Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma

Abstract

Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon β (IFNβ) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNβ production by repressing IRF7 and amplify prosurvival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies.

Figure 1. Lenalidomide induces a toxic type I interferon response in ABC DLBCL

(A) Viability (MTS assay) of ABC and GCB DLBCL cell lines treated with lenalidomide for 4 days. Error bars show the standard error of the mean (SEM) of triplicates. (B) Relative expression of interferon signature genes over a time course of lenalidomide (10μM) treatment. Gene expression changes induced by lenalidomide are depicted according to the color scale shown. Average relative expression of interferon signature genes is at the bottom. Yellow bars: genes with overlapping IRF4/SPIB ChIP-seq peaks. (C, D) IFNβ mRNA expression and secretion in lenalidomide-treated (10μM) cells. Error bars show the SEM of triplicates. (E) Activity of an ISRE-driven luciferase reporter in cells treated with lenalidomide (10μM) or vehicle control (DMSO) at the indicated times. Error bars show the SEM of triplicates. (F) Western blot analysis of the indicated proteins in lenalidomide-treated (10μM) ABC DLBCL cells. p-: phospho-. (G) Viability (MTS assay) of DLBCL cells treated with the indicated amount of human recombinant IFNβ for 4 days. Error bars show the SEM of triplicates. (H) Measurement of viability (MTS assay; right) and apoptosis (PARP cleavage and caspase-3 activation by FACS; left) in ABC DLBCL cells treated with control compounds (DMSO or isotype-matched antibody), lenalidomide (1μM), or lenalidomide plus the indicated blocking antibodies (2.5μg/ml) for 4 days. Error bars show the SEM of triplicates. (I) Viability (MTS assay) of OCI-Ly10 ABC DLBCL cells in which the indicated shRNAs were induced for 2 days before treatment with DMSO or lenalidomide, as indicated, for 4 days. Error bars show the SEM of triplicates. See also Figure S1 and Table S1.

Figure 2. IRF4 and SPIB are required for ABC DLBCL viability

(A) Toxicity of an IRF4 shRNA in a loss-of-function RNA interference screen of the indicated cell lines. Shown are the log2 ratios of shRNA abundance before induction (“uninduced d0”) versus abundance after 21 days in culture (“induced d21”). shRPS13 targets ribosomal protein S13, an essential gene in all cell types. Error bars show the SEM for quadriplicates. (B) Viability of shIRF4+ (GFP+) cells over time after induction as a percentage of live shIRF4+ cells following shIRF4 induction relative to day 0. The number of replicate infections is shown in parentheses. Error bars represent the SEM of replicates. (C) Overlap of IRF4 ChIP-Seq peaks in ABC DLBCL and multiple myeloma, based on all peaks (left), genes with an IRF4 peak within +/− 2kb of the TSS (middle), and genes with an IRF4 peak in a region encompassing the gene body and 10 kb upstream of the TSS (right). (D) Motif discovery using the Weeder and MEME algorithms based on the top 1000 ABC DLBCL IRF4 ChIP-Seq peaks by sequence tag abundance. The highest scoring motif is shown with core recognition motifs indicated. (E) Enrichment for the EICE motif in promoter-proximal peaks (+/− 2 kb from the TSS) based on IRF4 and SPIB ChIP-Seq data in ABC DLBCL or multiple myeloma, as a function of peak percentile, ranked by sequencing tag abundance. (F) Overlap of IRF4 and SPIB ChIP-seq peaks in ABC DLBCL, as in (C). (G) Crystal structure of the mouse IRF4 and PU.1 DNA binding domains interacting with an EICE, showing the interacting charged residues conserved in human IRF4 and SPIB. (H) Rescue experiment showing the viability of the indicated cell lines bearing empty vector, wild-type IRF4, or mutant IRF4 expression vectors, plotted as the fraction of shIRF4+ (GFP+) cells at times following shIRF4 induction relative to day 0. (I) Rescue experiment as in (H) with cells bearing the indicated SPIB expression vectors and an inducible SPIB-3′UTR-targeted shRNA. (J) Viability (FACS for live cells) of the indicated cell lines expressing an inducible IRF4-SPIB chimeric repressor, plotted as a percentage of live chimeric repressor+ cells relative to empty vector-bearing control cells, at various times following chimeric repressor induction. See also Figure S2 and Table S2.

Figure 3. IRF4 controls essential gene expression programs in ABC DLBCL

(A) IRF4 direct target genes grouped according to gene expression signatures (Shaffer et al., 2006). Signatures with significant enrichment for IRF4 targets were grouped by function (Table S3A). Genes that are induced or repressed by IRF4 are indicated in green and red, respectively. Asterisks indicate genes with an overlapping IRF4-SPIB ChIP-seq peak. (B) ISRE-driven luciferase reporter activity in ABC DLBCL lines with control or IRF4 shRNAs after 2 days of induction and subsequent addition of IFNβ (1000U). Error bars show the SEM of triplicates. See also Figure S3 and Table S3.

Figure 4. Lenalidomide toxicity in ABC DLBCL is opposed by IRF4 and SPIB

(A,C) Western blot of IRF4, SPIB and β-actin proteins in ABC DLBCL cell lines treated with lenalidomide (10μM) over time. (B,D) IRF4 and SPIB mRNA expression quantified by Q-PCR, normalized to β2-microglobulin(B2M) expression, in the ABC DLBCL line OCI-Ly10 treated with lenalidomide (10μM). Error bars show the SEM of triplicates. (E,F) IFNβ mRNA expression and protein secretion in the OCI-Ly10 ABC DLBCL line induced for IRF4 or control shRNA expression for 2 days and treated with lenalidomide (10μM). Error bars represent the SEM of triplicates. (G)Western blot of the indicated proteins in OCI-Ly10, following induction of IRF4 or control shRNAs for 2 days and treatment with lenalidomide (10μM)for the indicated times. p-: phospho-. (H)ISRE -driven luciferase reporter activity in OCI-Ly10 with control or IRF4 shRNAs after lenalidomide (10μM) treatment. Error bars represent the SEM of triplicates. (I)TRAIL mRNA quantified by Q -PCR, normalized to B2M, in OCI-Ly10 cells with shRNA induction for 2 days and lenalidomide (10μM)treatment for the indicated times. Error bars show the SEM of triplicates. (J) Western blot analysis of the indicated proteins in OCI-Ly10 cells transduced with a flag epitope-tagged IRF4 expression vector or an empty vector, induced for 48 h, then treated with lenalidomide (10μM)for the indicated times. The lower IRF4 band is endogenous IRF4; the upper band is FLAG-tagged exogenous IRF4. p-: phosphorylation. (K) Viability of ABC DLBCL lines induced to express control, IRF4 or SPIB shRNAs and the treated with DMSO or lenalidomide (10μM) over a time course of induction. See text for details. (L) Viability of OCI-Ly10 cells transduced with an IRF4 expression or empty vector, induced for 24h and then treated with DMSO or lenalidomide for 4 days. Error bars represent the SEM of triplicates. (M) Viability (MTS assay) of OCI-Ly10 cells induced to express the indicated shRNAs for 2 days and treated with lenalidomide at the indicated concentrations for 4 days. ctrl.: control. Error bars show the SEM of triplicates. (N) IFNβ mRNA expression, measured by Q-PCR, in OCI-Ly10 cells induced to express CRBN shRNAs for 2 days and treated with lenalidomide (10μM) for the indicated times. Error bars show the SEM of triplicates. (O) TMD8 ABC DLBCL cells expressing an IκBα-luciferase fusion protein were induced to express control or CRBN shRNAs for 2 days, then treated with lenalidomide at the indicated concentrations or DMSO for 2 days. Luciferase activity was normalized to the DMSO control. As a positive control, cells were treated with the IKKβ inhibitor MLN120B (10μM) for 2 days. Error bars show the SEM of triplicates. (P) IRF4 and SPIB mRNA expression, quantified by Q-PCR, in TMD8 cells transduced with the indicated shRNAs. shRNA expression was induced for 2 days followed by lenalidomide treatment (10μM) for 24 hours. Error bars show the SEM of triplicates. (Q) Western blot for the indicated proteins in TMD8 cells induced to express CRBN or control shRNAs for 2 days, followed by treatment with lenalidomide (10μM) for 24 hours. See also Figure S4.

Figure 5. IRF4-SPIB block interferon signaling by repressing IRF7

(A) UCSC browser depiction of ChIP-seq data from HBL1 ABC DLBCL cells showing IRF4 and SPIB-biotag binding at the IRF7 promoter. TSS (arrow). (B) IRF4 binding at the IRF7 locus by ChIP in OCI-Ly10 ABC DLBCL cells treated with DMSO (“−”) or lenalidomide (10μM) for 24 hours. Error bars show SEM of triplicates. (C) Q-PCR quantification of IRF7 mRNA levels, normalized to B2M, in OCI-Ly10 cells with control (ctrl.) or IRF4 shRNAs, treated with lenalidomide(10μM) or DMSO. Error bars show SEM of triplicates. (D) Western blot of the indicated proteins in cells from (C).(E) Viability (MTS assay) of OCI-Ly10 cells induced to express the indicated shRNAs for 2 days and then treated with DMSO or lenalidomide(10μM) for 4 days. Error bars show SEM of triplicates. (F) Western blot of the indicated proteins in ABC DLBCL lines induced to express control or IRF7 shRNAs for 2 days and then treated with DMSO or lenalidomide(10μM)for the indicated times. (G,H) Q -PCR analysis, normalized to B2M, of IFNβ (G) or TRAIL (H) mRNA levels in OCI-Ly10 cells induced to express control or IRF7 shRNAs for 2 days and treated with DMSO (“−”) or lenalidomide (10μM; “+”) for 24 hours. Error bars show SEM of triplicates.

Figure 6. IRF4-SPIB and CARD11 form an essential oncogenic loop in ABC DLBCL

(A) UCSC browser depiction of ChIP-seq data from the HBL1 ABC DLBCL line showing IRF4 (in triplicate) and SPIB-biotag binding at the CARD11 locus, with an evolutionarily conserved EICE binding motif indicated. Control (IRF4): OCI-Ly19 (IRF4-). Control (SPIB): HBL1 with empty biotag vector. TSS (arrow). (B) ChIP analysis in HBL1 cells for IRF4 and SPIB binding at the CARD11 peak identified in (A) or at a negative control (ctrl.) locus. IRF4 or control shRNAs were induced for 4 days. GCB DLBCL line: OCI-Ly19 (IRF4-) is IRF4-negative. Error bars show SEM of triplicates. (C) Relative CARD11 mRNA expression, depicted according to the color scale shown, from gene expression profiling of ABC DLBCL lines after induction of shIRF4 or the IRF4-SPIB chimeric repressor for 4 days. (D) IKK activity measured by an IκBα-luciferase reporter in TMD8 (ABC DLBCL) after induction of various shRNAs for 3 days (left) or the IRF4-SPIB chimeric repressor for 1 day (right). Also shown is the effect of 1 day exposure to an IKKβ inhibitor (MLN120B) or DMSO. (E) Western blot analysis of the indicated proteins following treatment of OCI-Ly10 cells with lenalidomide (10μM) for the indicated times. (F) IKK activity measured by an IκBα-luciferase reporter after treatment of TMD8 cells with lenalidomide at the indicated concentrations for 48 h. Error bars show the SEM of triplicates. See also Figure S5.

Figure 7. Synergy between lenalidomide and NF-κB pathway inhibitors in ABC DLBCL

(A) Western blot of the indicated proteins in OCI-Ly10 cells treated with lenalidomide (5μM) alone or with MLN120B (10μM), ibrutinib(5nM) or DMSO for the indicated times. p-: phospho-. (B) ISRE-driven luciferase activity in OCI-Ly10 cells treated with lenalidomide (5μM) +/− ibrutinib (5nM) for the indicated times. Error bars show the SEM of triplicates. (C) IKK activity measured by an IκBα-luciferase reporter in TMD8 cells treated with ibrutinib at the indicated concentrations +/− lenalidomide (1μM) for 48 hours. Error bars show the SEM of triplicates. (D) Viability (MTS assay) of OCI-Ly10 treated with MLN120B at the indicated concentrations +/− lenalidomide (1μM) for 4 Days relative to DMSO-treated cells. Error bars show the SEM of triplicates. (E) Viability (MTS assay) of DLBCL lines treated with ibrutinib, lenalidomide, or both for 4 days at the concentrations indicated. Error bars show the SEM of triplicates. (F) OCI-Ly10 ABC DLBCL cells were established as a subcutaneous tumor (average 80 mm³) in immunodeficient mice, and then treated daily for 20 days with DMSO, lenalidomide (10 mg/kg), ibrutinib (3mg/kg), or lenalidomide plus ibrutinib by intraperitoneal injection. Tumor progression was monitored as a function of tumor volume. Error bars show the SEM of 5 mice per group. See also Figure S6.

Figure 8. Exploiting synthetic lethality for the therapy of ABC DLBCL

Recurrent oncogenic mutations in ABC DLBCL activate both the BCR and MYD88 pathways to drive pro-survival NF-κB signaling. However, MYD88 signaling also induces IFNβ, which is detrimental to ABC DLBCL survival. IRF4 and SPIB lie at the nexus of both pathways, promoting ABC DLBCL survival by repressing IRF7, thereby blocking IFNβ, and transactivating CARD11, thereby increasing NF-κB signaling. NF-κB factors transactivate IRF4, creating a positive feedback oncogenic loop. Lenalidomide targets this circuitry by down modulating IRF4 and SPIB, thereby increasing toxic IFNβ secretion and decreasing NF-κB activity.