Abstract 5562: ML120B and Bortezomib (BTZ) inhibit both NF-κB and non NF-κB proteins in Primary Mediastinal B-cell Lymphoma (PMBL): Potential for future targeted therapy

Abstract

Abstract Background: PMBL has a significantly poor prognosis compared to other DLBCLs (Lones/Cairo, JCO, 2000; Patte/Cairo, Blood 2007). New chemotherapeutic agents are urgently needed to improve the current treatment protocol and to improve overall survival rates. Several groups have demonstrated that the NF-κB signaling pathway levels are upregulated in PMBL (Savage, Blood, 2003; Rosenwald, J Exp Med, 2003; Feuerhake, Blood, 2005). We recently demonstrated that the NF-κB inhibitor bortezomib (BTZ) and/or ML120B (I B kinase, IKK, inhibitor) increase apoptosis in PMBL (Waxman/Cairo, et al., Ann Onc, 2008a). Objective: To elucidate the underlying molecular mechanism(s) by which NF-κB blockade contributes to apoptosis in PMBL, we performed proteomic analyses and western blotting of PMBL cells after NF-κB Inhibitor BTZ and ML120B exposure in-vitro. Methods: PMBL cell line Karpas-1106P cells were incubated with BTZ (5ng/ml), ML120B (10μg/ml, generously supplied by Millennium Pharmaceuticals), or both for 24 hours. NF-κB binding activity was measured by TransAm kit (Active Motif). CASPASE-3 was measured by Human Apoptosis 3-plex kit (Invitrogen). Proteomic analysis were performed with cell lysates utilizing iTRAQ labeling followed by LC-MS/MS. Ratios between untreated and treated were quantile normalized and log2 transformed. False-discovery rates (FDR) were used to estimate the error among the selected proteins. All proteins with an FDR <40% were considered differentially expressed. Several of these proteins were confirmed by western blot. Student t-test was used for statistical analysis. Results: The combination of BTZ and ML120B significantly increased CASPASE 3 formation (330±53 vs. 55±5 pg/mg protein) (p<0.006) and significantly reduced activation of NF-κB members, p50 (45% decrease (dec) vs. untreated control (unt); 0.051±0.0015; p< 0.001), p52 (50% dec vs. unt, 0.022±0.0002; p<0.001), c-rel (41% dec vs. unt, 0.020±0.0008; p<0.03), p65 (RelA) (52% dec vs. unt, 0.019±0.0008; p<0.001) and Rel B (43% dec vs. unt, 0.023±0.0002; p<0.001). There was a significant decrease in NF-κB proteins including HSP90 (−4.0F), PSAT1 (−3.3F) and SND1 (−3.9F) and c-myc downstream proteins SNRPB (−5.3F) and PFKP (−3.9F) by proteomic analysis and similar results confirmed by western blot. Conclusions: BTZ and ML120B inhibit both NF-κB and non NF-κB pathways which contribute to promotion of apoptosis in PMBL. These results could provide the basis for future translational studies in PMBL and other tumors with constitutive activation of the NF-κB pathway. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5562.