Abstract A2-15: Gain-of-function kinase library screen identifies mechanisms of resistance to antiestrogen therapies in ER+ breast cancer

Estrogen receptor-positive (ER+) breast cancer is the most common clinical subtype of breast cancer. Endocrine therapies that target ER signaling have significantly reduced mortality in patients with ER+ breast cancer. Fulvestrant is a selective ER downregulator currently approved for treatment of patients with advanced hormone-dependent breast cancer. Previous data suggest co-targeting the ER and the cyclin D1/CDK4/E2F axis may act synergistically against ER+ breast cancer cells. CDK4/6 inhibitors in combination with endocrine therapies are currently in clinical trials and it anticipated they may be approved for treatment of this breast cancer subtype. LEE011 is a small molecule CDK4/6 inhibitor with an IC 50 against CDK4 and CDK6 of 10 and 39 nM, respectively. However, like for most targeted therapies, we anticipate that resistance to them will emerge in a fraction of these patients. The goals of this study are to identify potential mechanisms of resistance to ER inhibitors alone and in combination with CDK4/6 inhibitors. To achieve this, we independently expressed 559 human kinases open reading frames (ORFs) in MCF7 ER+ breast cancer cells treated with fulvestrant ± LEE011, using a lentiviral vector system. To quantitate the effect of treatment, we used high content image acquisition and automated image analysis of stained nuclei (Hoechst) to determine the percentage of cell number in drug-treated wells relative to untreated wells in three independent experiments. MCF7 cells treated with 3 nM fulvestrant or with 0.6 nM fulvestrant + 250 nM LEE011 exhibited a 22.4% and 21.7% cell viability compared to untreated controls, respectively. MCF7 cells stably expressing a constitutively active MEK1 mutant (S218/222D) (MEKDD) were used as a drug-resistant positive control. MCF7 MEKDD cells treated with fulvestrant displayed a 38.3% increase cell viability relative to fulvestrant-treated control cells. Their viability when treated with fulvestrant + LEE011 was 14.6% higher than control MCF7 cells treated with both drugs. Differential viability was normalized to MCF7 MEKDD cells. A Z-score was then generated for each ORF; kinase ORFs with Z-scores >2 were considered hits and were followed up in secondary confirmation assays with independent vectors. We identified 21 kinases (3.7% of the total) whose expression resulted in a >30% increase in cell viability compared to fulvestrant-treated control cells. Seventeen kinases induced a >30% increase in cell viability compared to control MCF7 cells treated with fulvestrant + LEE011. Interestingly, 11 of these kinases overlapped in both treatment groups: RAF1, FGR, FGFR1, DYRK1B, CRKL, FRK, HCK, MAP2K6, SYK, CDK6 and NTRK1. In secondary transduction experiments using V5-tagged expression lentiviruses, all 11 ORFs were confirmed to induce resistance to fulvestrant + LEE011. Ten of 11 kinases (except NTRK1) induced resistance to fulvestrant whereas 7 of 11 induced resistance to the CDK4/6 inhibitor used as a single agent. These results are currently being confirmed in HCC1500, HCC1428 and CAMA1 ER+ breast cancer cells, all with an IC 50 to fulvestrant Citation Format: Yao Lu, William Fry, Joshua A. Bauer, Carlos L. Arteaga. Gain-of-function kinase library screen identifies mechanisms of resistance to antiestrogen therapies in ER+ breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-15.