Abstract 323: Combined inhibition of STAT-3 & DNA repair in palbociclib resistant breast cancer
Authors: Kettner Nicole M. , Vijayaraghavan Smruthi , Durak Merih Guray , Bui Tuyen , Kohansal Mehrnoosh , Ha Min Jin , Liu Bin , Rao Xiayu , Yang Jing , Yi Min , Carey Jason P. , Chen Xian , Eckols T. Kris , Raghavendra Akshara S. , Ibrahim Nuhad K. , Karuturi Meghan , Watowich Stephanie S. , Sahin Aysegul A. , Tweardy David J. , Hunt Kelly K. , Tripathy Debu , Keyomarsi Khandan 
Published: August 2019

Abstract
The CDK4/6 inhibitor palbociclib is currently being used in combination with endocrine therapy to treat advanced ER positive breast cancer patients. While this treatment has shown great promise in the clinic, about 25-35% of the patients do not respond initially, and almost all patients eventually acquire resistance. Hence, understanding the mechanisms of acquired resistance to CDK4/6 inhibition is crucial to devise alternate treatment strategies.
To identify mechanisms of resistance to CDK4/6 inhibition we developed MCF-7 and T47D palbociclib resistant cells in a step-wise manner by gradually increasing concentrations of palbociclib. These cells are not only resistant to palbociclib, but exhibited resistance to the other approved CDK4/6 inhibitors; ribociclib and abemaciclib. Additionally, we assessed if these resistant cells have an altered response to endocrine therapy and observed that these cells are also resistant to treatment with tamoxifen or fulvestrant by about 16-fold. Multi-omics analyses revealed enrichment of pathways known to regulate EMT and promote stem-like properties, as well as, downregulation of estrogen response and DNA repair pathways.
Palbociclib resistant cells exhibited mammosphere formation and CD44high/CD24low population indicating the presence of increased breast cancer stem cell-like cells (B-CSC-L). Given the recently elucidated role of IL-6/STAT-3 mediated B-CSC-L phenotypes in drug resistance, we examined IL-6 mRNA levels, which increased by &gt;12-fold in the resistant cells. Treatment with STAT-3 inhibitors, napabucasin and C188-9, significantly decreased the B-CSC-L population and mammosphere formation, indicating a crucial role for the IL-6/STAT-3 pathway in driving B-CSC-L phenotype and palbociclib resistance.
Since DNA repair pathways were collectively downregulated in the palbociclib resistant cells, we examined their sensitivity to DNA damaging agents. Results showed that resistant cells were more sensitive to olaparib (PARP inhibition), with no effect on B-CSC-L population. Next, we examined if combined treatment with agents targeting STAT-3 and PARP would be synergistic in palbociclib resistant cells. Results show that combined treatment with olaparib and napabucasin or C-1889 significantly decreased B-CSC-L population, colony formation and increased cell death via apoptosis, when compared to no-treatment or single treatment controls of the palbociclib resistant cells.
Lastly, we interrogated matched tumor samples from breast cancer patients who progressed on palbociclib for deregulation of estrogen receptor, DNA repair, and IL-6/STAT3 signaling and found that these pathways are altered as compared to the pre-treatment samples.
Taken together, the results show that targeting IL-6/STAT-3 mediated cancer stem cells and DNA repair deficiency by PARP inhibitors in combination can effectively treat acquired resistance to palbociclib.
Citation Format: Nicole M. Kettner, Smruthi Vijayaraghavan, Merih Guray Durak, Tuyen Bui, Mehrnoosh Kohansal, Min Jin Ha, Bin Liu, Xiayu Rao, Jing Yang, Min Yi, Jason P. Carey, Xian Chen, T. Kris Eckols, Akshara S. Raghavendra, Nuhad K. Ibrahim, Meghan Karuturi, Stephanie S. Watowich, Aysegul A. Sahin, David J. Tweardy, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. Combined inhibition of STAT-3 & DNA repair in palbociclib resistant breast cancer [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 323.