Abstract 5366: The small molecule Bcl-2 antagonist obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC and ABC- sub types of DLBCL cells both in vitro and in vivo
Authors: Dasmahapatra Girija P. , Lembersky Dmitry , Friedberg Jonathan W. , Fisher Richard I. , Dent Paul , Grant Steven 
Published: June 2012

Abstract
Introduction: Based on previous studies showing synergistic interactions between proteasome inhibitors (e.g., bortezomib) and Bcl-2 antagonists, we have investigated whether the activity of the irreversible proteasome inhibitor carfilzomib (CFZ) could be enhanced in DLBCL cells by a clinically relevant Bcl-2 antagonist such as obatoclax (OBTX).
Experimental Procedures: SUDHL16, SUDHL4 (GC -subtype) and OCI-LY10, OCI-LY-3 (ABC -subtype) cell lines were used as model systems. CFZ and OBTX were provided by Onyx Pharmaceuticals and GeminX Inc, respectively. Apoptosis was monitored by Annexin V/PI following drug treatment with OBTX ± CFZ. Synergism was assessed by CalcuSyn software. Effects of drug treatment on protein expression was monitored by western blotting. Beige-nude-XID (NIH-III) mice were used for in vivo studies.
Results : Co-treatment (24- 48 hrs) with minimally toxic CFZ concentrations (i.e., 2-6 nM) and sub-toxic concentrations of either OBTX (0.05-2.0µM) or HA14-1 (2-10 µM) resulted in a striking increase in apoptosis in diverse DLBCL cells. Median Dose Effect analysis demonstrated that interactions were highly synergistic, with Combination Index values less than 1.0. The CFZ/OBTX regimen also strikingly induced apoptosis in 2 primary human DLBCL (1-GC and 1-ABC types) samples but exhibited minimal toxicity toward normal hematopoietic cells (i.e., CD34+ cells). Synergistic interactions were associated with a sharp increase in caspase-3 activation, PARP cleavage, induction of p-JNK, up-regulation of Noxa, and de-phosphorylation of AKT. Additionally, combined exposure diminished up-regulation of Mcl-1 induced by CFZ. Enhanced activity of this drug regimen was associated with Bax translocation, as well as Bax conformational change and dimerization. Notably, genetic interruption of JNK signaling (e.g., by shRNA knockdown) significantly attenuated CFZ/OBTX-mediated apoptosis, demonstrating a significant role for JNK activation in enhanced lethality. Additionally, shRNA knock down of Noxa or enforced activation of AKT significantly attenuated the lethality of the CFZ/ OBTX regimen in DLBCL cells. Notably, co-administration of CFZ/ OBTX resulted in sharp increases in apoptosis in 3 separate bortezomib-resistant DLBCL models. Finally, in vivo administration of CFZ and OBTX to mice inoculated with SUDHL4 cells substantially suppressed tumor growth compared to the effects of single agent treatment.
Conclusions: A strategy combining the carfilzomib with obatoclax is active against parental DLBCL cells, including both GC- and ABC-subtypes, their bortezomib-resistant DLBCL counterparts, as well as primary DLBCL cells. It also displays significant in vivo activity in a xenograft DLBCL model. This approach warrants further exploration as therapeutic strategy in DLBCL.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2011-5366