Oncogenic ras predicts lack of response to PX-866, an inhibitor of the phosphatidylinositol-3-kinase pathway in tumor xenograft models.

A257 The phosphatidylinositol-3-kinase (PI-3-K)/Akt signaling pathway plays a major in cell growth, survival and the resistance of cancer cells to therapy. Several inhibitors of PI-3-K signaling are rapidly moving towards or have reached early clinical trials. As the patient population begins receiving these agents, defining measurable patient parameters to predict a subset who will receive a maximal survival benefit will prove increasingly important. PX-866, a semisynthetic viridin inhibitor of PI-3-K, exhibits antitumor activity in a number of in vivo tumor models and has been shown to have a toxicity profile suitable for entry into clinical trials. We have screened PX-866 in vivo against multiple tissue tumor cell line-derived xenografts for its ability to inhibit tumor growth. PX-866, when administered at a schedule sufficient for the prolonged inhibition of AKT activity, showed pronounced single agent cytostatic effects in several different tumor types. Additionally, a subset of tumor types was identified which displayed little or no growth arrest after prolonged treatment with PX-866 at pharmacologically relevant doses. The magnitudes of the responses to PX-866 in vivo were not reflected in standard cell killing assays under monolayer culture conditions. An analysis of the mutations in the cell lines xenografts screened revealed that the five showing moderate to complete resistance to PX-866 all harbored an oncogenic, activating mutation in the K or N ras signaling protein at codons 12, 13, or 61 The activated ras protein has been shown to have the ability to simultaneously activate a number of signaling pathways involved in oncogenic processes, including the Ral, Raf and PI3K pathways. Through the use of reverse lysate protein arrays (RPPA) we investigated the activation of signaling pathways in cell lines responsive and resistant to PI-3-K inhibition in our in vivo model. There was a significant increase in cyclin B and decrease of E-cadherin in the resistant lines, both changes previously found associated with ras transformation. To further explore which signaling pathways was driving resistance we utilized a colon cancer line in which the oncogenic ras was replaced with a series of ras constructs that preferentially activate one of the three key identified effector pathways through modulation of domains on the enzyme. Clonogenic assays with cell lines harboring these constructs revealed the ability of ras to utilize raf for growth and survival independent of PI-3-K signaling. Identification of active ras and/or markers associated with its activation as an indicator of lack of response may serve as important tools in the selection of individuals best suited for treatment with inhibitors of the PI-3-K signaling pathway for cancer therapy. Additionally, identification of signaling pathways driving this resistance may serve as a guide for the rational combination of these inhibitors, including PX-866, with other current and emerging targeted therapies.