Ras-Related Protein Rab-32 and Thrombospondin 1 Confer Resistance to the EGFR Tyrosine Kinase Inhibitor Osimertinib by Activating Focal Adhesion Kinase in Non-Small Cell Lung Cancer

SIMPLE SUMMARY: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor and the standard of care therapy for non-small cell lung cancer patients harboring EGFR-activating mutations. However, even for patients treated with osimertinib, resistance inevitably occurs leading to disease progression. Here, we utilized two osimertinib-resistant cell lines and investigated their RNA profiles. We found that Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) were upregulated and associated with resistance in osimertinib-resistant cells as well as in liquid biopsies from patients with disease progression following osimertinib treatment. Moreover, we found RAB32 and THBS1 to be mechanistically linked to activation of the focal adhesion pathway where combination of osimertinib with a FAK inhibitor resulted in a synergistic suppression of viability of osimertinib-resistant cells. Our findings propose a potential therapeutic strategy for overcoming acquired resistance to osimertinib in non-small cell lung cancer. ABSTRACT: Treatment with the tyrosine kinase inhibitor (TKI) osimertinib is the standard of care for non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Osimertinib is also used in T790M-positive NSCLC that may occur de novo or be acquired following first-line treatment with other EGFR TKIs (i.e., gefitinib, erlotinib, afatinib, or dacomitinib). However, patients treated with osimertinib have a high risk of developing resistance to the treatment. A substantial fraction of the mechanisms for resistance is unknown and may involve RNA and/or protein alterations. In this study, we investigated the full transcriptome of parental and osimertinib-resistant cell lines, revealing 131 differentially expressed genes. Knockdown screening of the genes upregulated in resistant cell lines uncovered eight genes to partly confer resistance to osimertinib. Among them, we detected the expression of Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) in plasmas sampled at baseline and at disease progression from EGFR-positive NSCLC patients treated with osimertinib. Both genes were upregulated in progression samples. Moreover, we found that knockdown of RAB32 and THBS1 reduced the expression of phosphorylated focal adhesion kinase (FAK). Combination of osimertinib with a FAK inhibitor resulted in synergistic toxicity in osimertinib-resistant cells, suggesting a potential therapeutic drug combination for overcoming resistance to osimertinib in NSCLC patients.