather than oxidative phosphorylation as observed in normal cells. Unlike proliferating cancer cells, the internal environment of most solid tumors is hypoxic due to an inadequate or disorganized blood supply. This results in tumors generating ATP through anaerobic respiration, a process many times less efficient than oxidative phosphorylation. The importance of anaerobic glycolysis for cancer cell energy generation makes it an attractive target for inhibiting cancer growth. However, agents currently in use for regulating energy production in tumors are relatively ineffective analogs of glucose or intermediates of the glycolytic pathway. We performed a high throughput siRNA screen using an siRNA library targeting all known open reading frames of the human genome (Dharmacon Inc.) to identify genes that when inhibited would block hypoxia inducible factor-1 alpha (HIF-1α) transcription factor luciferase reporter activity. A significant number of genes selected and validated were members of the glycolysis pathway. It is known that many glycolysis enzymes are induced by HIF-1α providing a feed forward loop between glycolysis and HIF-1α. We have addressed two questions: (1) what is the biological mechanism by which these two signaling systems regulate one another?; and (2) can specific inhibitors of glycolysis be developed with the added effect of inhibiting HIF-1α? Our mechanistic studies have shown that of the 40 enzymes of glycolysis involving 11 enzymatic steps and various enzyme isoforms, siRNA knockdown of Aldolase A (ALDOA) produces the maximum inhibition of HIF-1α activity. Further work indicated that ALDOA siRNA inhibition of HIF-1α is mediated through AMPK, a sensor of low ATP levels in the cell, since siRNA knockdown of AMPK results in rescue of HIF-1α activity. The effect of ALDOA inhibition is not mediated through a reduction in HIF-1α protein suggesting that, contrary to the canonical AMPK pathway, mTOR and its regulation of translation is not involved. Instead, p300 is phosphorylated by AMPK thus inhibiting its function as a co-activator of HIF-1α. A structure based drug design approach was employed for lead identification and optimization using the crystal structure of ALDOA (4ALD) and GOLD virtual screening platform. Three compounds have been identified as having effects on the modulation of ALDOA activity in a protein based biochemical assay as well as inhibitory effects on glycolysis itself using the XF96 Analyzer from Seahorse Bioscience. Here, we present an overview of ALDOA target identification, its mechanism as a regulator of HIF-1α activity and virtual drug design methodologies used in the search for novel pharmacological probes which act as dual inhibitors of energy production and the HIF-1α survival pathway. Citation Format: Geoffrey V. Grandjean, John Kingston, John Kenneth Morrow, Shuxing Zhang, Garth Powis. Inhibiting glycolysis and HIF1α: a novel approach to cancer therapy. [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 2076. doi:10.1158/1538-7445.AM2013-2076

Oncogenic mutation of the KRas enzyme occurs frequently in human tumors and results in the constitutive activation of multiple signaling pathways. KRas mutation occurs in three specific codons, 12, 13, or 61, and involves the substitution of multiple amino acids. We observed in cancer cell lines containing activating mutations in KRas but not wild type KRas, that the Hypoxia Inducible Factor 1 α (HIF-1α) is expressed and functional under normoxic conditions. HIF-1α itself has been implicated in multiple aspects of tumorigenesis, including survival and invasion. We have found that exogenous expression of wild type KRas or the introduction of oncogenic KRas into transformed cells, causes the expression of HIF-1α in normoxia, but has little or no effect on HIF-1α induction by hypoxia. Additionally, in cancer cells, the deletion of the oncogenic KRas allele, while preserving the wild type allele, silenced the normoxic expression of HIF-1α. To determine the mechanisms by which KRas elicits the expression of HIF-1α, we quantitated the expression of 176 phospho or total proteins utilized a Reverse Phase Protein Array (RPPA) of 74 highly characterized non small cell lung cancer lines (NSCLC's) including 22 NSCLS with oncogenic KRas under three different culture conditions (with serum, without serum and starved then stimulated with serum for 30 minutes). From this RPPA, we found that NSCLC lines containing oncogenic KRas expressed active serine 473 Akt at differing levels, dependent upon which amino acid substitution was present in KRas. Additionally, we found that the KRas activation of Akt signaling, along with other KRas dependent signals, was responsible for HIF-1 stabilization in a hypoxia independent manner. As previously suggested we find that through KRas and Akt driven signaling, KRas is able to overcome the oxygen driven mechanisms responsible for HIF-1α degradation by increased HIF-1α protein translation However, we additionally found that aerobic glycolysis driven by KRas and Akt, specifically the accumulation of the products of glycolysis, also contribute to HIF-1α expression in normoxia. Finally, genome wide mRNA profiling of a KRas transformed line with a stable shRNA mediated knockdown of HIF-1α compared it a vector control showed that under normoxic conditions, knockdown of HIF-1α down regulates several genes important to transformation, including growth factors previously associated with KRas induced transformation. This data suggests inhibitors of HIF-1α may be effective in tumors harboring the KRas oncogenes. Additionally, Akt and HIF-1α inhibitors may be more active when combined with other agents targeted to other signaling pathways activated by KRas in order to bypass resistance mediated by redundant KRas driven signaling pathways. 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 306.

Thioredoxin (Txn), thioredoxin reductase (TxnR) and NADPH comprise a thioredoxin system which exists in nearly all living cells functioning in multi-facetted processes in mammalian cells. These include thiol-dependent thiol-disulfide exchange reactions crucial to control the redox homeostasis, cellular growth, defense against oxidative stress and control of apoptosis. Thioredoxin expression has been observed to be elevated in some types of human cancer and has been shown to confer resistance to chemotherapy or radiation-induced apoptosis. It has also been shown that elevated levels of thioredoxin contribute to the carcinogenic process and invasive phenotype of cancer. Identifying key inducers of thioredoxin expression in cancer cells will permit us to specifically target these components with the goal of improving the clinical outcome of treatment of patients with thioredoxin overexpressing cancers. Here, we describe a functional genomic screen designed to identify inducers of thioredoxin expression in which a library of 7040 small interfering RNAs (targeting human kinome and druggable genes) was screened for their ability to block the expression of endogenous thioredoxin in the pancreatic cancer cell line, MiaPaCa2. The primary screen identified numerous potential inducers of thioredoxin expression and, through the use of extensive validation of all these potential hits, yielded 21 high confidence genes that have not previously been associated with the induction of thioredoxin expression. Six of these target genes were associated with TGFβ signaling and selected for this study. Downregulation of these genes strongly inhibited the expression of endogenous thioredoxin in the MiaPaCa pancreatic cancer and other cancer cell lines. Moreover, treating MiaPaCa2 cells with TGF-β1 induced the expression of thioredoxin. TGF-β1-induced expression of thioredoxin is mediated through SMAD2, SNL2, ITGAV, ITGb3 and SP1 thus showing that our study uncovers new aspects of thioredoxin regulation. Collectively, these observations offer several insights on how thioredoxin mediates tumor growth and demonstrates the utility of large-scale RNAi screens in mammalian cells. 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 2188.