Abstract 1910: Resistance to anti-HER2 therapy in HER2+ breast cancer is mediated by genomic alterations that switch pathway dependence from PI3K/AKT to RAS/MAPK

Although HER2 overexpression activates both PI3K-AKT and RAS-MAPK signaling, HER2+ breast tumors are primarily driven by PI3K-AKT. Thus, tumor resistance to HER2-targeted agents has been thought to emerge via downstream reactivation of PI3K signaling. We performed genomic profiling on 272 HER2-amplified breast tumors to identify genetic alterations associated with de novo or acquired resistance to anti-HER2 therapies. Surprisingly, we identified enrichment of mutations in RAS-MAPK pathway members, including NF1, ERBB2, and KRAS, in patients who had received prior HER2-targeted therapies, raising the possibility that activation of this pathway contributes to therapy resistance. To determine the impact of RAS-MAPK activation on response to anti-HER2 therapy in HER2+ breast cancer, we generated isogenic HER2+ breast cancer cell line and patient derived xenograft models harboring MAPK pathway alterations enriched in patients. Depletion of NF1 expression in HER2+ breast cancer cell lines using shRNA or CRISPR/Cas9 indeed promoted resistance to the HER2 inhibitors (HER2i) lapatinib and neratinib in vitro, as measured by increased proliferation and markers of cell cycle progression compared to parental lines. Unexpectedly, resistant models exhibited diminished dependence on AKT (MK2206 IC50 increase from 200 nM to 4500 nM) and were sensitized to MEK/ERK inhibition (trametinib IC50 decrease from 740 nM to 13 nM and SCH772984 from 2900 nM to 250 nM). To establish the basis for this change in pathway dependence, we examined the cell cycle profile of the cell line models upon drug treatment. Both cell cycle analysis by DNA content and immunoblot analysis revealed that NF1 null cells arrested in G1 upon MEK inhibition (but not upon AKT inhibition). Given the known regulation of CDK4/6 and CDK2 kinases by the RAS-MAPK pathway, we used both shRNA knockdown and small molecular inhibitors to define the G1 CDK responsible and found CDK2 to be required for resistance to HER2i and MEK sensitivity. Further, in vitro kinase assays using a recombinant RB substrate showed CDK2 activity to be MEK dependent in resistant cells and not in parental. We observed similar phenomena in isogenic HER2+ models in which we introduced mutant BRAF, KRAS, and ERBB2, and found each of these mutations also promoted HER2i resistance and MAPK/CDK2 dependence. Finally, using lentiviral CRISPR/Cas9, we knocked out NF1 in a HER2+ patient derived xenograft model and again found that this alteration promoted profound MEK dependence in vivo. Overall, these data suggest that MAPK-activating mutations constitute an important potential mode of acquired resistance to HER2-targeted therapies and demonstrate the potential utility of MEK/ERK targeted therapies for a subset of anti-HER2 therapy resistant HER2+ breast cancers. Citation Format: Alison E. Smith, Sarat Chandarlapaty. Resistance to anti-HER2 therapy in HER2+ breast cancer is mediated by genomic alterations that switch pathway dependence from PI3K/AKT to RAS/MAPK [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1910.