Abstract PS18-30: Loss of NF1 leads to rho GTPase activation and sensitivity to multiple agents in breast cancer

Abstract

Background: The Ras signaling pathway is a key oncogenic growth signaling pathway. The neurofibromatosis gene (NF1) is a tumor suppressor gene and negative regulator of Ras. Approximately 20% of breast cancers experience loss of active NF1 protein, leading to unabated Ras activity. Germline inactivation of NF1 leads to neurofibromatosis, which can be treated with MEK inhibitors. The functional consequences of NF1 loss have not been thoroughly explored in breast cancer. Therefore, we created a cellular model for the loss of NF1 in breast cells in order to identify novel therapies that would target NF1 null breast cells. Methods: We used CRISPR CAS9 to knock out (KO) NF1 through targeted disruption of both NF1 alleles in the human non-tumorigenic breast cell line MCF-10A. Immunoblotting confirmed loss of the protein and this cell line was used to identify changes in cellular signaling that resulted from the loss of NF1. We then used a rational approach to select drugs that may target the cellular changes in NF1 null cells. Finally, the candidate drugs were tested in a tumor xenograft model in mice. Results: Loss of NF1 endowed cells with a more transformed phenotype, including EGF growth independence and anchorage-independent growth. We observed increased activation of MAPK and activated Rho GTPase. The MEK inhibitors trametinib and PD0325901 (IC50=0.25 nM and 0.16 nM, respectively) inhibited growth of NF1-null, but not parent cells. We explored inhibitors of other proteins in the MAPK signaling pathway, including the Raf inhibitors sorafenib and vemurafenib and the dual MAP3K1/MAP2K4 inhibitor LY2228820. However, these compounds did not selectively inhibit growth of NF1-null cells, suggesting that Raf and MAP3K1/MAP2K4 are not vulnerable targets in NF1-null cells. Vincristine and zoledronic acid can indirectly affect Rho GTPase function. NF1-null cells were more sensitive to both as single agents (IC50=1.1 nM and 6.9 μM, respectively) than parental cells. We also tested multiple combinations of these drugs and observed benefits from several combinations that exceeded single agent use. Similar sensitivity was not observed to docetaxel or ixabepilone. We then tested the drugs as single agents and in combination against mouse tumor xenograft models with the human breast cancer cell lines Hs578T (NF1 null) or MCF-7 (wild type NF1). Treatment with single agent vincristine or zoledronic acid for 19 days resulted in statistically-significant decreases of 37.3% and 38.9%, respectively (p<0.05) in Hs578T tumor xenografts. The combination treatment resulted in an enhanced reduction in tumor size of 48.8% with a higher level of significance (p<0.01). Treatment of mice with MCF-7 bearing tumors with single agent vincristine resulted in a statistically significant decrease of 35.2% (p<0.05); however, this effect was not observed using the combination treatment, as the change in growth compared to saline treatment was not statistically significant. Conclusions: Our results suggest that loss of NF1 results in a transformed phenotype. Our results provide the first evidence that vincristine in combination with zoledronic acid may be efficacious in treating breast cancers. Additionally, we provide evidence that other drugs and drug combinations may be more effective at treating breast cancers with NF1 loss than MEK inhibitors, warranting further exploration. Citation Format: Melody A. Cobleigh, Matthew Najor, Satnam Brar, Sanja Turturro, Liam Portt, Timothy Yung, Abde M Abukhdeir. Loss of NF1 leads to rho GTPase activation and sensitivity to multiple agents in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-30.