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.