Abstract P1-13-06: Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer
Authors: Perurena Naiara , Pilla Natalie , Schade Amy , Watanabe Marina , Loi Patrick , Rodriguez Carrie L. , Gardner Alycia M. , Cichowski Karen 
Published: March 2023

Abstract
Resistance to HER2 inhibitors remains a clinical challenge in HER2+ breast cancer. Therefore, there is an urgent need to 1) understand the mechanisms that underlie resistance to these current treatments and 2) develop improved, and more importantly, curative combination therapies. We recently discovered that two emerging tumor suppressor RasGAPs, DAB2IP and RASAL2, cooperatively drive metastatic breast cancer when lost or inactivated. Interestingly, we have now generated robust data demonstrating that the loss of these RasGAPs also induces resistance to HER2 inhibitors in breast cancer. First, we genetically ablated both RASAL2 and DAB2IP in multiple HER2+ breast cancer cell lines (SKBR3, EFM192A, SUM190, BT474) and performed manual counting experiments after 6 days of TKI (lapatinib, tucatinib) treatment. In all cell lines, RASAL2/DAB2IP knockdown conferred resistance to HER2 inhibitors. Moreover, loss of these RasGAPs prevented TKI-induced caspase-3/7 activation, measured by Incucyte live cell imaging, and enabled the regrowth of cells in long-term 10-day treatment and drug washout experiments, monitored by Incucyte or crystal violet staining. Next, we sought to investigate the individual contribution of each RasGAP to these phenotypes. Surprisingly, we found that RASAL2 and DAB2IP functioned quite differently in this context. Specifically, while RASAL2 loss prevented apoptosis, DAB2IP loss prevented irreversible cell cycle arrest (measured by functional long-term experiments and EdU staining assays). Mechanistically, RASAL2 loss uniquely impaired BIM induction at both mRNA and protein levels, which is required for lapatinib-induced cell death of HER2+ cancer cells. By contrast, cell cycle progression pathways were uniquely enriched in DAB2IP-deficient cells on lapatinib treatment and immunoblots revealed higher residual levels of pRb and low levels of p27 in these cells. These data suggest that RASAL2 and DAB2IP (loss) mediate resistance to HER2 inhibitors by differentially deregulating unique pathways/phenotypes. We next evaluated the relevance of these findings in a SUM190 orthotopic xenograft model. Importantly, while control tumors (expressing both RASAL2 and DAB2IP) regressed upon lapatinib treatment, DAB2IP- and RASAL2-deficient tumors did not regress and grew with kinetics comparable to vehicle-treated tumors after few days on treatment. These data suggest that the unique phenotypes/pathways induced by both RASAL2 and DAB2IP are important mediators of resistance to HER2 inhibitors. Further understanding the contribution of these pathways to anti-HER2 resistance and determining how RASAL2 and DAB2IP differentially function will be essential for developing effective therapeutic strategies to bypass each type of resistance.
Citation Format: Naiara Perurena, Natalie Pilla, Amy Schade, Marina Watanabe, Patrick Loi, Carrie L. Rodriguez, Alycia M. Gardner, Karen Cichowski. Loss of emerging tumor and metastasis suppressor RasGAPs mediates therapeutic resistance in HER2+ breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-06.