Abstract P4-03-15: Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer
Authors: Conlon N , Canonici A , Morgan C , Cremona M , Hennessey BT , Eustace A , O'Brien N , Slamon D , Crown J , O'Donovan N 
Published: February 2018

Abstract
Background: Afatinib is an irreversible pan-HER inhibitor approved for non-small cell lung cancer. We have previously shown that afatinib inhibits growth of HER2-positive breast cancer cells and enhances response to trastuzumab. However, we have also shown that long-term exposure to tyrosine kinase inhibitors leads to the development of acquired resistance. To determine if acquired afatinib resistance develops in HER2-positive breast cancer cells, we exposed a HER2-positive breast cancer cell line to afatinib for 6 months and investigated alterations in the cells following long-term exposure.
Methods: SKBR3 cells were treated with 150 nM afatinib twice-weekly for 6 months. Growth response to drug inhibitors was assessed by acid phosphatase assay. Drug sensitivity was examined in four HER2-positive cell lines (SKBR3, EFM192A, BT474 and HCC1954) and three acquired trastuzumab resistant cell lines (SKBR3-T, BT474-T and EFM192A-T). Reverse phase protein array (RPPA) was used to determine alterations in key signaling pathways. Src, p-Src, EGFR, p-EGFR, ERK1/2, p-ERK 1/2 levels were examined by Western blotting. To examine the prevention of the development of afatinib resistance, cells were treated twice weekly with afatinib, dasatinib, or the combination and stained with crystal violet when confluent.
Results: Following 6 months of afatinib treatment, the SKBR3-A cells were more resistant to afatinib compared to parental cells (IC50 SKBR3-A 284 ± 28.2 nM vs SKBR3-Par 10.9 ± 3.4 nM). Furthermore, the resistant cells were cross-resistant to lapatinib, neratinib and trastuzumab. RPPA interrogation of the SKBR3-A cells showed alterations in several pathways, including significantly increased levels of p-Src (Y416). SKBR3-A cells were more sensitive to Src inhibition with dasatinib compared to SKBR3-Par cells and the combination of afatinib and dasatinib was highly synergistic in SKBR3-A cells (CI value = 0.09 ± 0.06). The combination of afatinib and dasatinib was also synergistic in the trastuzumab resistant SKBR3-T cells (Table). Afatinib and dasatinib inhibited EGFR and Src activation and ERK 1/2 signalling in SKBR3-A cells.
Short-term resistance assays showed that the addition of dasatinib to afatinib blocks the emergence of resistant cells in three of four HER2 positive cell lines tested and two of the three acquired trastuzumab resistant cell lines tetsed.
Conclusion: HER2-positive breast cancer cells that are highly sensitive to afatinib can develop acquired resistance to afatinib within six months. Src is a potential target to prevent the development of afatinib resistance and thus combined treatment with afatinib and dasatinib may be beneficial in patients with HER2-positive breast cancer.
Percentage growth in HER2-positive cells treated with afatinib and/or dasatinib for 5 days. 20 nM Afatinib40 nM Dasatinib20 nM Afatinib + 40 nM DasatinibSKBR325.2 ± 6.1109.9 ± 6.222.6 ± 6.2SKBR3-A62.1 ± 0.787.3 ± 11.036.3 ± 0.3*SKBR3-T42.6 ± 4.9107.4 ± 9.729.3 ± 3.5*BT47418.2 ± 3.079.5 ± 5.213.3 ± 1.1BT474-T6.9 ± 2.795.3 ± 2.06.2 ± 2.3EFM192A39.1 ± 4.7105.7 ± 3.135.3 ± 2.7EFM192A-T30.4 ± 5.299.8 ± 2.528.5 ± 4.4HCC195461.8 ± 8.180.5 ± 8.219.4 ± 3.6** indicates enhanced anti-proliferative response for the combination compared to the single agents.
Citation Format: Conlon N, Canonici A, Morgan C, Cremona M, Hennessey BT, Eustace A, O'Brien N, Slamon D, Crown J, O'Donovan N. Targeting Src kinase blocks development of afatinib resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-15.