Cells, with a mechanism dependent on the activation of the NKG2D receptor, either using cultivated NK cells from healthy donors or autologous patient-derived NK cells (Fig. 3). Mechanistically, we found that treatment of MM cells with BETi upregulates the activity of the MICA promoter; moreover, using progressive deletions, we identified a minimal promoter fragment spanning from -270 bp still responsive to BETi (Fig. 4). Treatment of MM cells with BETi downregulated the expression of cMYC and IRF4 (Fig. 4 and MK-1439 cost Additional file 4), the latter being a transcriptional target of cMYC, and recently identified by our laboratory as a “IMiDs druggable” transcriptional repressor of MICA in MM cells [13]. In this context,cMYC shRNA-transduced cells expressed higher MICA surface levels, whereas MICB and PVR/CD155 membrane expression were unaffected. Accordingly, real-time qRT-PCR analysis showed that silencing of cMYC enhances MICA mRNA expression and represses IRF4 (Additional file 5). IRF4 can positively and negatively regulate different genes, in part by binding to distinct DNA binding motifs and through interaction with various additional transcription factors [27]. Its C-terminal transactivation domain is critical for gene activation, while the mechanism(s) responsible for gene repression are not well defined. In our experimental system, the overexpression of IRF4 by lentiviral-mediated transduction partially abrogated the induction of MICA after treatment with BETi. Moreover, the overexpression of a dominant negative form of IRF4 induced significant upregulation of MICA promoter, and the deletion of a putative IRF4 binding element in theAbruzzese et al. Journal of Hematology Oncology (2016) 9:Page 11 ofabcdefghFig. 7 The PROTAC/BRD4 degrader, ARV-825, upregulates MICA expression in SKO-007(J3) cells. a Treatment of SKO-007(J3) cells with ARV-825 induced a strong degradation of BRD4 that was reverted in the presence of lenalidomide. Lysates of SKO-007(J3) cells, untreated or treated with ARV-825 (0.2 M), lenalidomide (10 M), or the combination of the two drugs for 24 h, were subjected to Western blotting using anti-BRD4 or anti-p85 antibodies. b ARV-825 inhibits the expression of IRF4. Lysates of SKO-007(J3) cells, untreated or treated with the indicated (M) concentrations of ARV-825 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 for 48 h, were subjected to Western blotting using anti-IRF4 or anti-actin antibodies. The proteins transferred to nitrocellulose membranes were stained with Ponceau to verify that similar amounts of proteins had been loaded in each lane. Data are representative of one out of three independent experiments. c, d ARV-825 inhibits mRNA expression of IRF4 and cMYC. 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). e, f MICA, MICB, and PVR/155 cell surface expression were analyzed by flow cytometry on SKO-007(J3) cells treated with the indicated concentrations of ARV-825 for 72 h. The MFI of MICA, MICB, and PVR/155 were calculated based on at least four independent experiments and evaluated by paired Student t test (*P < 0.05). In e, a representative histogram of MICA upregulation is shown. The grey-colored histograms represent basal expression of the indicated ligand, while thick black hi.