The KRAS oncogene is found in 25% of patient tumors across many cancer types. Despite extensive effort since its discovery over 40 years ago there is no effective treatment for mutated KRAS, and an estimated 350,000 patients in the US in 2014 with mutated KRas in their tumors will die of their disease. Mutated KRAS plays a critical role in driving tumor growth and resistance to therapy. Its effects are so powerful that i overrides the activity of many of the new molecularly targeted signaling drugs being developed for cancer today. Thus, finding new agents that inhibit the effects of mutated KRas is a critical unmet need in cancer today. However, intracellular signaling by wild type KRas protein controls many aspects of normal cell function, so that a therapy directed at inhibiting mutant KRas should ideally leave wild type KRas function unaffected. Using a global siRNA functional screen and isogenic cell line pairs of mutant or wild type KRas we identified PLEKHA7 (pleckstrin homology domain containing, family A7) as a protein that when knocked down inhibits the proliferation of colon cancer cells with mutant KRas, but remarkably not wild type KRas cells. Other aspects of the mutant KRas phenotype were also inhibited including anchorage independent (3D) growth, cell invasion, and in vivo tumor growth. We have also shown that PLEKHA7 knockdown decreases the active (GTP bound) form of mutant but not of wild type KRas, with inhibition of downstream mutant KRas signaling. PLEKHA7 is normally found in the adherens junction of normal epithelial cells. In cancer cells it is found in plasma membrane tight junctions but its function is not known. PLEKHA7 is a member of a group of signaling proteins containing a distinctive 3D protein fold, the pleckstrin homology (PH)-domain, that binds to membrane phosphoinositides to position the parent proteins at specific sites on the membrane important for their function. Our previous studies have shown that PH domains can be selectively drugged by small molecules, thus inhibiting the signaling function of the proteins. We have evidence that in cancer cells PLEKHA7 is associated with proteins of the plasma membrane-bound KRas signaling nanocluster. We thus hypothesize that PLEKHA7 selectively regulates mutated KRas activity in the signaling nanocluster, and therefore that the PH- domain of PLEKHA7 is a target for small molecules inhibitors as pharmacological probes for mechanistic studies of PLEKHA7 function, as well as selectively blocking the growth of mutated KRas cancer cells as potential therapy. This represents a new paradigm for attacking Ras via inhibition of associated regulatory signaling nanocluster proteins. The objectives of our study are: 1) to investigate the mechanism for PLEKHA7's ability to selectively inhibit mutated KRas; 2) to conduct structural studies of the PLEKHA7 PH domain and two potential hinge regions, to delineate PLEKHA7's function in associating with the plasma membrane and other proteins of the KRas signaling nanocluster; and 3) to identify small molecule inhibitors of the PLEKHA7 PH- domain as pharmacological probes to study the role of PLEKHA7 in KRas regulation, and as leads for potential agents to treat mutated KRas tumors.
KRAS is the most studied human oncogene. Despite extensive efforts, KRas protein remains an undruggable cancer target. New approaches for identifying agents to inhibit KRas is a pressing medical need as an estimated 350,000 patents in the US in 2013 will have mutant KRas in their tumor and many will die of their disease. We have found that PLEKHA7, a protein with a membrane localizing PH domain, is an essential regulator of mutant but not wild type KRas activity and tumor growth. We will study how PLEKHA7 regulates KRas activity using molecular techniques and small molecule PH domain inhibitors as pharmacological probe compounds, and as potential lead agents to be developed into drugs to treat mutant KRAS tumors.
