Abstract 136: Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma
Authors: Larson Sarah , Peng Mao Yu , Mead Monica , Vandross Andrae , Conklin Dylan , Euw Erika Von , Slamon Dennis J. 
Published: July 2017

Abstract
Background: Copanlisib (BAY 80-6946) is a reversible, pan-class I PI3K inhibitor with preferential activity for the alpha isoform, which may be of particular importance in multiple myeloma. Here we demonstrated the in vitro efficacy of copanlisib in a panel of 20 multiple myeloma cell lines. Baseline levels of phospho-S6 (P-S6) correlated with sensitivity to copanlisib, resulting in a potential biomarker of response. In addition, the change of P-S6 post-treatment could be used as a pharmacodynamic biomarker for copanlisib treatment.
Methods: We screened a panel of 20 multiple myeloma cell lines and selected 3 sensitive: NCI-H929, MM.1S, L-363, and 3 resistant: AMO-1, JJN3, COLO-677 for further analysis. We performed apoptosis and cell senescence assays following 72 hours of 50nM and 100nM copanlisib exposure. Cell cycle analysis and induction of apoptosis were performed by FACS after propidium iodide or PI/ANX-V FITC staining, respectively. Cellular senescence was determined by measuring β-galactosidase activity in cells treated for 96 hours. Reverse phase protein array (RPPA) was performed at baseline and post treatment for proteomic analysis with confirmatory western blots. Flow cytometry was also performed to monitor the post-treatment P-S6 level changes.
Results: Copanlisib treatment induced apoptosis in the sensitive cell lines (50-80% AN-V+ cells) but not in resistant cell lines (1-5% AN-V+ cells). An increased cell cycle arrest in G1 was also observed in the sensitive cell lines but not in the resistant lines. The cell senescence assays confirmed apoptosis rather than cell senescence as the mechanism of inhibition of proliferation. RPPA analysis demonstrated lower baseline p-S6 (S235/236, S240/244) protein levels in sensitive compared to resistant cell lines and this was confirmed with western blot analysis. Treatment with copanlisib resulted in a greater decrease in p-S6 in the sensitive cell lines NCI-H929 and L363 (53-83%, 73-93% respectively) than in the resistant cell lines COLO-677 and JJN3 (5-27%, and 38-67%, respectively), which was validated by western blot and phospho-flow. We also showed by RPPA and WB that copanlisib down-regulates pro-survival and proliferation molecules including p-S6K1, p-S6 and p-4EBP1, and upregulates pro-apoptotic PDCD4 in all cell lines, but to a greater extent in sensitive cell lines. Finally, pharmacodynamic p-S6 response remained at different post-treatment time points.
Discussion: A differential response to copanlisib is seen in the myeloma cell line panel. A subgroup of multiple myeloma cell lines demonstrated median IC50 values in the low nanomolar range (5-100nM), and responses correlated with low baseline P-S6. This p-S6 stratified response was only observed with PI3K-alpha inhibitors, but not with inhibitors targeting other PI3K isoforms or pan-PI3K inhibitors. Further studies may allow development of a new patient screening method or companion diagnostic.
Citation Format: Sarah Larson, Mao Yu Peng, Monica Mead, Andrae Vandross, Dylan Conklin, Erika Von Euw, Dennis J. Slamon. Phospho-S6 levels correlate with response to Copanlisib (BAY 80-6946) in multiple myeloma [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 136. doi:10.1158/1538-7445.AM2017-136