American Society of Hematology

Figure 5.

$AZD8835 inhibits NF-κB signaling in ABC DLBCLs. (A) Treatment with AZD8835 for 6 and 24 hours decreases both AKT and PRAS40 phosphorylation levels in U2932 and HBL-1 cells measured by western blotting. (B) GEP following treatment with AZD8835 in HBL-1, OCI-Ly10, and U2932 cells. Changes of gene expression are profiled at the indicated time points following treatment with AZD8835. Gene expression changes are depicted according to the color scale shown. Genes that are involved in critical biological processes are highlighted. (C) Gene set enrichment analysis of a previously described NF-κB gene expression signature. The NF-κB signature is significantly enriched with genes that are downregulated after AZD8835 treatment. (D) NF-κB target genes are downregulated on mRNA level in HBL-1, OCI-Ly10, and TMD8 but not in OCI-Ly3 and BJAB cells after treatment with AZD8835. The BTK inhibitor, ibrutinib, that was used as a positive control, downregulates the NF-κB target genes IRF4, BCL-XL, IκB-ζ, JUNB, IκB-α, and TNFAIP3 to a similar degree. Representative results from at least 2 independent experiments are shown. Error bars indicate the standard deviation. (E) NF-κB target genes are downregulated on protein levels in HBL-1, OCI-Ly10, and TMD8 cells after treatment with AZD8835 and ibrutinib. No downregulation can be observed in insensitive control cell lines OCI-Ly3 and BJAB. Representative results from at least 3 independent experiments are shown. (F) Analyses of cytosolic and nuclear distribution of NF-κB subunits. Treatment with AZD8835 significantly decreases nuclear expression of the NF-κB subunits RelA and p50 in sensitive HBL-1, TMD8, and U2932 cells, whereas no changes are detectable in insensitive OCI-Ly3 and BJAB models. Successful nuclear and cytosolic fractionation is indicated by tubulin and poly(ADP-ribose) polymerase 1 (PARP) expression. Representative results from 2 independent experiments are shown. (G) AZD8835 treatment inhibits IκB-α phosphorylation in HBL-1 and TMD8 cells. In contrast, p-IκB-α levels are not changed in OCI-Ly3 cells following AZD8835 treatment. The IκB kinase inhibitor AFN700 reduces p-IκB-α levels in all 3 cell lines. Representative results from at least 3 independent experiments are shown. (H) The activating CARD11L244P mutant rescues HBL-1 and TMD8 cells from AZD8835- and ibrutinib-induced cytotoxicity. Data are expressed as means ± standard deviation of at least 2 independent experiments. (I) Exogenous expression of an MYC cDNA partially rescues TMD8 cells from AZD8835-induced cytotoxicity, whereas it has no effect on viability of HBL-1 cells. Representative results from at least 3 independent experiments are shown. **P < .01; ***P < .001.$

AZD8835 inhibits NF-κB signaling in ABC DLBCLs. (A) Treatment with AZD8835 for 6 and 24 hours decreases both AKT and PRAS40 phosphorylation levels in U2932 and HBL-1 cells measured by western blotting. (B) GEP following treatment with AZD8835 in HBL-1, OCI-Ly10, and U2932 cells. Changes of gene expression are profiled at the indicated time points following treatment with AZD8835. Gene expression changes are depicted according to the color scale shown. Genes that are involved in critical biological processes are highlighted. (C) Gene set enrichment analysis of a previously described NF-κB gene expression signature. The NF-κB signature is significantly enriched with genes that are downregulated after AZD8835 treatment. (D) NF-κB target genes are downregulated on mRNA level in HBL-1, OCI-Ly10, and TMD8 but not in OCI-Ly3 and BJAB cells after treatment with AZD8835. The BTK inhibitor, ibrutinib, that was used as a positive control, downregulates the NF-κB target genes IRF4, BCL-XL, IκB-ζ, JUNB, IκB-α, and TNFAIP3 to a similar degree. Representative results from at least 2 independent experiments are shown. Error bars indicate the standard deviation. (E) NF-κB target genes are downregulated on protein levels in HBL-1, OCI-Ly10, and TMD8 cells after treatment with AZD8835 and ibrutinib. No downregulation can be observed in insensitive control cell lines OCI-Ly3 and BJAB. Representative results from at least 3 independent experiments are shown. (F) Analyses of cytosolic and nuclear distribution of NF-κB subunits. Treatment with AZD8835 significantly decreases nuclear expression of the NF-κB subunits RelA and p50 in sensitive HBL-1, TMD8, and U2932 cells, whereas no changes are detectable in insensitive OCI-Ly3 and BJAB models. Successful nuclear and cytosolic fractionation is indicated by tubulin and poly(ADP-ribose) polymerase 1 (PARP) expression. Representative results from 2 independent experiments are shown. (G) AZD8835 treatment inhibits IκB-α phosphorylation in HBL-1 and TMD8 cells. In contrast, p-IκB-α levels are not changed in OCI-Ly3 cells following AZD8835 treatment. The IκB kinase inhibitor AFN700 reduces p-IκB-α levels in all 3 cell lines. Representative results from at least 3 independent experiments are shown. (H) The activating CARD11^L244P mutant rescues HBL-1 and TMD8 cells from AZD8835- and ibrutinib-induced cytotoxicity. Data are expressed as means ± standard deviation of at least 2 independent experiments. (I) Exogenous expression of an MYC cDNA partially rescues TMD8 cells from AZD8835-induced cytotoxicity, whereas it has no effect on viability of HBL-1 cells. Representative results from at least 3 independent experiments are shown. **P < .01; ***P < .001.

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