Abstract 5302: Characterization of tumor cell lines resistant to birinapant, a novel bivalent smac mimetic.
Authors: Neiman Eric M. , Benetatos Christopher A. , Kapoor Gurpreet S. , Mitsuuchi Yasuhiro , McKinlay Mark A. , Seipel Martin E. , Yu Guangyao , Condon Stephen M. , Chunduru Srinivas K. 
Published: August 2014

Abstract
Acquired resistance to cancer therapies remains a major therapeutic challenge. Identification of resistance mechanisms and biomarkers of acquired resistance are important for management of patients with drug refractory tumors. The present study was aimed to understand the potential mechanism(s) of acquired resistance to birinapant (TL32711), a bivalent Smac Mimetic (SM), currently in clinical trials for the treatment of solid and hematological malignancies. We generated SM resistant cell lines from four tumor cell lines (SKOV-3, EFM192A, EVSA-T and MDA-MB-231) which were initially sensitive to birinapant. Resistant variants of each cell line were resistant to 10μM concentration of birinapant, equivalent to 1000-10,000x IC50 values. Variants were also cross-resistant to other, structurally diverse, SM compounds suggesting common mechanisms of resistance. Sensitivity to birinapant in combination with TRAIL and TNF was also measured. These experiments revealed a range of sensitivity among the resistant variants. In order to identify changes which occurred during development of resistance, total mRNAs were extracted from SM sensitive cell lines and their resistant variants and were subjected to gene expression analyses using commercially available apoptosis pathway real-time PCR arrays. Preliminary data analysis identified several candidate genes that were down- or up-regulated in the SM resistant cell line relative to their sensitive counterparts. Three of four SM-resistant subclones exhibited decreased expression of genes including NAIP, BCL2A1, DAPK1, TNF, cFLIP, Smac, and TNFSF10 or increased expression of BCL2L2, or BAK1 (Table 1).
Table 1 - Resistant cell line gene expression changes Cell Line EVSA-T EFM192A MDA-MB-231 SKOV-3 Gene Fold change from sensitive NAIP −1.60 −6.95 −42.07 −31.4 BCL2A1 ND −6.30 −11.37 −55.61 DAPK1 −3.09 −4.86 −189.2 −2.80 TNF 1.39 −3.27 −8.41 −70.71 cFLAR (cFLIP) −1.17 −3.25 −3.02 −6.64 DIABLO (Smac) −2.31 −2.27 −2.53 −3.52 TNFSF10 (TRAIL) −3.38 −1.46 −17.95 −67.64 BCL2L2 4.94 5.75 1.43 3.46 BAK1 3.92 14.15 1.66 4.65
Identification of genes modulated during acquired resistance provides a starting point for the discovery of novel biomarkers that may explain mechanism(s) of acquired resistance, and could potentially be exploited to achieve maximum anti-tumor response with birinapant as a single agent or in combination with other treatment regimens.
Citation Format: Eric M. Neiman, Christopher A. Benetatos, Gurpreet S. Kapoor, Yasuhiro Mitsuuchi, Mark A. McKinlay, Martin E. Seipel, Guangyao Yu, Stephen M. Condon, Srinivas K. Chunduru. Characterization of tumor cell lines resistant to birinapant, a novel bivalent smac mimetic. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5302. doi:10.1158/1538-7445.AM2013-5302