Abstract LB228: Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells

Abstract Background: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinases, which is approved for HER2-positive metastatic breast cancer as second line treatment. Significant proportion of patients experience disease progression due to acquired resistance. Activation of DNA damage repair (DDR) is one of the drug-resistance mechanism, however, the impact of DDR on sensitivity to lapatinib is unclear. Thus, lapatinib resistance mechanism was explored from the standpoint of DDR activation. Methods: Acquired lapatinib-resistant (LR) SK-BR-3 cell lines were generated by continuously exposing to lapatinib, starting with 30 nmol/L and incrementally increasing to 10 μmol/L over 7 months. MTT assay was used to confirm lapatinib sensitivity. Cell cycle progression was analyzed using BrdU assay. Replication fork speed and stalled fork were analyzed by DNA fiber assay. Expression of the molecules was examined using Western blotting, immunofluorescence assay and transcriptome data analysis. DNA strand breaks and repair efficacy were evaluated through alkaline comet assay. Results: G1/S phase transition was increased and early/late S phase population was increased in SK-BR-3 LR cells. Replication fork speed was accelerated and replication stress are elevated in SK-BR-3 LR cells. p-Chk1, Rad51, Rad51B, Rad51C and XRCC3 were upregulated in SK-BR-3 LR cells. After irradiation or treatment with ATR inhibitor, γ-H2AX was continuously increased in parental cells. In contrast, γ-H2AX did not change significantly in SK-BR-3 LR cells, and Chk1 was activated after irradiation or treatment with ATR inhibitor. Tail of comet disappeared at early time point in in SK-BR-3 LR cells after induction of DNA damage compared with parental cells. These results demonstrated that DDR was activated and DNA damage repair capacity was enhanced in LR cells. Conclusion: Replication stress is elevated in SK-BR-3 LR cells. Up-regulation of molecules involved in the homologous recombination repair pathway was observed in SK-BR-3 LR cells. Moreover, DNA repair capacity was increased in SK-BR-3 LR cells. These data suggested that activation of DNA damage repair pathway caused by replication stress contributes to lapatinib resistance. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yu Jin Kim, Sujin Ham, Hae Min Hwang, Minyoung Lee, Changyun Lee, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB228.