Correlation Between Increases in Apoptosis in Primary Mediastinal B-Cell Lymphoma (PMBL) and NF-κB Pathway Blockade: Bortezomib (BTZ), Small Molecule IKK Inhibitor ML120B and Combination Therapy Significantly Decrease NF-κB Family Member Protein Activation

Primary mediastinal B-cell lymphoma (PMBL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) with a significantly lower event-free survival in children than other identically treated DLBCLs (Lones/Cairo, JCO, 2000). It has a unique gene expression profile with upregulation of a subset of NF-κB signal pathway genes (Rosenwald, J Exp Med, 2003). We previously reported significant increases in apoptosis in a PMBL cell line with NF-κB pathway blocking agents bortezomib (BTZ) and small molecule IKK inhibitor ML120B (supplied by Millennium Pharmaceuticals, MA) used alone (BTZ alone: 5.25%±1.43 increase in apoptosis vs. control; ML120B alone: 4.0%±1.13 increase in apoptosis vs. control, p<.0005) and in combination (ML120B+BTZ vs. ML120B single agent therapy; 17-fold increase in apoptosis, p<.01; ML120B+BTZ vs. BTZ single agent therapy; 4-fold increase in apoptosis, p<.01) (Waxman, Ann Onc, 2008a). We now report the effect of single and combination treatment in PMBL on expression of NF-κB family member protein expression and its correlation with apoptosis.

PMBL line Karpas-1106P was incubated with BTZ (5 ng/ml), ML120B (10 μg/ml), and ML120B+BTZ for 24h. Activation of NF-κB transcription factors was measured by ELISA (TransAM Kit, Active Motif) and results were correlated with changes in apoptosis, determined by flow cytometry with Annexin V-FITC detection kit (BD Pharmingen) using identical drug doses and incubation times.

In untreated PMBL, p50 had the highest activation (0.093±0.005OD/μg protein), followed in order of decreasing activation by P52 (0.045±0.0006), P65 (RelA) (0.040±0.0019), RelB (0.039±0.0016) and c-Rel (0.028±0.0036). ML120B monotherapy was associated with inhibition of the activation of P50 (32% decrease vs. untreated control, 0.064±0.0046; p<0.01), c-Rel (31% decrease vs. untreated control, 0.019±0.0004; p<0.05), P52 (16% decrease vs. untreated control, 0.038±0.0019; p<0.01) and P65 (RelA) (55% decrease vs. untreated control, 0.018±0.0006; p<0.001). BTZ monotherapy inhibited the activation of P52 (9% decrease vs. untreated control, 0.041±0.0006; p<0.01) and RelB (14% decrease vs. untreated control, 0.034±0.0009; p<0.05). ML120B+BTZ combination therapy strongly inhibited the activation of P50 (45% decrease vs. untreated control, 0.051±0.0015; p<0.001), P52 (50% decrease vs. untreated control, 0.022±0.0002; p<0.001) and RelB (43% decrease vs. untreated control, 0.023±0.0002; p<0.001).

These results indicate that increases in apoptosis in PMBL previously reported for ML120B monotherapy, BTZ monotherapy, and ML120B+BTZ combination therapy are associated with inhibition of members of the NF-κB family of transcription factors. These results suggest that ML120B and BTZ increase apoptosis in PMBL by preventing activation of NF-κB transcription factors and subsequent transcription of anti-apoptotic genes that would otherwise promote cell survival. As ML120B+BTZ combination therapy leads to greater inhibition of P50 and P52 than single-agent therapy, there appears to be an additive effect when both agents are used together. Furthermore, as only ML120B inhibited c-Rel and RelA activation and only BTZ inhibited RelB activation, it is clear that each NF-κB blocker does not affect all 5 NF-κB transcription factor family members and gene expression profiling may therefore play a role in choosing optimal therapy. Finally, our findings suggest that ML120B and BTZ inhibit will also increase apoptosis in tumors other than PMBL with constitutive activation of the NF-κB pathway and these drugs should therefore be studied in other tumor types with upregulation of NF-κB genes.