The NF-κB pathway as a target to increase apoptosis in primary mediastinal B-cell lymphoma (PMBL): Results of studies with rituximab (RTX), bortezomib (BTZ) and small molecule IKK inhibitor ML120B Free

2994

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, including anti-apoptotic genes (Rosenwald, J Exp Med, 2003). We studied the effect of treatment with 3 NF-κB pathway blocking agents, RTX, BTZ and ML120B (supplied by Millennium Pharm, MA) on apoptosis in a PMBL cell line in hopes of finding agents that increase cell death.
 PMBL line Karpas-1106P was incubated with RTX (5-40 μg/ml), BTZ (5-50 ng/ml) or ML120B (10 ng/ml-10 μg/ml) at dose-escalated concentrations (conc.) for 24h. Cells were also incubated with RTX (5-40 μg/ml) for 4 and 14h to determine time-dependent response. % of cells induced to undergo apoptosis was measured with Annexin V-FITC. GCB DLBCL line SU-DHL-6, with low expression of NF-κB genes, was used for comparison in all experiments. RS4;11 CD20(-) ALL line was used as a negative control in RTX experiments.
 Significant increases in apoptosis were found in the PMBL line at 24h with max conc. RTX (4.5±0.2%, p<.003) and ML120B (4.5±1.6%, p<.05). A nearly significant increase was found in the PMBL line at 24h with 5 μg/ml BTZ (14.4±4.3%, p<.08). Significant increases were found in the GCB line at 24h with RTX 20 μg/ml and 40 μg/ml (38.3±4.3%, p<.02 and 42.5±2.6%, p<.004, respectively) and with all BTZ conc. (5 ng/ml: 54.7±6.4%, p=.0004; 50 ng/ml: 62.0±6.6%, p=.0002; 500 ng/ml: 55.9±10.0%, p=.03, 5 μg/ml: 51.2±6.3%, p=.015), but not with max conc. ML120B. No significant difference in apoptosis was found in GCB or PMBL lines with 4 vs 14h incubation at any RTX conc., but a significant increase was found in the PMBL line with 4 vs 24h and 14 vs 24h incubation with RTX 20 μg/ml (4h: 1.9±0.7%, 14h: 0.3±0.3%, 24h: 11.1±3.2%; p [4 vs. 24h] <.05, p [14 vs. 24h] < .03) and RTX 40 μg/ml (4h: 2.3±0.3%, 14h: 0.9±0.7%, 24h: 4.5±0.2%; p [4 vs 24h] <.004, p [14 vs 24h] =.008) and in the GCB line with 4 vs 24h incubation at all conc. and with 14 vs 24h incubation for RTX 40 μg/ml (4h: 15.4±5.4%, 14h: 20.1±5.8%, 24h: 42.5±2.6%; p [4 vs 24h]=.01, p [14 vs 24h]<.04). RS4;11 showed no time-dependent response and no increase in apoptosis at max RTX conc.
 Significant time-dependent increases in apoptosis occurred in PMBL and GCB lines after incubation with RTX. Significant dose-dependent responses occurred in the PMBL line after incubation with RTX and ML120B and in the GCB line after incubation with RTX. These results suggest that agents which target NF-κB can increase apoptosis in PMBL, but concurrent increases in apoptosis in the RTX- and BTZ-treated GCB line indicate that NF-κB pathway blockade may not be the sole mechanism. Studies to assess downregulation of NF-κB genes in PMBL after incubation with RTX, BTZ and ML120B are underway.