Stograms represent the expression after treatment with the drug. Data are representative of one out of four independent experiments. g ARV-825 increases mRNA expression of MICA in SKO-007(J3) cells. Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with the indicated (M) concentrations of ARV-825 for 48 h. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells considered as calibrator and represent the mean of three experiments (*P < 0.05). h MICA cell surface expression was analyzed by flow cytometry on patient-derived MM cells treated with the indicated ARV-825 for 72 h. The grey-colored histograms represent basal expression, while thick black histograms represent the expression after treatment with the drugminimal MICA/-270 bp promoter fragment enhanced its activity, further confirming the repressive activity of IRF4 at this level (Fig. 5).Interestingly, the expression of IRF4 has been linked to the activity of the transcription factors IKZF1 and IKZF3 [28, 29, 47], two recently identified repressors ofAbruzzese et al. Journal of Hematology Oncology (2016) 9:Page 12 ofabcdefFig. 8 CBP/EP300-BRi upregulate MICA expression in SKO-007(J3) MM cells. a, b MICA, MICB, and PVR/CD155 cell surface expression were analyzed by flow cytometry on SKO-007(J3) cells treated with CBP30 (1 or 2 M) for 72 h. In a, a representative histogram of MICA upregulation AZD3759 web compared to MICB and PVR/CD155 is shown (2 M). The MFI of MICA, MICB, and PVR/CD155 was calculated based on at least four independent experiments and evaluated by paired Student t test (*P < 0.05). c Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with CBP30 or JQ1 as described above for 48 h. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells considered as calibrator and represent the mean of three experiments (*P < 0.05). d, e CBP/EP300-BRi inhibits mRNA expression of IRF4 and cMYC. Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with CBP30 or JQ1 as described above, for 48 h. Data, expressed as fold change units, were normalized with GAPDH and referred to the untreated cells considered as calibrator and represent the mean of three experiments (*P < 0.05). f MICA cell surface expression was analyzed by flow cytometry on SKO-007(J3) cells treated with C646 (5 M) for 72 h. The MFI of MICA was calculated based on at least three independent experiments and evaluated by paired Student t test (*P < 0.05)MICA gene expression [13]. However, in our experimental system, treatment of SKO-007(J3) cells with JQ1 only slightly decreased the expression of the IKZF1 and IKZF3 (Additional file 6A ); as a control, lenalidomide completely abrogated the expression of these two transcription factors, further suggesting that the upregulation of MICA mediated by BETi was mainly dependent on the repression of IRF4. Noteworthy, as shown in Additional file 6E, the combination of the two treatments (lenalidomide + BETi) further increased the expression of MICA, suggesting the possibility that low-dose combinations ofIMiDs with BETi could display additional or synergistic activities in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/25432023 MM, as already shown in primary effusion lymphoma (PEL) preclinical data, where the simultaneous targeting of IKZF1-IRF4-MYC increased the cytotoxic effect as compared with either agent alone [48]. In this context, in.