Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response and is often genetically activated in Non-Small Cell Lung Cancers (NSCLCs) by, for instance, mutations in the interacting protein KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these was NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain and demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable, transcriptional regulator that supports NRF2-dependent lung cancers.

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser 473 -Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Rasdependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

Purpose: The purpose of the study was to evaluate the stability of phosphoprotein as a marker of signaling activity in human tumors using clinical samples and xenografts. Experimental Design: The expression of phospho-Ser473-Akt (p-Akt) was assessed by immunohistochemistry in paraffin-embedded samples from patients enrolled in a Southwest Oncology Group clinical trial of gastroesophageal junction tumors and by immunohistochemistry and Western blotting in human colon tumor xenografts at various times after removal from the animal. Results: Clinical samples had evaluable p-Akt staining only when obtained as biopsies (9 of 13) and no staining was observed in tumors obtained as surgically resected samples (0 of 15). In HT-29 colon cancer xenografts, p-Akt staining was present in fresh sample but not in tissue that had been allowed to stand for 30 minutes at room temperature. Western blotting of HT-29 tumor xenografts at room temperature showed a slow decrease in total Akt with a half-life of 180 minutes and a rapid decrease in p-Akt with a half-life of 20 minutes. Conclusions: Caution should be used when using phosphoprotein levels in human tumor specimens to measure intrinsic signaling activity or drug effects because of the potential for rapid dephosphorylation. Rapid processing of biopsies is essential and postoperative surgical samples may be of limited value because of the time to fixation.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is currently one of the most exciting drug targets in oncology. However, only a short time ago, the paradigm existed that drugs targeted to the four PI3K class

Epidermal growth factor receptor (EGFR) inhibitors such as gefitinib show antitumor activity in a subset of non -small cell lung cancer (NSCLC) patients having mutated EGFR. Recent work shows that phosphatidylinositol-3-kinase (PI3-K) is coupled to the EGFR only in NSCLC cell lines expressing ErbB-3 and that EGFR inhibitors do not inhibit PI3-K signaling in these cells. The central role PI3-K plays in cell survival suggests that a PI3-K inhibitor offers a strategy to increase the antitumor activity of EGFR inhibitors in resistant NSCL tumors that do not express ErbB-3. We show that PX-866, a PI3-K inhibitor with selectivity for p110A, potentiates the antitumor activity of gefitinib against even large A-549 NSCL xenografts giving complete tumor growth control in the early stages of treatment. A-549 xenograft phospho-Akt was inhibited by PX-866 but not by gefitinib. A major toxicity of PX-866 administration was hyperglycemia with decreased glucose tolerance, which was reversed upon cessation of treatment. The decreased glucose tolerance caused by PX-866 was insensitive to the AMP-activated protein kinase inhibitor metformin but reversed by insulin and by the peroxisome proliferator-activated receptor-; activator pioglitazone. Prolonged PX-866 administration also caused increased neutrophil counts. Thus, PX-866, by inhibiting PI3-K signaling, may have clinical use in increasing the response to EGFR inhibitors such as gefitinib in patients with NSCLC and possibly in other cancers who do not respond to EGFR inhibition. [Mol Cancer Ther 2005;4(9):1349 -57]

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.

AKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non -lipid-based compounds that bind selectively to the PH domain of AKT, with ''in silico'' calculated K D values ranging from 0.8 to 3.0 Mmol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide -dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro K D s for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 Mmol/L. Some of the compounds exhibited PH domain -binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide -dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT. [Mol Cancer Ther 2008; 7(9):2621 -32]