Blockade of oncogenic IκB kinase activity in diffuse large B-cell lymphoma by bromodomain and extraterminal domain protein inhibitors

Abstract

In the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), NF-κB activity is essential for viability of the malignant cells and is sustained by constitutive activity of IκB kinase (IKK) in the cytoplasm. Here, we report an unexpected role for the bromodomain and extraterminal domain (BET) proteins BRD2 and BRD4 in maintaining oncogenic IKK activity in ABC DLBCL. IKK activity was reduced by small molecules targeting BET proteins as well as by genetic knockdown of BRD2 and BRD4 expression, thereby inhibiting downstream NF-κB-driven transcriptional programs and killing ABC DLBCL cells. Using a high-throughput platform to screen for drug-drug synergy, we observed that the BET inhibitor JQ1 combined favorably with multiple drugs targeting B-cell receptor signaling, one pathway that activates IKK in ABC DLBCL. The BTK kinase inhibitor ibrutinib, which is in clinical development for the treatment of ABC DLBCL, synergized strongly with BET inhibitors in killing ABC DLBCL cells in vitro and in a xenograft mouse model. These findings provide a mechanistic basis for the clinical development of BET protein inhibitors in ABC DLBCL, particularly in combination with other modulators of oncogenic IKK signaling.

Keywords: cancer therapy; drug synergism.

Fig. 1.

JQ1 toxicity in ABC DLBCL. (A) Viability of ABC DLBCL cell lines after JQ1 treatment, as assessed by MTS assay at day 4 posttreatment. The multiple myeloma line LP1 is shown as positive control for toxicity. (B) Apoptotic cells (percentage of total), as assessed by a dual intracellular-flow assay for active Caspase 3 and cleaved Parp-1, after JQ1 treatment for the indicated times. See Fig. S1C for a representative staining. (C) Shown is the fraction of live, shRNA-expressing (GFP⁺) cells over time after shRNA induction, compared with the day 0 preinduction values. Error bars represent SEM of triplicates.

Fig. 2.

Mechanism of JQ1 toxicity in ABC DLBCLs. (A) Heat maps of BRD4 ChIP-seq in HBL1 and LP1 cells, after 3-h treatment with either DMSO or 500 nM JQ1. For each cell line, heat maps were ranked based on BRD4 occupancy in the DMSO sample and peaks were centered on a ±2-kb window from their apex. See Materials and Methods for details. (B) Genes down-regulated by JQ1 in the HBL1 ABC DLBCL line were selected (less than −1 log₂ fold change, at least two out of four time points), and heat maps generated to compare the effect of JQ1 treatment among HBL1 and LP1 cells. (C) Top enriched signatures among genes down-regulated by JQ1 in the HBL1 cells were selected. Enrichment ratios are shown for both HBL1 and LP1 cells. See Table S2 for a detailed signature definition. (D) Relative activity of an NF-κB–dependent luciferase reporter in the indicated ABC DLBCL lines treated overnight (16 h) with either DMSO, JQ1, or the IKKβ inhibitor MLN120b. (E) Relative activity of an NF-κB–dependent luciferase reporter in HBL1 and TMD8 cells, after induction of either control shRNA or BRD4 shRNA for the indicated time points. (F) Top enriched signatures among genes down-regulated after both BRD2 and BRD4 knockdown were selected. Enrichment ratios are shown for shBRD2, shBRD4, and shBDR2+shBRD4 combined analyses. See Table S3 for details. Error bars represent SEM of triplicates. For signature enrichment analyses, error bars represent an estimate of the SE. See Materials and Methods for details.

Fig. 3.

JQ1 affects NF-κB signaling in ABC DLBCLs. (A) ABC DLBCL and GCB DLBCL cell lines were treated for 16 h with either DMSO or JQ1 (500 nM). Whole-cell lysates were analyzed by Western blot. (B) ABC DLBCL cell lines were either left untreated or treated for 16 h with DMSO, 500 nM JQ1, or 500 nM I-BET 151. Whole-cell lysates were analyzed by Western blot. (C) ABC DLBCL cell lines were treated with 500 nM JQ1 for the indicated times. Whole-cell lysates were analyzed by Western blot. (D) ABC DLBCL cell lines were treated with 500 nM JQ1 for the indicated times. IκBα mRNA levels where analyzed by TaqMan Q-PCR. IκBα protein levels were analyzed in parallel by Western blot. (E) Relative IκBα-luciferase activity in TMD8 cells, after treatment with DMSO, 100 nM JQ1, or 500 nM JQ1 for the indicated times. (F) Relative IκBα-luciferase activity in TMD8 cells. Cells were infected with the indicated shRNAs, selected, and then induced for the indicated times before measuring the IκBα-luciferase activity. (G) Relative IκBα-luciferase activity in TMD8 cells. Cells were infected with the indicated expression vectors, selected, and then induced for 2 d before a 6-h JQ1 treatment. (H) HBL1 and TMD8 ABC DLBCL cell lines were infected with either empty, IKKβ WT, or IKKβ EE expression vectors, selected, and preinduced for 2 d before seeding for MTS assay. Shown is the relative cell viability at day 4 after drug treatment. Error bars represent SEM of triplicates, except for D, OCI-LY10 cells, n = 2.

Fig. 4.

JQ1 synergize with ibrutinib in killing ABC DLBCLs. (A) Combination responses for the BET inhibitor JQ1 and the IKKβ inhibitor SPC-839 as judged by a 10 × 10 viability matrix. TMD8 cells viability was measured at 48 h posttreatment by CellTiterGlo assay (Left). An isobologram analysis of viability data are shown on the Right. (B) Same as in A, but the combination between JQ1 and the BTK inhibitor ibrutinib was analyzed. (C) The indicated cell lines were treated with a large concentration range of BTK inhibitor ibrutinib, in combination with either DMSO or three different concentration of JQ1. Shown is the relative viability of each combination subset, as assessed by MTS assay at day 4 posttreatment. (D) Relative IκBα-luciferase activity in the ABC DLBCL TMD8 cell line. Cells were treated overnight (16 h) with indicated concentration of ibrutinib, in combination with either DMSO or three different concentration of JQ1. Error bars represent SEM of triplicates.

Fig. 5.

Combination therapy in a xenograft model of ABC DLBCL. (A) Human TMD8 ABC DLBCL cells were established as a s.c. tumor in NOD/SCID mice and treated by i.p. injection with either vehicle or BET Inhibitor CPI203 (5 mg/kg). Mice were treated for 21 d, and tumor growth was measured as a function of tumor volume. (B) Human TMD8 ABC DLBCL cells were established as a s.c. tumor in CB17 SCID mice and treated by i.p. injection with vehicle, BET inhibitor CPI203 (5 mg/kg), BTK inhibitor ibrutinib (2 mg/kg), or with a combination of CPI203 and ibrutinib. Mice were treated for 12 d, and tumor growth was measured as a function of tumor volume. (C) Total RNA was extracted from xenograft biopsies. Human IL6, IL10, and MYC mRNA levels were measured by TaqMan RT Q-PCR. For each gene, the relative abundance was calculated vs. B2M values, arbitrarily set to 1,000. Error bars represent SEM. (A) n = 3 mice per group. (B) n = 9 mice per group. (C) n = 7 for vehicle group, n = 8 for combination group, and n = 9 for single-arm groups.