Kazi, Armina A.

Abstract Treatment of estrogen receptor positive (ER+) breast cancer have included single-agent endocrine therapy, such as with aromatase inhibitors (AIs) and, more recently, AIs in combination with the cyclin-dependent kinase 4/6 inhibitor palbociclib. While combination therapy is effective, drug resistance is a well-known phenomenon and breast cancer cells can acquire resistance to aromatase inhibitor and palbociclib individually. In anticipation of patient who may become resistant to combination therapy with aromatase inhibitor (letrozole) and palbociclib, this current study has 1) developed MCF7Ca cells resistant to both letrozole and palbociclib (MCF7Calet+palb); 2) characterized them in comparison to drug naïve (MCF7Ca) and letrozole alone-resistant (LTLTCa) cells; and 3) explored potential treatments. Characterization of MCF7Calet+palb cells by phase contrast microscopy, mammosphere assays, and invasion assays, indicate that they have more basal/mesenchymal morphology, produce more mammospheres (an indicator of cancer stem cells; 150±10 vs. 80±10 in MCF7Ca and 123± 8 in LTLTCa cells), and are more invasive (1200±50 cells vs. 791±15 in MCF7Ca cells). The more aggressive phenotype of MC7Calet+palb correlated with changes in protein expression. Compared to the other cell lines, MCF7Calet+palb expressed significantly lower fold expression levels of cell cycle cyclin D1 (0.2±0.1), phosphorylated CDK9 (0.5±0.1), and phosphoRb and total Rb (0.4±0.2), but higher levels of phosphoCDK1 and CDK2 (1.4±0.1 and 3.1±0.6). Regarding growth pathway proteins, MCF7Calet+palb comparably had increased HER2 and phosphorylated Akt (6.2±1.3 and 3.3±1.2, resp.) as LTLTCa vs. MCF7Ca cells, but had even more reduced levels of ERα (0.04 vs. 0.1±0.4 in LTLTCa and 1±0.01 in MCF7Ca). Cancer stem cell marker breast cancer resistance protein (BCRP) was increased further in MCF7Calet+palb cells (2.11-fold). Based on these results, the effect of dinaciclib, a broader range CDK inhibitor, was studied. In MTT assays, dinaciclib was shown to be effective in inhibiting cell viability in all three cell types. Dinaclicib’s IC50s were 1.4 nM (MC7Ca) 9.5 nM (LTLTCa) and 12.4 nM in MCF7Calet+palb cells, while palbociclib IC50s were 1.5 μM, 11.2 μM, and 118 μM, respectively. Dinaciclib also significantly reduced mammosphere formation 0.01-fold and cell invasion/migration by 0.3-fold vs. untreated. Lastly, preliminary exome sequencing data suggested highly mutated FGFRL1, ARL11, and FCGBP genes, among others, when MCF7Calet+palb cells were compared to MCF7Ca cells. Overall, these results suggest that acquired resistance to combination therapy with letrozole and palbociclib induces genetic, molecular, and phenotypic changes in breast cancer cells, and that dinaciclib may be an effective treatment to either treat and/or prevent this resistance. Citation Format: Armina Kazi, Emily Shukdinas, Saranya Chumsri, Gauri J. Sabnis. Characterization of breast cancer cells resistant to letrozole and palbociclib combination therapy and the potential of dinaciclib to treat such resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1014.

Abstract Estrogen receptor-positive (HR+) breast cancer is the most common form of breast cancer that is accountable for the majority of breast cancer mortality. Currently, the cyclin dependent kinase inhibitor palbociclib in combination with endocrine therapy represents the new standard first and second line therapy for patients with metastatic HR+ breast cancer. While palbociclib has been shown to significantly improved progression free survival in combination with aromatase inhibitor (AI) and fulvestrant, resistance will inevitably occur in patients with metastatic breast cancer. However, the mechanisms of resistance to the combination of palbociclib and AI remain largely unknown. It is also unclear whether resistance mechanisms would be the same if palbociclib was given as first line treatment in combination with letrozole vs. if palbociclib was given as second line treatment after letrozole resistance had already occurred. To address this, previously well established and well characterizied letrozole-sensitive MCF7Ca cells and letrozole-resistant LTLTCa cells were subjected to continuous, long term treatment with increasing doses of letrozole and palbociclib until resistance to both drugs was achieved (MCF7Calet+palb and LTLTCapalb, respectively). Preliminary MTT cell viability assays indicate palbociclib IC50s of 750 nM in MCF7Ca and LTLTCa cells vs. 21 uM in palbociclib and letrozole-resistant cells. First line and second line palbociclib resistance correlated with changes in morphology, protein expression, and cancer stem cell characteristics. Under phase contrast microscopy, first line and second line palbociclib-resistant MCF7Ca and LTLTCa cells were larger in size, more irregular in shape, and tended not to grow in epithelial cell-like groups compared to palbociclib-sensitive MCF7Ca and letrozole cells, with second-line palbociclib resistant cells exhibiting these characteristics the most. Western blot showed that ER protein expression in ER+/HER2- MCF7Ca cells decreased with first line palbociclib and letrozole resistance, and that both ER and HER2 protein expression were decreased with second line palbociclib resistance in ERlow/HER2+ LTLTCa cells. Lastly, mammosphere assays demonstrated increasing percentage of cancer stem cells with letrozole resistance alone (4 per 1000 cells plated MCF7Ca vs. 58 per 1000 planted LTLTCa) and with palbociclib and palbociclib resistance (317 per 1000 cells plated MCF7Ca and 202 per 1000 cells plated LTLTCa cells). Overall, these results indicate that 1) resistance to palbociclib, whether as first line or second line treatment has significant effects on breast cancer cells that may be relevant to patient diagnosis and treatment. Citation Format: Armina A. Kazi, Antony Sare, Saranya Chumsri, Angela Brodie. Mechanisms of resistance to palbociclib and aromatase inhibitors in hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2053. doi:10.1158/1538-7445.AM2017-2053