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Letters to the Editor
Interleukin-1 receptor associated kinase 1/4 and bromodomain and extra-terminal inhibitions converge on NF-κB blockade and display synergistic antitumoral activity in activated B-cell subset of diffuse large B-cell lymphoma with MYD88 L265P mutation
The outcome of patients with diffuse large B-cell lym- phoma (DLBCL) is very heterogeneous and is most likely dictated by their cell of origin (COO), defining two main molecular subtypes, i.e., germinal center B-cell (GCB) and activated B-cell (ABC).1 Upon treatment with multi-agent chemotherapy (cyclophosphamide, doxorubicin, vin- cristine and prednisone) combined with the monoclonal anti-CD20 antibody rituximab (R-CHOP), almost a third of the patients, corresponding mainly to the ABC subtype of the disease, does not achieve complete remission (CR) or relapses shortly after CR.2 However, the COO does not fully account for the different outcomes. Massive sequenc- ing analyses recently uncovered molecular subtypes of DLBCL with distinct outcomes. In this regard, Chapuy et al. have described five different molecular subtypes with dis- tinct pathogenic mechanisms and prognosis, independent- ly of the COO. Interestingly, the C5 cluster (mostly ABC subtypes), enriched in MYD88 L265P and CD79B muta- tions, maintained a shorter survival compared to the other ABC cluster.3
ABC-DLBCL tumors rely almost exclusively on constitu- tive nuclear transcription factor κB (NF-κB) signaling for their survival, a phenomenon that has been linked to a vari- ety of genetic alterations that aberrantly activate the B-cell receptor (BCR) and the Toll-like receptor (TLR) signaling pathways.1 Within the TLR axis, mutations in the gene codifying for the adaptor protein myeloid differentiation primary response gene 88 (MYD88) enhance interleukin-1 receptor-associated kinase 1 and 4 (IRAK1 and IRAK1) activity, providing sustained activation of NF-κB through most of the TLR. The p.L265P mutation, characterized by a change from leucine (CTC) to proline (CCG) in the MYD88 Toll/interleukin (IL)-1 receptor domain, recruits MYD88 to the cytoplasmic tail of TLR to form an active complex. Beside NF-κB, this complex promotes Janus kinase-signal transducer and activator of transcription 3 (JAK-STAT3) signaling through a pathway involving inter- leukin (IL)-6 and IL-10 secretion.4
Preclinical data have indicated that MYD88-mutant ABC-DLBCL cells were sensitive to pharmacological blockade of IRAK4 kinase activity, being IRAK4-compro- mised cells especially responsive to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or the BCL-2 antagonist venetoclax, as almost all ABC-DLBCL display BCL2 amplification/overexpression.5,6 Considering that both IRAK1 and IRAK4 are required for ABC-DLBCL cell sur- vival,4 we investigated the effect of a 24-72 hour treat- ment with a selective and orally bioavailable IRAK1/4 inhibitor (IRAKi, Merck),7 in three well-characterized MYD88-mutated cell lines, OCI-LY3, OCI-LY10, HBL-1, using proliferation as a read out. Three germinal center B-cell (GCB)-DLBCL cell lines (SUDHL-4, SUDHL-8 and OCI-LY8) with wild-type MYD88 (MYD88wt) were ana- lyzed in the same settings, as a control. We observed a partial and transitory response to IRAKi in ABC-DLBCL cells only, when using the compound at the physiological dose of 50 mM (Figure 1A). Treatment-related cytotoxicity decreased from 25.5% at 24 hours to 19% at 72 hours, respectively, despite an efficient blockade of IRAK1 and IRAK4 phosphorylation at Thr209 and Thr345 residues, in the three MYD88-mutated cell lines (Figure 1B).
Interestingly, the destabilization of the anti-apoptotic pro- tein and key mediator of IRAKi activity, MCL-1,8 was not sufficient to confer a significant cytotoxicity to the com- pound (Figure 1B). A gene expression profiling (GEP) analysis in the three MYD88-mutated cell lines exposed for 6 hours to the inhibitor, further showed that IRAK1/4 blockade significantly altered the expression of the top NF-κB gene signatures associated to B-cell lymphoma,9 namely NFKB_ALL_OCI_LY10 and NFKB_BOTH OCILY3ANDLY10, with normalized enrichment score (NES) values reaching 1.8, while in contrast a third gene set, NFKB_OCILY10_ONLY, was slightly upregulated (NES: -1.20), according to GSEA analysis (Figure 1C; Online Supplementary Table S1). In agreement, the tran- scription of several NF-κB-regulated genes known to pro- mote ABC-DLBCL pathogenesis, including IL6, IL10, IRF4 and CCL3, were either unaffected or even increased after treatment with IRAKi (Figure 1D, Online Supplementary Table S1). Consistently, in an OCI-LY3 mouse xenograft model the compound failed to elicit a significant tumor growth inhibition (Online Supplementary Figure S1A).
We then considered the possibility to enhance IRAKi activity in MYD88 L265P ABC-DLBCL by combining the compound with the BET bromodomain inhibitor CPI203 (kindly provided by Constellation Pharmaceuticals), as this BRD4 antagonist has been shown to effectively sup- press a NF-κB gene signature that includes IL6, IL10 and IRF4, in ABC-DLBCL.10 After exposing the same ABC- DLBCL cell lines as above to a 50 mM dose of IRAKi, fol- lowed by a 24-hour treatment with 0.5 mM CPI203, a new GEP analysis was performed. As shown in Figure 2A, IRAKi-CPI203 combination induced a significant down- regulation of NF-κB-related genes when compared to IRAKi single agent, with NES comprised between 1.46 and 1.99. Of note, the combination therapy allowed to a significant disruption of NFKB_OCILY10_ONLY gene sig- nature with a NES of 1.89. Among the genes included in the NFKB_ALL_OCILY3_LY10 gene set, a selected list of nineteen factors underwent a ≥2-fold increase in their rank metric score between this analysis and the previous one (Online Supplementary Table S2), suggesting that their improved modulation may be associated with the combi- national effect of IRAKi and CPI203. From this list, we identified only four genes (LTA, MARCKS, CD44 and HEATR1) that were not included in the core component of the NF-κB target genes affected by either IRAKi or CPI203 as single agents, but which underwent a significant down- regulation upon treatment with the drug combination. Among these genes, we were unable to detect significant levels of LTA and HEATR1 transcripts in the three ABC- DLBCL cell lines (data not shown). In contrast, upon expo- sure of the three MYD88-mutated cell lines to the IRAKi we observed a 1.2- to 2-fold transcriptional increase of MARCKS and CD44, together with IL6 and IL10 used here as hallmarks of NF-kB activation. These genes were all reduced down to 0.5-fold in cells treated with the drug combination (Figure 2B). Accordingly, IRAKi-CPI203 treat- ment led to the accumulation of the intracellular inhibitor of NF-κB, IκB, and to the consequent reduction in CD44 and MARCKS protein levels, while IRAKi and CPI203 sin- gle agents slightly affected the expression of these factors (Figure 2C). As expected, in two out of the three cell lines, CPI203-based treatments led to the decrease in MYC pro- tein and mRNA, used here as hallmarks of BRD4 inhibition (Figures 2B and C). Also confirming a previous report link- ing bromodomain inhibitor therapy with IRAK1 downreg- ulation in B-cell lymphoma,11 IRAK1-pThr209 levels under- went a slight downregulation after CPI203 treatment and this effect was remarkably potentiated upon addition of
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