ATRA activates the expression of IFN-β response genes that are important for promoting Cfz-induced MM-killing effect. (A) Microarray data of MM.1S cells treated with ATRA+Cfz or Cfz for 12 hours were analyzed by GSEA. The top 6 representative Hallmark gene sets (with indicated colors) based on normalized enrichment score (NES) from data of ATRA+Cfz (positively correlated, red)- vs Cfz (negatively correlated, blue)-treated MM.1S cells are shown. (B) Summarized flow data showing apoptosis of MM.1S or ARP-1 cells pulsed with 1-hour Cfz treatment followed by 50 U/mL IFN-β (12.5 pM) or IFN-γ (150 pM) for 24 hours. (C) Histogram showing CI value for the combination of 50 U/mL IFN-β or IFN-γ with 30 nM Cfz in MM.1S or 100 nM ARP-1 cells. Each column represents the mean CI value obtained from 3 independent experiments plotted against the corresponding fraction affected (Fa) by the combination treatment in panel B. Apoptotic rates of MM cells treated with single-agent Cfz, IFN-β, or IFN-γ in serial dilution were obtained for calculating CI value of their combination therapy shown in supplemental Table 4. CI < 1 represents synergism; CI = 1 represents additive effect; and CI > 1 represents antagonism. (D) Heat map illustrating the log2-fold change of the genes involved in IFN-β response pathways. The color bar indicates gene expression value. (E) Western blot displaying protein expression of IRF1 and OAS1-3 in MM.1S or ARP-1 cells with indicated treatment for 14 hours. (F) Western blot showing IRF1 expression in Ctrl-KD or IRF1-KD MM cell lines with DMSO or ATRA treatment for 14 hours. (G) Summarized data showing apoptosis of Ctrl-KD or IRF1-KD MM cells with indicated treatment of 24 hours. Student t test was used to compare 2 samples, *P < .05; **P < .01.