e24001 Background: p21-activated kinase 1 (PAK1) stimulates growth and metastasis in several types of tumors, including NSCLC. Protein kinase C iota (PKCi) is highly expressed in NSCLC and regulates PAK1 signaling. We have shown that cancer pathway-specific intervention, like EGFR inhibitors in EGFR mutant NSCLC, results in parallel compensatory activation of other pathways, including the receptor tyrosine kinases AXL and MET, the transmembrane protein CUB domain-containing protein-1 (CDCP1) or the transcriptional regulators STAT3 and YAP1. We have now explored whether a non-pathway-specific approach can be efficient in three classes of NSCLC. Methods: Three lung cancer cell lines were used: HCC827 and H23 lung adenocarcinoma cells with EGFR and KRAS mutations respectively, and H520 PAK1 amplified squamous NSCLC cells. Cell viability assays and western blotting were applied to evaluate the effect of IPA-3 plus auranofin and OTSSP167 plus auranofin. The Chou-Talalay modified method was used for drug combination studies (combination index [CI] = 1), synergism (CI < 1), and antagonism (CI > 1). Results: We found a differential PAK1 expression or activation profile in the three models, with H520 cells being the ones with the highest PAK1 expression and activation. IPA-3 plus auranofin was highly synergistic in HCC827, H23 and H520 cells with CIs of less than 0.4. In the HCC827 cells, the combination of IPA-3 plus auranofin abrogated EGFR and downstream signaling (ERK, AKT, STAT3, YAP1) and inhibited the expression and activation of AXL, MET and CDCP1. IPA-3 plus auranofin was also highly synergistic in H23 (CI = 0.3) and H520 (CI = 0.3) cells. In addition, the combination of OTSSP167 plus auranofin was synergistic in the three cell lines (0.7 in each cell line). Specifically, we noted that OTSSP167 ablated PAK1 phosphorylation in Ser144, Thr423 and Thr212. Conclusions: To date there are no PAK1 inhibitors available. Phase I trials of OTSSP167 in breast cancer (NCT02926690) and leukemia are ongoing. Hence, the combination of OTSSP167 and auranofin could be effective and of interest to be tested in EGFR, KRAS mutant and squamous NSCLC patients.