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. 2021 Oct 1;106(10):2749-2753.
doi: 10.3324/haematol.2020.278258.

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

Ivan Dlouhy  1 , Marc Armengol  2 , Clara Recasens-Zorzo  3 , Marcelo L Ribeiro  4 , Patricia Pérez-Galán  3 , Francesc Bosch  5 , Armando López-Guillermo  1 , Gaël Roué  6
Affiliations

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

Ivan Dlouhy et al. Haematologica. .

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Figures

Figure 1.
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 incomplete 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 (RQPCR) 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).
Figure 2.
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 combination 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).
Figure 3.
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. 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. (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.
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