Abstract 309: Mechanisms of resistance to trastuzumab emtansine in gastric cancer
Authors: Sauveur Juliette , Chettab Abdelkamel , matera Eva , Cleret Aurore , Savina Ariel , Dumontet Charles 
Published: July 2016

Background: Overexpression of Human Epidermal growth factor Receptor 2 (HER2) in cancer was associated with poor outcome and high chances of recurrence before the development of anti-HER2 agents. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate targeting HER2 based on its antibody component trastuzumab. After internalization of T-DM1/HER2, Lys-MCC-DM1, a tubulin binding agent is released in the cytoplasm. In spite of therapeutic advances, acquired resistance to treatment remains a major obstacle. A better understanding of the mechanisms of resistance to T-DM1 is necessary to improve treatment regimens for HER2-overexpressing cancer patients. We have developed and characterized resistance models to T-DM1 in order to describe underlying resistance mechanisms and develop alternative strategies.

Methods: To develop in vitro T-DM1 resistance models, HER2-overexpressing esophageal adenocarcinoma cell line OE-19 was exposed to increasing doses of T-DM1 several months in the absence or presence of cyclosporin A (CsA, an inhibitor of MDR1 transporter). Resistance models were called OE-19 TR (exposed to T-DM1) and OE-19 TCR (exposed to T-DM1 and CsA). To characterize the resistance models, sensitivity to T-DM1 was assessed by MTT assay, Annexin V-PI staining and xCELLigence. Cell cycle distribution was assessed by propidium iodide using flow cytometry. Cross-resistance was studied by MTT assay. Expression of HER2, ABC transporters and tubulin was studied by rt-qPCR, flow cytometry and/or Western Blot. An expression profile of sensitive and resistant cells to T-DM1 was performed using a pan-genomic Illumina cDNA array.

Results: After prolonged exposure of OE-19 cells to T-DM1, IC50 value is 18-fold higher in TR cells and 21-fold higher in TCR cells and both models become less sensitive to T-DM1-induced apoptosis. T-DM1 induced G2/M arrest in OE-19 sensitive cells but not in resistant cells. We found that resistant cells become less sensitive to trastuzumab but remain sensitive to other HER2-targeted therapies such as lapatinib, as well as to a variety of chemotherapy agents. Among the resistant cells, a subpopulation with increased expression MDR1 was identified in OE-19 TR and TCR. Concerning the expression of the main targets of T-DM1 (HER2 and tubulin); we found that HER2 expression and total α and β tubulin expression and content remained unchanged. However the isoforms β2 and β3 were overexpressed in both resistant models. A transcriptomic approach of T-DM1 resistant cells showed modification in adhesion junctions and focal adhesions.

Conclusion: Prolonged exposure of the OE-19 cell line to T-DM1 resulted in resistance to this immunoconjugate. We are working on describing the resistance mechanisms to T-DM1, which are important to understand cancer progression in patients receiving this therapy. In the long term, we hope this study will allow proposing agents that benefit T-DM1-refractory cancer patients.

Citation Format: Juliette Sauveur, Abdelkamel Chettab, Eva matera, Aurore Cleret, Ariel Savina, Charles Dumontet. Mechanisms of resistance to trastuzumab emtansine in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 309.