Fig. 8: Targeting the NF-κB inducing kinase (NIK) to overcome tumor microenvironment (TME) resistance in Diffuse large B cell lymphoma (DLBCL).

A Overview of signaling between TME, NF-κB, and BCL2-family proteins indicating interactions revealed here with dashed lines, and NIK-inhibitor Amgen16 displayed in red. B Schematic of the co-culture system used, following 24 h of treatment with 0.0001–100 μΜ of venetoclax (ABT199) or A1331852 ± 50 μM of the NIK inhibitor Amgen16. C Western blot analysis of p100 and p52 levels in RIVA cells under monoculture, post a 24-h hCD40-3T3 co-culture or post a 24-h hCD40L-3T3 co-culture with the addition of 50 = μM of Amgen16. Quantification of p52:p100 ratio from western blot data, normalized to total protein across conditions. Bar graphs depict the p52:p100 ratio of two independent experiments. Uncropped blots are presented in the supplementary material. D Fold change to monoculture in MCL1 and BCLXL protein levels in RIVA cells under the indicated conditions. Data points represent paired measurements from individual biological replicates. Statistical analysis was performed using paired T tests with significance indicated as *P < 0.05, ns = not significant. E Cell viability of RIVA cells in response to 0.0001–100μΜ of the BCL-2 inhibitor ABT199 following a 24-h treatment under three conditions: monoculture, co-culture with hCD40L-3T3 cells, and co-culture with the addition of 50 μM of the NIK inhibitor Amgen16. LC₅₀ values for each condition are shown (right) with error bars representing the mean ± standard deviation of three independent experiments, normalized to the untreated control under four conditions: monoculture, co-culture with hCD40L-3T3 cells, co-culture with the addition of 50 μM of the NIK inhibitor Amgen16, and co-culture with the addition of 0.001 μM of the NIK inhibitor CW15337 (*P < 0.05, **P < 0.01, one-way ANOVA with Tukey’s comparisons test). F Cell viability of U2932 cells in response to 0.0001–100 μΜ of ABT199 following a 24-h treatment under three conditions: monoculture, co-culture with hCD40L-3T3 cells, and co-culture with the addition of 50 μM of the NIK inhibitor Amgen16. LC₅₀ values for each condition are shown (right) with error bars representing the mean ± standard deviation of three independent experiments, normalized to the untreated control (*P < 0.05, **P < 0.01, unpaired Ttest). G Schematic representation of the proposed mechanism by which NIK inhibition via Amgen16 disrupts CD40-mediated NF-κB crosstalk, thereby restoring sensitivity to A1331852 (BCLXL inhibitor) by reducing BCLXL and MCL1 levels in the presence of TME signals. H Fold change to monoculture in MCL1 and BCLXL protein levels in SUDHL8 cells cultured under co-culture and co-culture with Amgen16 conditions. Data points represent paired measurements from individual biological replicates. Statistical analysis was performed using paired T tests (*P < 0.05, **P < 0.01). I Cell viability of SUDHL8 cells in response to 0.0001–100μΜ of the BCLXL inhibitor A1331852 following a 24-h treatment under three conditions: monoculture, co-culture with hCD40L-3T3 cells, and co-culture with the addition of 50 μM of the NIK inhibitor Amgen16. LC₅₀ values for each condition are shown (right) with error bars representing the mean ± standard deviation of three independent experiments, normalized to the untreated control (***P < 0.001; one-way ANOVA with Tukey’s comparisons test).