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Letters to the Editor
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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.
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haematologica | 2021; 106(10)