Letters to the Editor Interestingly, the destabilization of the anti-apoptotic pro- Interleukin-1 receptor associated kinase 1/4 and tein and key mediator of IRAKi activity, MCL-1,8 was not bromodomain and extra-terminal inhibitions sufficient to confer a significant cytotoxicity to the com- converge on NF-κB blockade and display synergistic pound (Figure 1B). A gene expression profiling (GEP) antitumoral activity in activated B-cell subset of analysis in the three MYD88-mutated cell lines exposed diffuse large B-cell lymphoma with MYD88 L265P for 6 hours to the inhibitor, further showed that IRAK1/4 mutation blockade significantly altered the expression of the top NF-κB gene signatures associated to B-cell lymphoma,9 The outcome of patients with diffuse large B-cell lym- namely NFKB_ALL_OCI_LY10 and NFKB_BOTH phoma (DLBCL) is very heterogeneous and is most likely OCILY3ANDLY10, with normalized enrichment score dictated by their cell of origin (COO), defining two main (NES) values reaching 1.8, while in contrast a third gene molecular subtypes, i.e., germinal center B-cell (GCB) and set, NFKB_OCILY10_ONLY, was slightly upregulated activated B-cell (ABC).1 Upon treatment with multi-agent (NES: -1.20), according to GSEA analysis (Figure 1C; chemotherapy (cyclophosphamide, doxorubicin, vin- Online Supplementary Table S1). In agreement, the tran- cristine and prednisone) combined with the monoclonal scription of several NF-κB-regulated genes known to pro- anti-CD20 antibody rituximab (R-CHOP), almost a third of mote ABC-DLBCL pathogenesis, including IL6, IL10, IRF4 the patients, corresponding mainly to the ABC subtype of and CCL3, were either unaffected or even increased after the disease, does not achieve complete remission (CR) or treatment with IRAKi (Figure 1D, Online Supplementary relapses shortly after CR.2 However, the COO does not Table S1). Consistently, in an OCI-LY3 mouse xenograft fully account for the different outcomes. Massive sequenc- model the compound failed to elicit a significant tumor ing analyses recently uncovered molecular subtypes of growth inhibition (Online Supplementary Figure S1A). DLBCL with distinct outcomes. In this regard, Chapuy et al. We then considered the possibility to enhance IRAKi have described five different molecular subtypes with dis- activity in MYD88 L265P ABC-DLBCL by combining the tinct pathogenic mechanisms and prognosis, independent- compound with the BET bromodomain inhibitor CPI203 ly of the COO. Interestingly, the C5 cluster (mostly ABC (kindly provided by Constellation Pharmaceuticals), as subtypes), enriched in MYD88 L265P and CD79B muta- this BRD4 antagonist has been shown to effectively sup- tions, maintained a shorter survival compared to the other press a NF-κB gene signature that includes IL6, IL10 and ABC cluster.3 IRF4, in ABC-DLBCL.10 After exposing the same ABC- ABC-DLBCL tumors rely almost exclusively on constitu- DLBCL cell lines as above to a 50 mM dose of IRAKi, fol- tive nuclear transcription factor κB (NF-κB) signaling for lowed by a 24-hour treatment with 0.5 mM CPI203, a new their survival, a phenomenon that has been linked to a vari- GEP analysis was performed. As shown in Figure 2A, ety of genetic alterations that aberrantly activate the B-cell IRAKi-CPI203 combination induced a significant down- receptor (BCR) and the Toll-like receptor (TLR) signaling regulation of NF-κB-related genes when compared to pathways.1 Within the TLR axis, mutations in the gene IRAKi single agent, with NES comprised between 1.46 codifying for the adaptor protein myeloid differentiation and 1.99. Of note, the combination therapy allowed to a primary response gene 88 (MYD88) enhance interleukin-1 significant disruption of NFKB_OCILY10_ONLY gene sig- receptor-associated kinase 1 and 4 (IRAK1 and IRAK1) nature with a NES of 1.89. Among the genes included in activity, providing sustained activation of NF-κB through the NFKB_ALL_OCILY3_LY10 gene set, a selected list of most of the TLR. The p.L265P mutation, characterized by nineteen factors underwent a ≥2-fold increase in their a change from leucine (CTC) to proline (CCG) in the rank metric score between this analysis and the previous MYD88 Toll/interleukin (IL)-1 receptor domain, recruits one (Online Supplementary Table S2), suggesting that their MYD88 to the cytoplasmic tail of TLR to form an active improved modulation may be associated with the combi- complex. Beside NF-κB, this complex promotes Janus national effect of IRAKi and CPI203. From this list, we kinase-signal transducer and activator of transcription 3 identified only four genes (LTA, MARCKS, CD44 and (JAK-STAT3) signaling through a pathway involving inter- HEATR1) that were not included in the core component of leukin (IL)-6 and IL-10 secretion.4 the NF-κB target genes affected by either IRAKi or CPI203 Preclinical data have indicated that MYD88-mutant as single agents, but which underwent a significant down- ABC-DLBCL cells were sensitive to pharmacological regulation upon treatment with the drug combination. blockade of IRAK4 kinase activity, being IRAK4-compro- Among these genes, we were unable to detect significant mised cells especially responsive to the Bruton's tyrosine levels of LTA and HEATR1 transcripts in the three ABC- kinase (BTK) inhibitor ibrutinib or the BCL-2 antagonist DLBCL cell lines (data not shown). In contrast, upon expo- venetoclax, as almost all ABC-DLBCL display BCL2 sure of the three MYD88-mutated cell lines to the IRAKi amplification/overexpression.5,6 Considering that both we observed a 1.2- to 2-fold transcriptional increase of IRAK1 and IRAK4 are required for ABC-DLBCL cell sur- MARCKS and CD44, together with IL6 and IL10 used here vival,4 we investigated the effect of a 24-72 hour treat- as hallmarks of NF-kB activation. These genes were all ment with a selective and orally bioavailable IRAK1/4 reduced down to 0.5-fold in cells treated with the drug inhibitor (IRAKi, Merck),7 in three well-characterized combination (Figure 2B). Accordingly, IRAKi-CPI203 treat- MYD88-mutated cell lines, OCI-LY3, OCI-LY10, HBL-1, ment led to the accumulation of the intracellular inhibitor using proliferation as a read out. Three germinal center of NF-κB, IκB, and to the consequent reduction in CD44 B-cell (GCB)-DLBCL cell lines (SUDHL-4, SUDHL-8 and and MARCKS protein levels, while IRAKi and CPI203 sin- OCI-LY8) with wild-type MYD88 (MYD88wt) were ana- gle agents slightly affected the expression of these factors lyzed in the same settings, as a control. We observed a (Figure 2C). As expected, in two out of the three cell lines, partial and transitory response to IRAKi in ABC-DLBCL CPI203-based treatments led to the decrease in MYC pro- cells only, when using the compound at the physiological tein and mRNA, used here as hallmarks of BRD4 inhibition dose of 50 mM (Figure 1A). Treatment-related cytotoxicity (Figures 2B and C). Also confirming a previous report link- decreased from 25.5% at 24 hours to 19% at 72 hours, ing bromodomain inhibitor therapy with IRAK1 downreg- respectively, despite an efficient blockade of IRAK1 and ulation in B-cell lymphoma,11 IRAK1-pThr209 levels under- IRAK4 phosphorylation at Thr209 and Thr345 residues, went a slight downregulation after CPI203 treatment and in the three MYD88-mutated cell lines (Figure 1B). this effect was remarkably potentiated upon addition of haematologica | 2021; 106(10) 2749 Letters to the Editor IRAKi to the cell cultures (Figure 2C). In line with the of cells with high contents of IL6 mRNA, a percentage increased blockade of NF-κB signaling, the addition of superior to what observed upon treatment with each drug CPI203 synergistically improved IRAKi cytostatic effect in alone (Figure 3B, right panel). the three cell lines, as attested by an 86% blockade in cell Among the above mentioned genes, CD44 expression proliferation, significantly higher than the 19% activity and IL-6 serum levels have been described as prognostic achieved by IRAKi alone (combination index [CI]: 0.52, markers in DLBCL.13,14 In order to investigate the role of Figure 2D). Importantly, the co-operation between the these two factors in the response of ABC-DLBCL cell lines IRAKi and CPI203 involved a remarkable downregulation to IRAKi-based treatment, HBL-1 and OCI-LY3 cells were of MCL-1 (Figure 2C), which was accompanied by a 36% stimulated with 0.5 mM of the CD44 ligand, hyaluronic increase in the relative apoptosis rate when compared with acid (HA), or exposed to a 5 mg/mL dose of the IL-6 block- IRAKi and CPI203 used separately (Figure 2F). ing antibody tocilizumab, prior to a 72-hour treatment In order to further validate the activity of the drug com- with the drugs. In the case of HA, cells were exposed to bination, primary lymph node biopsies from DLBCL IRAKi (50 mM) +/- CPI203 (0.5 mM), while effect of patients with either MYD88wt or MYD88 L265P were co- tocilizumab pretreatment was evaluated in IRAKi-treated cultured in the presence of a feeding stromal monolayer as cells. Cell response was determined by fluorescence previously reported,12 and treated with the different drugs microscopy recounting of cells with high contents in F- as above. While IRAKi-CPI203 was almost inactive in actin and by MTT assay, respectively. As shown on Figure MYD88wt cells, the combination induced a 16% augmenta- 3B, both IRAKi and CPI203 were able to block actin poly- tion in relative apoptotic cell death in the merization by 50.4% and 54.5%, respectively, while the MYD88 L265P primary co-culture (Figure 3A, left panel), drug combination achieved a total 77.9% decrease in cells which was accompanied by a 12% decrease in the fraction with high contents in F-actin following stimulation of A B C D Figure 1. Limited activity of IRAKi single agent in activated B-cell - diffuse large B cell lymhoma cell lines with MYD88 L265P in relation with incom- plete inhibition of NF-κB gene signatures. (A) MTT assay showing that IRAKi (50 mM) elicited a partial and transitory response in activated B-cell - diffuse large B cell lymhoma (ABC-DLBCL) cell lines, while germinal center B-cell (GCB)-DLBCL cell lines were almost completely resistant to the compound. (B) IRAKi efficiently blocked the phosphorylation of IRAK1 at Thr29 and IRAK4 at Thr345, in the three ABC-DLBCL cell lines with MYD88 L265P. b-actin was used as a loading control. (C) Gene expression signatures of NF-κB in HBL-1, OCI-Ly3 and OCI-Ly10 cell lines exposed to IRAKi as above, highlighting that IRAKi treatment slightly affects this pathway (note that only 2 out 3 gene sets showed a false discovery rate [FDR] below 0.05). NES: normalized enrichment score. (D) Quantitative reverse transcriptase polymerare chain reaction (RQ- PCR) analysis of the predominant NF-κB-regulated genes IL6, IL10, CCL3, and IRF4, and the IRAK1/4 target gene MCL-1 in ABC-DLBCL cell treated by IRAKi as before (*P=0.01; **P<0.001). 2750 haematologica | 2021; 106(10) Letters to the Editor A B C D E Figure 2. The BETi CPI203 synergizes with IRAKi in activated B-cell - diffuse large B cell lymphoma mediated by the inhibition of NF-κB downstream pathways. (A) Enrichment plots from gene set enrichment analysis (GSEA) analysis comparing IRAKi single agent vs. IRAKi-CPI203 combo in the 3 cell lines treated for 6 hours (Affymetrix HG-U219; GSEA), showing a significant improvement of NF-κB signature decrease by the addition of CPI203 to IRAKi. (B) Quantitative reverse transcriptase polymerare chain reaction (RQ-PRC) analysis of NF-κB downstream genes in the three cell lines exposed to IRAKi, CPI203 or CPI203-IRAKi combo as before. (*P=0.01; **P<0.001). (C) CPI203-IRAKi combination led to intracellular accumulation of IκB, and subsequent downregulation of IRAK1, MYC, CD44, MARCKS and MCL-1 proteins in activated B-cell - diffuse large B cell lymphoma (ABC-DLBCL) cells with MYD88L265P. (D) OCI-Ly3, OCI-Ly10, HBL-1 cells were exposed for 24 hours to 0.1-0.5 mM CPI203 and/or 50-500 mM IRAKi. Cytotoxicity was evaluated by MTT assay and combination index (CI) was determined using the Calcusyn software. Shown are the cytotoxicity and the mean CI value calculated for cell treatment with 0.5 mM CPI203 and 50 mM IRAKi. (E) The drug com- bination led to a synergistic antitumoral effect in vitro in these 3 cell lines, inducing a median 36% increase in apoptosis rate when compared to single agent treatments (*P<0.04). haematologica | 2021; 106(10) 2751 Letters to the Editor A B C D Figure 3. IRAKi and CPI203 combination is active in activated B-cell - diffuse B-cell lymphoma primary cultures and impairs tumor growth in vivo. (A) Left panel: antitumoral activity of CPI203 (0.5 mM) and/or IRAKi (50 mM) was evaluated after a 24-hour culture of primary lymph node biopsies from activated B-cell - diffuse B-cell lymphoma patients with either MYD88wt or MYD88 L265P by cytofluorimetric quantification of AnnexinV+ cells. Cell of origin (COO) and MYD88 mutational status of the patients were determined by allele-specific polymerase chain reaction and gene expression analysis, as previously.15 Right panel: DLBCL cultures treated as above were labeled with an IL-6 Hu-Cyanine 5 SmartFlare RNA detection probe (Merck Millipore), and percentage of viable cells with high contents in IL6 mRNA was determined by flow cytometry, as previously.16 (B) HBL-1 y OCI-LY3 cell lines were preincubated for 24 hours with 0.5 mM CPI203 and/or 50 mM IRAKi, followed by a 24-hour stimulation with 0.5 mM HA, labeling with 50 mM Phalloidin-TRITC (Sigma-Aldrich) and recounting of red fluorescent cells on a Nikon H5505 microscope by means of a 20X/1.30 NA oil objective (Nikon) with the use of Isis Imaging System v5.3 software (MetaSystems GmbH) (***P<0.001). (C) NOD/SCID IL2Rγ-null (NSG) mice were inoculated subcutaneously with 107 OCI-LY3 cells and after 13 days, tumor-bearing animals (n=5 mice per group) received intraperitoneal (i.p.) injection of 2.5 mg/kg CPI203 (BID) and/or i.p. administration of 7.5 mg/kg IRAKi (BID), or an equal volume of vehicle, for 11 days, in a five/two (on/off) schedule. Tumor volumes were measured each 2-3 days with external calipers. (D) Immunohistological analysis of consecutive tumor sections from representative animals reveals a notable decrease in mitotic index and in the NF-κB-regulated CD44, as well as a strong downregulation of MCL-1 and induction of apoptosis in IRAKi-CPI203 combo group. 2752 haematologica | 2021; 106(10) Letters to the Editor CD44 by HA. In contrast, the anti-IL-6 antibody failed to Disclosures: GR received research support from Celgene Corp and sensitize ABC-DLBCL cells to IRAKi-based treatment TG Therapeutics. CPI203 was kindly provided by Constellation (Online Supplementary Figure S1B). Thus, these results sug- Pharmaceuticals. gest a significant activity of the drug combination towards Contributions: ID and MA performed experiments, analyzed data CD44 downstream signaling, while IL-6 expression may not be directly involved in the effect of these agents. and co-wrote the manuscript; CRZ provided support in animal studies; Finally, in order to assess the efficacy of the drug combi- MLR interpreted the results and reviewed the manuscript; PP-G nation in vivo, NSG mice were subcutaneously injected helped in analyzing the gene expression data; FB evaluated the data with OCI-LY3 cells, and tumor-bearing animals received and reviewed the manuscript critically; AL-G and GR conceived and daily doses of either IRAKi (5 mg/kg, intraperitoneal [i.p.], designed the study, analyzed data and wrote the manuscript. BID), CPI203 (2.5 mg/kg, i.p., BID), the combination of both agents, or the equivalent volume of vehicle, for 11 References days. Figure 3C shows that CPI203 and IRAKi single agents induced a 31.5% and 46.3% tumor growth 1. Pasqualucci L. Molecular pathogenesis of germinal center-derived B inhibition (TGI), respectively, while the combination of cell lymphomas. Immunol Rev. 2019;288(1):240-261. both drugs significantly improved this effect with a 65.6% 2. Miyazaki K. Treatment of diffuse large B-cell lymphoma. J Clin Exp TGI, when compared to vehicle-receiving animals Hematopathol. 2016;56(2):79-88. (*P=0.011; **P=0.007). No significant toxicity was observed 3. Chapuy B, Stewart C, Dunford AJ, et al. Molecular subtypes of dif- fuse large B cell lymphoma are associated with distinct pathogenic in any of the treatment arms. Histological analysis of the mechanisms and outcomes. Nat Med. 2018;24(5):679-690. corresponding tumors revealed an improved reduction of 4. Ngo VN, Young RM, Schmitz R, et al. Oncogenically active MYD88 mitotic index together with an accumulation of apoptotic mutations in human lymphoma. Nature. 2010;470(7332):115-119. cells by the combination therapy, as assessed by phospho- 5. Kelly PN, Romero DL, Yang Y, et al. Selective interleukin-1 receptor– histone H3 and activated-caspase-3 staining (Figure 3D). In associated kinase 4 inhibitors for the treatment of autoimmune dis- agreement with the orders and lymphoid malignancy. J Exp Med. 2015;212(13):2189- in vitro results, an enhanced reduction in the levels of CD44 2201. and MCL-1, and an improved downregulation of nuclear 6. Knittel G, Liedgens P, Korovkina D, et al. B-cell–specific conditional p50 used as a read of NF-κB activity, was observed in the expression of Myd88p.L252P leads to the development of diffuse combination group when compared with the other arms large B-cell lymphoma in mice. Blood. 2016;127(22):2732-2741. (Figure 3D). 7. Wang Z, Sun D, Johnstone S, et al. Discovery of potent, selective, and Collectively, our results suggest that IRAK1/4 inhibition orally bioavailable inhibitors of interleukin-1 receptor-associate is modestly effective in in vitro and in vivo models of ABC- kinase-4. Bioorg Med Chem Lett. 2015;25(23):5546-5550. DLBCL with MYD88 L265P, achieving only a partial inhi- 8. Li Z, Younger K, Gartenhaus R, et al. Inhibition of IRAK1/4 sensitizes bition of NF-κB signaling. We confirm that BET inhibition T cell acute lymphoblastic leukemia to chemotherapies. J Clin Invest. 2015;125(3):1081-1097. is an efficient strategy to counteract NF-κB over-activation 9. Lam LT, Davis RE, Pierce J, et al. Small molecule inhibitors of IκB in these models, offering synergistic anti-tumoral and pro- kinase are selectively toxic for subgroups of diffuse large B-cell lym- apoptotic activities with IRAK inhibition, mediated by the phoma defined by gene expression profiling. Clin Cancer Res. downregulation of the NF-κB-regulated factors, CD44 and 2005;11(1):28-40. MCL-1, and the consequent blockade of cell motility and 10. Ceribelli M, Kelly PN, Shaffer AL, et al. Blockade of oncogenic IκB triggering of tumor cell death. kinase activity in diffuse large B-cell lymphoma by bromodomain and extraterminal domain protein inhibitors. Proc Natl Acad Sci U S Ivan Dlouhy,1,2* Marc Armengol,3* Clara Recasens-Zorzo,2 A. 2014;111(31):11365-11370. Marcelo L. Ribeiro,3,4 Patricia Pérez-Galán,2 Francesc Bosch,5 11. Boi M, Gaudio E, Bonetti P, et al. The BET bromodomain inhibitor Armando López-Guillermo1,2# and Gaël Roué3# OTX015 affects pathogenetic pathways in preclinical B-cell tumor 1 Department of Hematology, Hospital Clínic, BarceIona, Spain; models and synergizes with targeted drugs. Clin Cancer Res. 2 2015;21(7):1628-1638. Division of Hematology and Oncology, Institut d’Investigacions 12. Esteve-Arenys A, Valero JG, Chamorro-Jorganes A, et al. The BET Biomèdiques August Pi i Sunyer (IDIBAPS), CIBERONC, Barcelona, bromodomain inhibitor CPI203 overcomes resistance to ABT-199 Spain; 3Lymphoma Translational Group, Josep Carreras Leukemia (venetoclax) by downregulation of BFL-1/A1 in in vitro and in vivo Research Institute (IJC), Badalona, Spain; 4Post Graduate Program in models of MYC+/BCL2+ double hit lymphoma. Oncogene. Health Science, Universidade São Francisco (USF), Bragança Paulista, 2018;37(14):1830-1844. Brazil and 5Laboratory of Experimental Hematology, Department of 13. Wei X, Xu M, Wei Y, et al. The addition of rituximab to CHOP ther- Hematology, Vall d'Hebron Institute of Oncology (VHIO), Vall apy alters the prognostic significance of CD44 expression. J Hematol d’Hebron University Hospital, Universitat Autònoma de Barcelona, Oncol. 2014;7:34. Barcelona, Spain 14. Dlouhy I, Filella X, Rovira J, et al. High serum levels of soluble inter- *ID and MA contributed equally as co-first authors. leukin-2 receptor (sIL2-R), interleukin-6 (IL-6) and tumor necrosis fac- # tor alpha (TNF) are associated with adverse clinical features and pre- ALG and GR contributed equally as co-senior authors. dict poor outcome in diffuse large B-cell lymphoma. Leuk Res. Correspondence: 2017;59:20-25. 15. Rovira J, Karube K, Valera A, et al. MYD88 L265P mutations, but no GAËL ROUÉ - groue@carrerasresearch.org other variants, identify a subpopulation of DLBCL patients of acti- doi:10.3324/haematol.2020.278258 vated B-cell origin, extranodal involvement, and poor outcome. Clin Received: December 25, 2020. Cancer Res. 2016;22(11):2755-2764. 16. Vaidyanathan S, Friend S, Weldon D, Morrissey P. Measurement of Accepted: May 5, 2021. IL-6 levels in live cells using RNA detection probe with Imaging Flow Pre-published: May 13, 2021. Cytometer. J Immunol. 2016;196(Suppl 1):S196. haematologica | 2021; 106(10) 2753