Abstract 4041: The EML4-ALK fusion protein mediates reduced sensitivity to the combination of neratinib and dasatinib
Authors: Castel Myra E. , Conlon Neil T. , Eli Lisa D. , Wong Alvin , Crown John , Collins Denis M. 
Published: April 2023

Abstract
Introduction: The anaplastic lymphoma kinase (ALK) gene encodes a receptor tyrosine kinase involved in cellular proliferation, differentiation, and cell death. ALK becomes oncogenic when it forms a fusion gene. The EML4-ALK fusion gene has been identified mainly in non-small cell lung cancer (NSCLC). It is unknown whether neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI) targeting EGFR, HER2 and HER4, is effective in cancers harboring EML4-ALK gene fusion. The objective of this study was to assess in vitro efficacy of neratinib in combination with dasatinib (Src/Abl TKI) or crizotinib (ALK TKI) in EML4-ALK+ NSCLC.
Methods: The antiproliferative effects of neratinib, dasatinib and crizotinib were assessed in the CRISPR/Cas9-modified EML4-ALK fusion-A549 (EML4-ALK+) and parental A459 (A549-Par) NSCLC cell lines by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between neratinib and dasatinib, and neratinib and crizotinib, matrix assays were performed and analyzed using Combenefit software. To further assess the efficacy of the TKIs, changes in signaling pathways were assessed by Western blotting, and apoptosis induction and cell migration were measured with the Incucyte® S3 imaging system.
Results: Neratinib, crizotinib, and dasatinib displayed nanomolar IC50 values in both cell lines. As expected, EML4-ALK+ cells were more sensitive to crizotinib than A549-Par cells (IC50 = 595 nM vs 1 µM, p ≤ 0.05). EML4-ALK expression led to numerical increases in neratinib IC50 value (326.37 ± 44.34 nM in EML4-ALK+ vs 247 ± 32.65 nM in A549-Par) and dasatinib IC50 value (39.95 ± 5.67 nM in EML4-ALK+ vs 27.75 ± 18.53 nM in A549-Par) but these changes were not statistically significant. Matrix assays showed that neratinib-crizotinib (NC) was more effective than crizotinib alone in both cell lines, with more potent sensitivity in EML4-ALK+ cells. The neratinib-dasatinib (ND) combination was synergistic in the A549-Par cell line but only additive in the EML4-ALK+ cell line, suggesting a reduced sensitivity to the ND combination. EML4-ALK+ cells had sustained pERK1/2 levels after treatment with all TKIs, except NC, compared to A549-Par cells. Neratinib alone was significantly less effective at inhibiting pAkt (99.2 ± 5.3 % in EML4-ALK+ vs 64.9 ± 19.0 %, p ≤ 0.05) and pERK1/2 (91.3 ± 4.6 % in EML4-ALK+ vs 54.9 ± 15.9 %, p ≤ 0.01) in EML4-ALK+ cells than in A549-Par cells at 24h. In A549-Par cells, ND caused the highest apoptotic induction, followed by NC. Apoptosis levels induced by ND in EML4-ALK+ cells were 4-fold lower at 72hr than in A549-Par cells (p ≤ 0.0001). Dasatinib and ND were more effective than crizotinib or NC at preventing cell migration in both cell lines.
Conclusion: EML4-ALK potentially decreases sensitivity to the neratinib-dasatinib combination. This warrants further investigation in other EML4-ALK+ models.
Citation Format: Myra E. Castel, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. The EML4-ALK fusion protein mediates reduced sensitivity to the combination of neratinib and dasatinib. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4041.