To address the need for improved systemic therapy for non–small‐cell lung cancer ( NSCLC ), we previously demonstrated that mesenchymal NSCLC was sensitive to polo‐like kinase (Plk1) inhibitors, but the mechanisms of resistance in epithelial NSCLC remain unknown. Here, we show that cM et was differentially regulated in isogenic pairs of epithelial and mesenchymal cell lines. Plk1 inhibition inhibits cM et phosphorylation only in mesenchymal cells. Constitutively active cM et abrogates Plk1 inhibitor–induced apoptosis. Likewise, cM et silencing or inhibition enhances Plk1 inhibitor–induced apoptosis. Cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cM et activation after Plk1 inhibition. In four animal NSCLC models, mesenchymal tumors were more sensitive to Plk1 inhibition alone than were epithelial tumors. The combination of cM et and Plk1 inhibition led to regression of tumors that did not regrow when drug treatment was stopped. Plk1 inhibition did not affect HGF levels but did decrease vimentin phosphorylation, which regulates cM et phosphorylation via β1‐integrin. This research defines a heretofore unknown mechanism of ligand‐independent activation of cM et downstream of Plk1 and an effective combination therapy.

Mouse models are powerful tools to study lung cancer initiation and progression in vivo and have contributed significantly to recent advances in therapy. Using micro-computed tomography to monitor and study parenchymal and extra-parenchymal metastases in existing murine models of lung cancer is challenging owing to a lack of radiographic contrast and difficulty in achieving respiratory gating. To facilitate the analysis of these in vivo imaging studies and study of tumor progression in murine models we developed a novel, rapid, semi-automated method of calculating thoracic tumor burden from computed tomography images. This method, in which commercially available software is used to calculate the mass of the thoracic cavity (MTC), takes into account the aggregate tumor burden in the thoracic cavity. The present study showed that in tumor-free mice, the MTC does not change over time and is not affected by breathing, whereas in tumor-bearing mice, the increase in the MTC is a measure of tumor mass that correlates well with tumor burden measured by lung weight. Tumor burden calculated with our MTC method correlated with that measured by lung weight as well as or better than that calculated using four established methods. To test this method, we assessed metastatic tumor development and response to a pharmacologic PLK1 inhibitor in an orthotopic xenograft mouse model. PLK1 inhibition significantly inhibited tumor growth. Our results demonstrate that the MTC method can be used to study dynamic changes in tumor growth and response to therapeutics in genetically engineered mouse models and orthotopic xenograft mouse models of lung cancer.

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Introduction: Plk1 is a serine-threonine protein kinase that is overexpressed in cancer cells, and plays a major role in regulating tumor growth. Plk1 inhibitors are well tolerated, but only a few unselected patients with non-small cell lung cancer (NSCLC) respond to single-agent therapy. Our lab discovered that mesenchymal NSCLC cell lines are more sensitive to Plk1 inhibitors than epithelial cell lines in vitro and in vivo. However, mechanisms of resistance to Plk1 inhibitors have not been elucidated and this unknown is a major gap in knowledge. Experimental procedure: To study the mechanisms of Plk1 inhibitor-induced apoptosis we used 3 pairs of isogenic epithelial NSCLC cell lines induced to a mesenchymal phenotype with TGF-β. These isogenic pairs were treated with the Plk1 inhibitor volasertib for 24 h and levels of 301 proteins and phosphoproteins were simultaneously measured using reverse phase protein array (RPPA). Volasertib acquired resistance (VAR) cell lines were generated by exposing cells to increasing doses of volasertib. Results: The induction of a mesenchymal phenotype using TGF-β increased Plk1 inhibition-induced apoptosis in all 3 cell lines. To further elucidate mechanisms of resistance, we compared protein expression in these isogenic cell lines, 24 h after Plk1 inhibition. There were 33 proteins differentially regulated following Plk1 inhibition in parental vs TGF-β induced isogenic cells (p-value < 0.05). Notably, phosphorylated c-Met (Y1234/1235), FAK (Y397) and Src (Y416) were consistently inhibited following Plk1 inhibition in the mesenchymal lines. These changes were confirmed by Western blotting. Total c-Met, FAK and Src protein levels were not affected, implicating a post-translational changes. Likewise, VAR cell lines exhibited an epithelial phenotype and c-Met phosphorylation was persistent even after Plk1 inhibition. Simultaneous c-Met and Plk1 inhibition or silencing increased apoptosis in NSCLC cell lines tested compared to single agent inhibition or silencing. Combination of Plk1 and c-Met inhibitors decreased tumor volume and increased mouse survival in vivo in patient derived and cell line xenograft models. Similarly VAR cells also showed more apoptosis when treated with combination of Plk1 and c-Met inhibitors. Levels of the c-Met ligand HGF were unchanged after Plk1 inhibition and further mechanistic studies are on-going. Conclusion: NSCLC cell lines have diverse sensitivities to Plk1 inhibition, which is consistent with the results of clinical trials of Plk1 inhibitors in solid tumors. This study reveals a novel mechanism of non-canonical c-Met activation in resistant epithelial NSCLC after Plk1 inhibition. We demonstrate a profound effect of combination Plk1 and c-Met inhibition in vivo in multiple mouse models that could be a novel therapy for NSCLC patients. Citation Format: Ratnakar Singh, Pavitra Viswanath, Shaohua Peng, Vaishnavi Sambandam, Li Shen, Lerong Li, Jing Wang, Bingliang Fang, Faye M. Johnson. Noncanonical c-Met activation mediates de novo and acquired resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 895.