Ession [22, 23]. Accordingly, lentiviralmediated overexpression of order A-836339 miR-125b-5p inhibits IRF4 and increases MICA expression in MM cells, extending the possible immunoregulatory role of miR-125b-5p as promising anti-MM effector.Finally, selective CREB-binding protein/E1A interacting protein of 300 kDa (CBP/EP300) bromodomain inhibition, already shown to affect MM viability through transcriptional suppression of IRF4 [24], recapitulated the observations obtained using pan-BETi on MICA expression, adding novel information on the possible immuno-therapeutic applications of this class of bromodomain inhibitors and transcriptional coactivators in MM. In conclusion, these findings provide new insights on the immuno-mediated antitumor activities of BETi and further elucidate the molecular mechanisms that regulate NK cell-activating ligand expression in MM.ResultsBETi upregulate MICA expression on human multiple myeloma cells and enhance their recognition by NK cellsWe investigated the ability of BETi to regulate the expression of NK cell-activating ligands in MM. To this purpose, a panel of human MM cell lines [SKO-007(J3), RPMI-8226, U266, ARP-1, and JJN3] were treated with a range of concentrations and at different times with JQ1 or I-BET151, two small molecule bromodomain inhibitors displaying potent binding affinity to BET family proteins [25], and analyzed for the expression of NKG2DLs and DNAM-1Ls, by FACS analysis and qRT-PCR. As shown in Fig. 1a (and Additional file 1), these drugs upregulated the basal expression of the NKG2D ligand MICA in different MM cell lines, with no significant effects on MICB and PVR/CD155 levels. Concerning the other NKG2D and DNAM-1/ CD226 ligands, SKO-007(J3) cells express low or undetectable levels of ULBP2/5/6 or ULBP1, ULBP3, and Nec-2 respectively and the treatment with BETi did not modify their cell surface levels (Additional file 2). These treatments did not affect the cell viability of these cell lines after 72 h treatment, as assessed by PI staining (data not shown). To examine whether the upregulation of MICA by BETi could be associated with increased mRNA levels, total RNA was isolated from the different cell lines exposed to JQ1 or I-BET151 and analyzed by real-time qRT-PCR. As shown in Fig. 1b and Additional file 3, we noticed a significant PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26866270 increase of MICA mRNA levels in treated cells, in particular after 48 h. In addition, we confirmed these results also in CD138+ MM cells isolated from the bone marrow of MM patients (Table 1), showing higher cell surface and mRNA levels of MICA following treatment with JQ1 or I-BET151 (Fig. 2a, b). Of note, these drugs did not show a significant effect on either MICB or PVR/CD155, independently from the clinical status of the disease and from basal expression levels. To investigate the functional consequences of BETiinduced changes of MICA expression, we analyzed the lysosomal marker CD107a (a surrogate marker for granule mobilization [26]) on NK cells isolated from healthy donors against SKO-007(J3) cells, untreated or treated with JQ1 orAbruzzese et al. Journal of Hematology Oncology (2016) 9:Page 4 ofa80 60 40 20MICAJQSKOMICAI-BETMFI*n.s.MICA MICBMFIUntreated JQ1 n.s.60 40 20*Untreated I-BET n.s. n.s.PVR/CDMICAMICBPVR/CDbUntreatedJQ1 24hMICAUntreated* *0 1 2 3 4I-BET 24h* *0 1 2 3 4JQ1 48hI-BET 48hmRNA (fold change)mRNA (fold change)Fig. 1 BETi upregulate MICA expression in SKO-007(J3) human MM cells. a MICA, MICB, and PVR/CD155 cell surface expression wer.
