title: Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay.
abstract: Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells.
  
  
  
  Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138+MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays.
  
  
  
  Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi-mediated inhibition of cMYC correlates with the upregulation of miR-125b-5p and the downregulation of the cMYC/miR-125b-5p target gene IRF4, a transcriptional repressor of MICA.
  
  
  
  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.
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Abruzzese
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Bilotta
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Fionda
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Zingoni
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Soriani
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Vulpis
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.|Center for Life Nano Science @ Sapienza, Italian Institute of Technology, Sapienza University of Rome, Rome, Italy.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Borrelli
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Zitti
authors.affiliation: Division of Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Petrucci
authors.affiliation: Hematology, Department of Clinical and Molecular Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Ricciardi
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Molfetta
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Paolini
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy. angela.santoni@uniroma1.it.|Istituto Pasteur-Fondazione Cenci Bolognetti, Rome, Italy. angela.santoni@uniroma1.it.|Istituto Mediterraneo di Neuroscienze Neuromed, Pozzilli, Italy. angela.santoni@uniroma1.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Santoni
authors.affiliation: Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy. marco.cippitelli@uniroma1.it.
authors.forename: Marco
authors.identifier: 0000-0002-9620-538X
authors.initials: M
authors.lastname: Cippitelli
mesh_terms: D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic; D015972:Gene Expression Regulation, Neoplastic; D015395:Histocompatibility Antigens Class I / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D050835:Interferon Regulatory Factors / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology*; D035683:MicroRNAs / Q000378:metabolism; D009101:Multiple Myeloma / Q000276:immunology / Q000378:metabolism*; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D000099218:Interferon Regulatory Factor-4; D000092468:Bromodomain Containing Proteins
chemical_list: D015395:Histocompatibility Antigens Class I; D050835:Interferon Regulatory Factors; D035683:MicroRNAs; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; D000099218:Interferon Regulatory Factor-4; C527085:KLRK1 protein, human; C099187:MHC class I-related chain A; C531888:MIRN125 microRNA, human; C489427:MYC protein, human; C000624329:bromodomain and extra-terminal domain protein, human; D000092468:Bromodomain Containing Proteins
keywords: Bromodomain; IRF4; Multiple myeloma; NKG2DLs; Natural killer