Differentiating Ensartinib from Lorlatinib and Alectinib for First Line Use in an ALK+ Non-Small Cell Lung Cancer Preclinical Model (Rescu)

e20607 Background: The objective of this study was to determine whether Ensartinib with its high potency and resistance suppressing properties creates a more favorable treatment profile when used as first-line therapy in ALK+ non-small cell lung cancer compared to two other ALK inhibitors, Alectinib and Lorlatinib. Methods: The ResCu system, a novel culturing system, allows for long term culture of cells without the need for passaging, allowing determination of treatment resistance that can develop over time in a physiologically relevant system that conserves the resistance pathways found in patients. Two ALK-dependent lung adenocarcinoma cell lines, H3122 and H2228 were used to derive starting populations for this study. Using ResCu, we drove resistance to three ALK inhibitors: alectinib, lorlatinib, and ensartinib by using a clinically equivalent dose. The evolved cells were characterized for cross-sensitivity and resistance to a representative panel of 21 drugs. Cell viability was assessed by luminescence after 7 days of exposure to these secondary drugs. Additionally, BulkRNA Seq and WES were performed on the resistant evolved cells. Results: The ResCu system developed resistant cell populations by evolutionary steering of H3122 and H2228 cell lines treated with ensartinib, alectinib, and lorlatinib. Genetic background had a significant effect on which ALKi exhibited the most durable response. The H2228 derived cell population became resistant to alectinib and lorlatinib by 14 days while ensartinib remained effective for at least 42 days. In contrast, H3122 derived cells became resistant to all ALK inhibitors tested by 14 days. Rate of cross-resistance generation also varied between ALK inhibitors. Alectinib led to the highest levels of cross-resistance to other ALKi, approximately 3x higher. Lorlatinib led to highest levels of cross-resistance to other drug classes. Treatment with lorlatinib led to resistance in 13 other drug classes. Ensartinib led to least cross-resistance to ALKi and other drug classes. While the H3122 derived cells more rapidly evolved ALKi resistance, first line treatment with ensartinib sensitized them to lorlatinib, extending overall response to ALKi. Conclusions: The ResCu system developed two ALKi resistant cell populations from distinct genetic backgrounds under physiologically relevant conditions. In one of the two genetic background ensartinib response was durable for greater than 42 days compared to 14 days with alectinib and lorlatinib. Alectinib led to the most cross-resistance to other ALKi while lorlatinib led to the most cross-resistance to other drug classes in both backgrounds. Biomarkers of ALKi escape and cross-resistance were derived from RNA-seq, and we are currently validating these alterations against clinical samples.