Abstract PR-2: KRAS mutation and amplification status predicts sensitivity to antifolate therapies in non-small cell lung cancer.

Abstract

Somatic genetic mutation in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene has been linked to poor prognosis and 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 investigated the relationship between antifolate sensitivity and KRAS mutation/amplification status in NSCLC.

Human NSCLC cell lines (KRAS wild type, KRAS mutant non-amplified and KRAS mutant amplified) were treated with Methotrexate (MTX) or Pemetrexed (PEM) and assayed for proliferation after 72h. In these studies, 5 out of 7 KRASwt (wildtype) cells and all KRASmut (mutant) amplified cells showed resistance to MTX treatment (IC50 >10μM). In contrast, growth of all KRASmut non-amplified cell lines studied was inhibited with MTX treatment (IC50 <100nM). Similar effects were observed for PEM in this study. Interrogation of the NCI Developmental Therapeutics Program cancer drug screen database for the relationship between KRAS mutation status and drug efficacy also revealed a similar trend in other NSCLC cell lines for MTX and other anti-folates. Cell cycle analysis of MTX treated cells demonstrated induction of S phase growth arrest in MTX treated KRASmut non-amplified NSCLC cells, an effect that is not observed in KRASwt or KRASmut amplified NSCLC cell lines. qRT-PCR analysis demonstrated a dramatic downregulation of KRAS gene expression in KRASwt and KRASmut cells with antifolate treatment. However, KRAS gene expression was less affected in antifolate treated KRASmut amplified cells. Co-treatment of KRASmut cells with antifolates and hypoxanthine/thymidine (which compensate for folate pathway inhibition) prevented downregulation of KRAS gene expression and rescued KRASmut cells. qPCR array analysis of miRNA expression in antifolate treated cells revealed increased expression of KRAS specific miRNAs including miR-181 and miR-143 with treatment compared to untreated controls. Finally, we present patient data describing unprecedented rapid and durable radiographic responses in KRAS mutant NSCLC cancer patients.

Collectively, these studies identify increased sensitivity to antifolates in KRASmut non-amp NSCLC cell lines. Anti-folate therapies decrease KRAS gene expression in KRASwt and KRASmut cells but do not do so in KRASmut amplified cells. We propose that decreased KRAS gene expression is detrimental to KRASmut cells due to their dependency on this pathway for survival. We also propose that decreases in KRAS gene expression are mechanistically linked to stress (folate inhibition) induced miRNA expression which target KRAS gene expression. Overall, antifolates represent a novel method to target KRAS and as such should be investigated further for use in this subtype of NSCLC. As clinical evidence emerges, it is apparent that both KRAS mutation and amplification status should be considered for patient stratification prior to antifolate treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-2.