Abstract 4529: KRAS mutation status predicts sensitivity to Anti-DHFR therapy (methotrexate) in non small cell lung cancer cell lines
Authors: Trusk Patricia , Moran Diarmuid M. , Shell Scott A. , Bacus Sarah 
Published: June 2012

Abstract
Somatic genetic mutation in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been linked to resistance to various targeted therapeutics in Non Small Cell Lung Cancer (NSCLC). Therapeutic strategies that target tumors harboring these mutations represent an unmet medical need. In this study, we have identified higher sensitivity to a dihydrofolate reductase (DHFR) inhibitor, methothrexate (MTX), in KRAS mutant (mut) versus wild type (wt) NSCLC cell lines. Ten human NSCLC cell lines (4 KRASwt / 6 KRASmut) were treated with MTX and assayed for proliferation after 72h. KRASwt cells did not show response to MTX treatment, however, growth of 4 out of 6 KRASmut cell lines was significantly inhibited. Interrogation of the NCI Developmental Therapeutics Progam cancer drug screen database also revealed a similar trend in MTX treated NSCLC cell lines. Previous reports describe decreased KRAS activity in MTX treated cells. However, in this study KRAS activity (as measured by substrate pull-down assay) was not altered in MTX treated KRASmut cells. Furthermore, phosphorylation of signaling proteins downstream of KRAS (ERK and AKT) was maintained or increased in MTX treated KRASmut cells. This suggests that MTX does not inhibit KRASmut as a mechanism of action in these cells and cannot account for differences between wt and mut cells. To further investigate the differences in response to MTX in KRASwt versus KRASmut cells, we transfected KRAS in A549 cells and examined the expression of proteins related to folate metabolism and cell cycle progression. We observed increased expression of DHFR, Thymidylate Synthase, E2F and phospho-Retinoblastoma (Rb) protein upon KRAS transfection. Conversely, siRNA knockdown of KRAS in KRASmut cells decreases phospho-Rb and E2F transcription factors. We propose that mutant KRAS drives expression and release of E2F transcription factors which may in turn lead to increased expression of DHFR and potential dependency on this pathway. Moreover, a link between active E2F transcription factors and MTX sensitivity has been previously described. To investigate this further we propose to examine the expression of these and other cell cycle regulatory proteins that control Rb phosphorylation (e.g Cyclin D, CDK4) in KRASwt versus mut NSCLC cell lines. In addition, we will also examine the responsiveness of KRASmut NSCLC cell lines to MTX upon siRNA knockdown of KRAS dependent pathways. Furthermore, we aim to examine the proliferation of wt versus mut cells under conditions of DHFR knockdown using siRNA which will determine dependency on this specific pathway. Collectively, these studies highlight increased sensitivity to MTX in KRASmut NSCLC cell lines. The remainder of this study aims to characterize the underlying molecular mechanisms leading to this anti-DHFR sensitivity and ultimately help to direct the treatment of this subtype of NSCLC tumors in humans.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2011-4529