Abstract P5-08-01: Systematic interrogation of resistance to HER2-directed therapy identifies a survival pathway activated by PRKACA and PIM1

Amplification and/or overexpression of the receptor tyrosine kinase HER2 occurs in 20-25% of breast cancers, and is associated with poor prognosis. Targeting of HER2 with drugs such as trastuzumab, lapatinib, or pertuzumab has led to clinical benefit in patients with both metastatic and early-stage HER2-amplified breast cancer. However, resistance and disease progression always occurs in patients with metastatic disease, and many patients with early-stage breast cancer experience recurrences despite adjuvant anti-HER2 therapy. As such, understanding the mechanisms of resistance to anti-HER2 therapy has important clinical implications. Recent studies have identified mutations in PIK3CA, the gene encoding the catalytic subunit of Phosphatidylinositol 3 kinase (PI3K), as one mechanism of resistance to trastuzumab. However, such mutations are present in only a fraction of trastuzumab-resistant breast cancers. We therefore sought to uncover novel mechanisms of resistance to anti-HER2 therapy through an unbiased screen for kinases and kinase-related molecules that are able to rescue HER2-amplified breast cancer cells from HER2 inhibition. We utilized a library of nearly 600 lentivirally-delivered open reading frames (ORFs) to constitutively express the coding sequence of each molecule individually in HER2-amplified BT474 breast cancer cells in arrayed high-throughput format. We conducted two parallel screens for the ability of each of these molecules to rescue cells from anti-HER2 therapy: one in which we treated the cells with a lapatinib-like drug that inhibits the kinase activity of HER2 and EGFR, and one in which we lentivirally delivered a short hairpin RNA that suppresses expression of HER2. We identified those ORFs that restored viability of BT474 cells to greater than two standard deviations above the median of all ORFs in each screen. Multiple members of the MAPK and PI3K signaling pathways scored in both screens, serving to validate the approach. In addition, the survival kinases PIM1 and PRKACA scored robustly. Mechanistic studies suggest that these kinases may confer resistance by restoring the phosphorylation of, and thereby inactivating, the pro-apoptotic protein BAD. Consistent with this finding, overexpression of Bcl-xl, which is inhibited by BAD, also conferred resistance to lapatinib in HER2-amplified breast cancer cells. Furthermore, pharmacological blockade of Bcl-xl and Bcl-2 with ABT-263 enhanced lapatinib-induced killing of HER2-amplified breast cancer cells in vitro, and partially abrogated the rescue conferred by both PRKACA and PIM1. These findings suggest that combined inhibition of HER2 and the anti-apoptotic molecules Bcl-xl and Bcl-2 could enhance tumor cell eradication and prevent or delay the emergence of resistant disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-01.