Abstract 2753: Targeting bromodomain and extra-terminal (BET) family member BRD4 for treatment of pediatric sarcomas.
Authors: Xiao Linlin , Phelps Doris A. , Baker Laurence A. , Qi Jun , Bid Hemant K. , Houghton Peter J. 
Published: August 2014

Abstract
Background. Potent inhibitors of histone binding modules have the potential to suppress, selectively, transcription of specific genes, including MYC family of transcription factors. JQ1 binds competitively to acetyl-lysine recognition motifs, or bromodomains. JQ1 inhibits BET bromodomain, and down-regulates MYC transcription. Ewing sarcoma (EWS) and alveolar rhabdomyosarcoma (ARMS) are associated with high expression of c-MYC and MYCN, respectively (Delmore JE, Cell 2011; Filippakopoulos P, Nature 2010). Consequently, we have evaluated sensitivity of EWS and ARMS cell lines and xenograft models to JQ1.
Methods. EWS and ARMS cells were incubated with JQ1, or JQ1R (non-active enantiomer) for 96 hrs. Cell viability was determined by Alamar Blue staining. C-MYC and MYCN were determined by immunoblotting. Xenograft tumor lines were grown in CB17 female SCID mice. JQ1 was administered P.O. at 30 mg/kg (BID) or 50 mg/kg (SID) for up to 3 weeks. Assessment of JQ1 activity was as described (Houghton P, Ped Blood & Cancer, 2007). The effect of JQ1 on proliferation and tube formation of human umbilical vascular endothelial cells (HUVECs) was as described (Bid H, Mol Cancer Ther, 2012).
Results. For all cell lines, the IC50 concentration for JQ1R (inactive enantiomer) was &gt; 10 microM. EWS cell lines (n=9) segregated into two distinct groups with IC50 &lt; 1 microM, (ES-1, ES-2, ES-3, ES-4, ES-7, ES-8) and cell lines with significantly higher IC50s (IC50 &gt; 7 microM ES-6, EW8, CHLA-258). Seven of 9 ARMS cell lines demonstrated sensitivity to JQ1 (IC50 &lt; 1 microM) whereas 2 lines were insensitive (IC50 &gt; 10 microM Rh18, Rh30). JQ1 (500 nM, 24 hr) markedly down-regulated c-MYC in Rh41, and to a lesser extent in Rh36. MYCN expression in Rh5 ARMS cell line was also suppressed by JQ1. Of interest, JQ1 did not suppress c-MYC in any EWS cell line. In vivo, BID and SID dosing was well tolerated (mortality 1.2%) and gave similar antitumor activity. JQ1 significantly retarded growth of all 4 sarcoma models (Rh10, Rh28 ARMS and EW5, EW8 EWS lines. T/C at day 21 range 0.30 – 0.43). Of note, while EW8 cells in vitro were resistant to JQ1 (IC50 &gt; 10 microM), the xenografts were equally sensitive as other lines to JQ1 treatment, suggesting JQ1 exerted effects on stroma/angiogenesis. In vitro, JQ1 (500 nM) suppressed tube formation of HUVECs without decreasing viability.
Conclusions. JQ1 potently inhibited proliferation of EWS and ARMS cells in vitro, with distinct groups demonstrating either sensitivity or resistance. Sensitivity was associated with decreased levels of MYCN or c-MYC in some cell lines. In vivo, JQ1 demonstrated significant antitumor activity in 4 xenograft lines, including EW8 derived from cells resistant to JQ1 in vitro. The potential for JQ1 to exert anti-angiogenic effects is suggested by its potent inhibition of HUVECs differentiation. Supported through a grant from SWOG
Citation Format: Linlin Xiao, Doris A. Phelps, Laurence A. Baker, Jun Qi, Hemant K. Bid, Peter J. Houghton. Targeting bromodomain and extra-terminal (BET) family member BRD4 for treatment of pediatric sarcomas. [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 2753. doi:10.1158/1538-7445.AM2013-2753