Abstract A42: Quantification of MYC expression and mTORC signaling as biomarkers of BET inhibition in multiple myeloma.
Authors: Kalota Anna , Garfall Alfred , Carroll Martin P. , Stadtmauer Edward A. , Dang Chi , Vogl Dan T. 
Published: August 2015

Abstract
Dysregulation of the MYC oncogene has been implicated in the pathogenesis of myeloma. Novel small molecule inhibitors of bromodomain extraterminal (BET) transcriptional readers inhibit MYC expression in pre-clinical settings and are currently being evaluated in phase 1 trials for myeloma. However, biomarkers of response to BET inhibitors have not been developed or validated. Here we report a strategy for flow cytometric measurements of Myc protein and phosphorylation of ribosomal S6 kinase (pS6), a marker of cell growth activation, as potential biomarkers of response to BET inhibition. We evaluated our methodology in the P493 cell line, a model of lymphoplasmacytic malignancies carrying two trans-genes that enable experimental manipulation of MYC expression through regulation of the endogeneous promoter or an ectopic promoter. As predicted, exposure to BET inhibitors: JQ1 and CPI-0610 (both 1µM for 24h) significantly decreased Myc protein levels in P493 cells expressing endogenous MYC activated by the EBNA2-ER fusion protein but had no effect on ectopic MYC under the control of the tetracycline-responsive promoter. Interestingly, in P493 cells expressing endogenous MYC, the decrease in Myc expression highly correlated with a decrease in pS6. We confirmed our results in 4 human myeloma cell lines known to express different levels of MYC and previously shown to have varying responses to BET inhibition (Delamore J.E; Cell 2011). The results from these cell lines represent various states of responses that we expect to see in patient samples and provide a rationale for Myc and pS6 measurements as biomarkers for BET inhibition. Finally, to demonstrate clinical applicability of our assay, we incorporated measurement of MYC and pS6 into a multiparameter flow cytometry panel. This panel allows identification of plasma cells based on CD45, CD38 and CD138 expression, and specific identification of malignant plasma cells based on expression of CD56, CD19, and intracellular immunoglobulin light chain restriction. Using this panel, we successfully identified malignant plasma cells and detected changes in Myc and pS6 in a fresh patient bone marrow aspirate treated ex vivo with CPI-0610. Our experimental results show that Myc protein levels can be reliably quantitated by flow cytometry as a marker of the efficacy of BET inhibitors in suppressing MYC expression and that flow cytometric quantification of pS6 may be a marker of suppression of cell growth signaling due to MYC suppression. Our strategy can be applied to fresh whole bone marrow aspirates with minimal ex vivo processing and can identify changes in MYC and pS6 levels in malignant plasma cells, with resolution at the single cell level. Our data thus provide a solid foundation for reliable and clinically applicable measurements of Myc and pS6 in myeloma cells as readout for BET inhibitor efficacy.
Citation Format: Anna Kalota, Alfred Garfall, Martin P. Carroll, Edward A. Stadtmauer, Chi Dang, Dan T. Vogl. Quantification of MYC expression and mTORC signaling as biomarkers of BET inhibition in multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A42.