Abstract #LB-11: Combination therapies with small molecule kinase inhibitors for PKC\#946; and c-met are more effective together than each alone in lung cancer cells whose receptors, EGFR and c-met, are activated but without mutations

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The epithelial growth factor receptor (EGFR) in lung cancer has served as a successful target for therapy particularly in patients whose EGFR genes exhibit activating mutations. Our studies have indicated that EGFR chains are promiscuous and can associate physically with other growth factor receptors including c-met, as well as erbB2, and erbB3. As a result, intracellular signaling pathways that are triggered by a given growth factor become amplified to potentially include several alternative pathways. A single kinase inhibitor targeting one pathway thus, is no longer sufficient to inhibit growth of tumor cells where multiple pathways are initiated. Using non-small cell lung cancer (NSCLC) cell lines, we demonstrate that the combination of a PKC\#946; kinase inhibitor with a c-met kinase inhibitor greatly diminishes signal transduction through Akt and decreases cell viability in a cell line where multiple pathways are activated. A similar effect was not observed in a NSCLC cell line (H1975) bearing an activating mutation in EGFR. Lysates were prepared for immunoprecipitation and Western blotting from serum-starved Calu3 and H1975 cell lines after incubation with kinase inhibitors, including Enzastuarin for PKC\#946;, SU 11274 for c-met, and AG 1478 for EGFR. Specific site directed anti-phospho-antibodies were used to probe Western blots to determine the effect of respective inhibitors on downstream kinase activation. Proliferation and cell viability assays were performed with 10,000 cells seeded into 96-well plates and incubated with inhibitors in serum-starved cultures for 72 hours. Cell viability was determined by the addition of Calcein-AM (calcein-acetoxymethyl ester, diacetate) for the final 2 hours and plates were read at 517 nm. c-Met was co-immunoprecipitated with EGFR from lysates of Calu3 cells. These cells were particularly sensitive to the PKC\#946; kinase inhibitor Enzastuarin. In fact, a combination of Enzastuarin with the c-met inhibitor, SU 11274 appreciably decreased cell viability over either inhibitor used alone. Western blotting with antibodies directed against phosphorylated downstream products revealed that the combination of Enzastuarin with SU 11274 decreased EGFR (Y1068) and Akt (ser473) phosphorylation at low doses where Enzastuarin or SU 11274 alone had only minimal effects. The dual combination demonstrated an Enzastuarin concentration dependent decrease in EGFR and Akt phosphorylation. In H1975, c-met was not co-immunoprecipitated with EGFR and the low-dose combination therapy of Enzastuarin and SU 11274 did not inhibit phosphorylation of either EGFR or Akt. Clinical evaluation of a combination therapy to include Enzastuarin with a c-met kinase inhibitor should be undertaken in lung cancer patients lacking EGFR mutations and with tumors that reveal activated c-met. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-11.