Abstract The identification of oncogenic molecular drivers in non-small cell lung cancer (NSCLC) has allowed biologically targeted therapies to improve clinical outcomes in the treatment of NSCLC. ALK gene rearrangements are observed in a sub-set of NSCLC patients who demonstrate high response rates to treatment with the oral kinase inhibitor, crizotinib. Unfortunately, disease progression is inevitable, either through intrinsic or acquired resistance. A recently completed series of crizotinib-resistant, ALK+ NSCLC patients by our group demonstrates two broad categories of resistance: (1) continued reliance on ALK signaling (via secondary mutations in the ALK kinase domain or ALK gene fusion copy number gain) or (2) loss of reliance on ALK signaling (e.g. via reliance on alternate oncogene signaling). Here we investigate potential therapeutic strategies when ALK signaling is retained. In an effort to minimize drug resistance to crizotinib in ALK+ NSCLC, we performed a genome-wide shRNA synthetic lethal screen to identify genes whose depletion synergizes with crizotinib in ALK+ NSCLC lines. We identified several genes involved in nucleotide synthesis and DNA metabolism, including dihydrofolate reductase (DHFR) and the trifunctional enzyme encoded by GART, which are substrates for inhibition with pemetrexed. Indeed, ALK+ NSCLC cell lines show increased sensitivity to pemetrexed in vitro. Identification of this cellular process as critical for ALK+ NSCLC is also consistent with clinical data demonstrating that patients with ALK+ NSCLC exhibit an increased clinical benefit on pemetrexed compared to other molecular subgroups of NSCLC. We also identified genes involved in chaperone function in our synthetic lethal screen. As such, we investigated whether proper folding and prevention of degradation was essential for continued reliance on ALK signaling. Both non-mutated EML4-ALK wild-type and EML4-ALK containing kinase domain mutations (including a novel mutation, G1269A) are sensitive to HSP inhibition with 17AAG. By identifying critical cellular processes in ALK+ lung cancer and employing therapies that target these pathways, improved treatment strategies can be derived to treat and prevent resistance in this molecular subtype of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5594. doi:1538-7445.AM2012-5594