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Figure S3. MEL-treated MM cells induce NK cells entry in the S phase of the cell cycle.

Fig S2. MM patients’ malignant PCs treated with Bortezomib and Lenalidomide do not express augmented levels of the IL-15/IL-15RA complex

Fig S4. MEL-treated ARK cells elicit NK cell activation and proliferation by IL-15 trans-presentation

Treatment of multiple myeloma (MM) cells with sublethal doses of genotoxic drugs leads to senescence and results in increased NK cell recognition and effector functions. Herein, we demonstrated that doxorubicin- and melphalan-treated senescent cells display increased expression of IL15, a cytokine involved in NK cell activation, proliferation, and maturation. IL15 upregulation was evident at the mRNA and protein level, both in MM cell lines and malignant plasma cells from patients’ bone marrow (BM) aspirates. However, IL15 was detectable as a soluble cytokine only in vivo, thus indicating a functional role of IL15 in the BM tumor microenvironment. The increased IL15 was accompanied by enhanced expression of the IL15/IL15RA complex on the membrane of senescent myeloma cells, allowing the functional trans-presentation of this cytokine to neighboring NK cells, which consequently underwent activation and proliferation. We demonstrated that MM cell–derived exosomes, the release of which was augmented by melphalan treatment in senescent cells, also expressed IL15RA and IL15, and their interaction with NK cells in the presence of exogenous IL15 resulted in increased proliferation. Altogether, our data demonstrated that low doses of chemotherapeutic drugs, by inducing tumor cell senescence and a senescence-associated secretory phenotype, promoted IL15 trans-presentation to NK cells and, in turn, their activation and proliferation, thus enhancing NK cell–tumor immune surveillance and providing new insights for the exploitation of senescence-based cancer therapies. Cancer Immunol Res; 6(7); 860–9. ©2018 AACR.

Fig S5. MEL-treated SKO-007(J3) and ARK exosomes display a similar IL-15RA expression.

Fig S1. MM cells acquire a senescent phenotype upon genotoxic drug treatment.

Supplemental Table 2. Patient characteristics. Analysis of patient characteristics used in the set of experiments in Figure 6. Patients were classified according to the state of the disease. PC=plasma cell.

Table S1. Patient characteristics Analysis of patient characteristics used in the set of experiments in Figure 3. Ig=immunoglobulin; PC=plasma cell.

New technologies with the capacity to tune immune system activity are highly desired in clinical practice and disease management. Here we demonstrate that nanoparticles with a protein corona enriched with gelsolin (GSN), an abundant plasma protein that acts as a modulator of immune responses, are avidly captured by human monocytic THP-1 cells in vitro and by leukocyte subpopulations derived from healthy donors ex vivo. In human monocytes, GSN modulates the production of tumor necrosis factor alpha (TNF-α) in an inverse dose-dependent manner. Overall, our results suggest that artificial coronas can be exploited to finely tune the immune response, opening new approaches for the prevention and treatment of diseases.

The success of senescence-based anticancer therapies relies on their anti-proliferative power and on their ability to trigger anti-tumor immune responses. Indeed, genotoxic drug-induced senescence increases the expression of NK cell-activating ligands on multiple myeloma (MM) cells, boosting NK cell recognition and effector functions. Senescent cells undergo morphological change and context-dependent functional diversification, acquiring the ability to secrete a vast pool of molecules termed the senescence-associated secretory phenotype (SASP), which affects neighboring cells. Recently, exosomes have been recognized as SASP factors, contributing to modulating a variety of cell functions. In particular, evidence suggests a key role for exosomal microRNAs in influencing many hallmarks of cancer. Herein, we demonstrate that doxorubicin treatment of MM cells leads to the enrichment of miR-433 into exosomes, which in turn induces bystander senescence. Our analysis reveals that the establishment of the senescent phenotype on neighboring MM cells is p53- and p21-independent and is related to CDK-6 down-regulation. Notably, miR-433-dependent senescence does not induce the up-regulation of activating ligands on MM cells. Altogether, our findings highlight the possibility of miR-433-enriched exosomes to reinforce doxorubicin-mediated cellular senescence.

Immune checkpoint inhibitors (ICIs) are largely used in the treatment of patients with advanced non-small-cell lung cancer (NSCLC). Novel biomarkers that provide biological information that could be useful for clinical management are needed. In this respect, extracellular vesicles (EV)-associated microRNAs (miRNAs) that are the principal vehicle of intercellular communication may be important sources of biomarkers. We analyzed the levels of 799 EV-miRNAs in the pretreatment plasma of 88 advanced NSCLC patients who received anti-PD-1 therapy as single agent. After data normalization, we used a two-step approach to identify candidate biomarkers associated to both objective response (OR) by RECIST and longer overall survival (OS). Univariate and multivariate analyses including known clinicopathologic variables and new findings were performed. In our cohort, 24/88 (27.3%) patients showed OR by RECIST. Median OS in the whole cohort was 11.5 months. In total, 196 EV-miRNAs out 799 were selected as expressed above background. After multiplicity adjustment, abundance of EV-miR-625-5p was found to be correlated with PD-L1 expression and significantly associated to OR by RECIST (p = 0.0366) and OS (p = 0.0031). In multivariate analysis, PD-L1 staining and EV-miR-625-5p levels were constantly associated to OR and OS. Finally, we showed that EV-miR-625-5p levels could discriminate patients with longer survival, in particular in the class expressing PD-L1 ≥50%. EV-miRNAs represent a source of relevant biomarkers. EV-miR-625-5p is an independent biomarker of response and survival in ICI-treated NSCLC patients, in particular in patients with PD-L1 expression ≥50%.

Natural killer (NK) cells are innate lymphoid cells that play a major role in the immune surveillance against tumors and their activity is regulated through signals derived by a number of NK cell inhibitory and activating receptors as well as cytokines and other soluble factors released in the tumor microenvironment. Extracellular vesicles (EVs) are membrane-enclosed particles secreted by all cell types, both in healthy and diseased conditions, and are important mediators of intercellular communication. Depending on the molecular cargo, tumor-derived extracellular vesicles have the capability to either promote or suppress NK cell-mediated functions. Anti-cancer therapies designed to sustain host anti-tumor immune response represent an appealing strategy to control tumor growth avoiding tumor immune escape. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Moreover, the activation of the DNA damage response (DDR) and the induction of senescence represent two crucial modalities aimed at promoting the clearance of drug-treated tumor cells by NK cells. Herein, we will address the main mechanisms used by cancer-derived extracellular vesicles to modulate NK cell activity, and we will discuss how anti-cancer therapies might impact on the secretion and the immunomodulatory function of these vesicles.

For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes’ synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or “proteoDNAsome”, which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity.

Trogocytosis modulates immune responses, with still unclear underlying molecular mechanisms. Using leukemia mouse models, we found that lymphocytes perform trogocytosis at high rates with tumor cells. While performing trogocytosis, both Natural Killer (NK) and CD8+T cells acquire the checkpoint receptor PD-1 from leukemia cells. In vitro and in vivo investigation revealed that PD-1 on the surface of NK cells, rather than being endogenously expressed, was derived entirely from leukemia cells in a SLAM receptor–dependent fashion. PD-1 acquired via trogocytosis actively suppressed NK cell antitumor immunity. PD-1 trogocytosis was corroborated in patients with clonal plasma cell disorders, where NK cells that stained for PD-1 also stained for tumor cell markers. Our results, in addition to shedding light on a previously unappreciated mechanism underlying the presence of PD-1 on NK and cytotoxic T cells, reveal the immunoregulatory effect of membrane transfer occurring when immune cells contact tumor cells.

RIG-I is a cytosolic RNA sensor that recognizes short 5 ' triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)-calreticulin, HMGB1 and ATP-and high levels of ICD-related cytokines CXCL10, IFN beta, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.

Nanomedicine has demonstrated a substantial role in vaccine development against severe acute respiratory syndrome coronavirus (SARS-CoV-2 and COVID-19). Although nanomedicine-based vaccines have now been validated in millions of individuals worldwide in phase 4 and tracking of sex-disaggregated data on COVID-19 is ongoing, immune responses that underlie COVID-19 disease outcomes have not been clarified yet. A full understanding of sex-role effects on the response to nanomedicine products is essential to building an effective and unbiased response to the pandemic. Here, we exposed model lipid nanoparticles (LNPs) to whole blood of 18 healthy donors (10 females and 8 males) and used flow cytometry to measure cellular uptake by circulating leukocytes. Our results demonstrated significant differences in the uptake of LNP between male and female natural killer (NK) cells. The results of this proof-of-concept study show the importance of recipient sex as a critical factor which enables researchers to better consider sex in the development and administration of vaccines for safer and more-efficient sex-specific outcomes.

Leucocytes often perform trogocytosis, the process by which cells acquire parts of the plasma membrane from interacting cells. Accumulating evidence indicates that trogocytosis modulates immune responses, but the underlying molecular mechanisms are unclear. Here, using two mouse models of leukemia, we found that cytotoxic lymphocytes perform trogocytosis at high rates with tumor cells. While performing trogocytosis, both Natural Killer and CD8+ T cells acquire the checkpoint receptor PD-1 from leukemia cells. In vitro and in vivo investigation revealed that PD-1 protein found on the surface of Natural Killer cells, rather than being endogenously expressed, was derived entirely from leukemia cells. Mechanistically, SLAM receptors were essential for PD-1 trogocytosis. PD-1 acquired via trogocytosis actively suppressed anti-tumor immunity, as revealed by the positive outcome of PD-1 blockade in PD-1-deficient mice. PD-1 trogocytosis was corroborated in patients with clonal plasma cell disorders, where Natural Killer cells that stained for PD-1 also stained for tumor cell markers. Our results, in addition to shedding light on a previously unappreciated mechanism underlying the presence of PD-1 on Natural Killer and cytotoxic T cells, reveal the immune-regulatory effect of membrane transfer occurring when immune cells contact tumor cells.Once sentence summaryNatural Killer cells are inhibited by PD-1 acquired from the surface of tumor cells via trogocytosis.

Treatment of multiple myeloma (MM) cells with sublethal doses of genotoxic drugs leads to senescence and results in increased NK cell recognition and effector functions. Herein, we demonstrated that doxorubicin- and melphalan-treated senescent cells display increased expression of IL15, a cytokine involved in NK cell activation, proliferation, and maturation. IL15 upregulation was evident at the mRNA and protein level, both in MM cell lines and malignant plasma cells from patients’ bone marrow (BM) aspirates. However, IL15 was detectable as a soluble cytokine only in vivo, thus indicating a functional role of IL15 in the BM tumor microenvironment. The increased IL15 was accompanied by enhanced expression of the IL15/IL15RA complex on the membrane of senescent myeloma cells, allowing the functional trans-presentation of this cytokine to neighboring NK cells, which consequently underwent activation and proliferation. We demonstrated that MM cell–derived exosomes, the release of which was augmented by melphalan treatment in senescent cells, also expressed IL15RA and IL15, and their interaction with NK cells in the presence of exogenous IL15 resulted in increased proliferation. Altogether, our data demonstrated that low doses of chemotherapeutic drugs, by inducing tumor cell senescence and a senescence-associated secretory phenotype, promoted IL15 trans-presentation to NK cells and, in turn, their activation and proliferation, thus enhancing NK cell–tumor immune surveillance and providing new insights for the exploitation of senescence-based cancer therapies. Cancer Immunol Res; 6(7); 860–9. ©2018 AACR.

NK cells are innate lymphocytes with a key role in the immune response against cancer cells. NK cell killing of tumor cells is activated by the integration of inhibitory and activating intracellula...

Natural killer (NK) cells are innate lymphoid cells that play a pivotal role in tumor surveillance. Exosomes are nanovesicles released into the extracellular environment via the endosomal vesicle pathway and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Moreover, the activation of the DNA damage response (DDR) and the induction of senescence represent two crucial modalities aimed at promoting the clearance of drug-treated tumor cells by NK cells. Emerging evidence has shown that stress stimuli provoke an increased release of exosome secretion. Remarkably, tumor-derived exosomes (Tex) produced in response to stress carry distinct type of DAMPs that activate innate immune cell populations. Moreover, stress-induced ligands for the activating receptor NKG2D are transported by this class of nanovesicles. Here, we will discuss how Tex interact with NK cells and provide insight into their potential role in response to chemotherapy-induced stress stimuli. The capability of some “danger signals” carried by exosomes that indirectly affect the NK cell activity in the tumor microenvironment will be also addressed.

In vivo liposomes, like other types of nanoparticles, acquire a totally new 'biological identity' due to the formation of a biomolecular coating known as the protein corona that depends on and modifies the liposomes' synthetic identity. The liposome-protein corona is a dynamic interface that regulates the interaction of liposomes with the physiological environment. Here we show that the biological identity of liposomes is clearly linked to their sequestration from peripheral blood mononuclear cells (PBMCs) of healthy donors that ultimately leads to removal from the bloodstream. Pre-coating liposomes with an artificial corona made of human plasma proteins drastically reduces capture by circulating leukocytes in whole blood and may be an effective strategy to enable prolonged circulation in vivo. We conclude with a critical assessment of the key concepts of liposome technology that need to be reviewed for its definitive clinical translation.

Natural Killer (NK) cells play a pivotal role in the immunosurveillance of Multiple Myeloma (MM), but it is still undefined whether the NK cell functional properties underlying their protective activity against MM are confined to distinct NK cell populations. Interestingly, herein we report that the CD56lowCD16low NK cell subset displayed higher cytolytic activity compared to the other NK cell subsets (i.e., CD56highCD16+/−, CD56lowCD16high) against MM cells and its activity was impaired in MM patients. Decreased DNAM-1 expression levels were observed on the CD56lowCD16low NK cells during MM progression. Evaluating NK cell subset frequency after autologous hematopoietic stem cell transplantation, we found that CD56lowCD16low NK cells recovered earlier after transplantation. Overall, our data denote a key role of CD56lowCD16low subpopulation in the killing of MM cells and suggest that the reconstitution of CD56lowCD16low subpopulation after HSCT could be a useful approach of adoptive immunotherapy in the treatment of relapsed/refractory MM patients.

Treatment of multiple myeloma (MM) cells with sublethal doses of genotoxic drugs leads to senescence and results in increased NK cell recognition and effector functions. Herein, we demonstrated that doxorubicin- and melphalan-treated senescent cells display increased expression of IL15, a cytokine involved in NK cell activation, proliferation, and maturation. IL15 upregulation was evident at the mRNA and protein level, both in MM cell lines and malignant plasma cells from patients' bone marrow (BM) aspirates. However, IL15 was detectable as a soluble cytokine only

Exosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNγ production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-κB pathway in a TLR2/HSP70-dependent manner. Interestingly, HSP70+ exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNγ production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors.

Natural killer (NK) cells are critical immune effector cells capable of mediating antitumor responses. These cytotoxic lymphocytes recognize transformed cells through a mechanism mainly dependent on the engagement of several activating receptors. However, many tumors have developed strategies to evade immunosurveillance and detection by NK cells. A relevant immune escape mechanism is the down regulation of NK cell activating ligands on the surface of tumor cells by proteolytic shedding mediated by different members of metalloproteinase families. Here, we consider two important NK activating receptors, namely NKG2D and NKp30, the ligands (i.e., MICA/B, ULBPs, and B7-H6) of which can be released by cancer cells through proteolytic cleavage. Modulation of ligand shedding in response to cancer therapy is also examined, and we discuss how metalloproteinases implicated in the ligand cleavage could be targeted in novel therapeutic schemes to counteract tumor escape from stress-elicited immune responses.

Natural Killer (NK) cells play a pivotal role in the immunosurveillance of Multiple Myeloma (MM), but it is still undefined whether the NK cell functional properties underlying their protective activity against MM are confined to distinct NK cell populations. Interestingly, herein we report that the CD56lowCD16low NK cell subset displayed higher cytolytic activity compared to the other NK cell subsets (i.e., CD56highCD16+/&minus, CD56lowCD16high) against MM cells and its activity was impaired in MM patients. Decreased DNAM-1 expression levels were observed on the CD56lowCD16low NK cells during MM progression. Evaluating NK cell subset frequency after autologous hematopoietic stem cell transplantation, we found that CD56lowCD16low NK cells recovered earlier after transplantation. Overall, our data denote a key role of CD56lowCD16low subpopulation in the killing of MM cells and suggest that the reconstitution of CD56lowCD16low subpopulation after HSCT could be a useful approach of adoptive immunotherapy in the treatment of relapsed/refractory MM patients.

Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells. Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells.

Cardiac dysfunction of Fabry disease (FD) has been associated with myofilament damage and cell death as result of α-galactosidase A deficiency and globotriaosylceramide accumulation. We sought to evaluate the role of oxidative stress in FD cardiomyocyte dysfunction. Myocardial tissue from 18 patients with FD was investigated for the expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine by immunohistochemistry. Western blot analysis for nitrotyrosine was also performed. Oxidative damage to DNA was investigated by immunostaining for 8-hydroxydeoxyguanosine (8-OHdG), whereas apoptosis was evaluated by in situ ligation with hairpin probes. iNOS and nitrotyrosine expression was increased in FD hearts compared with hypertrophic cardiomyopathy and normal controls. Remarkably, immunostaining was homogeneously expressed in FD male cardiomyocytes, whereas it was only detected in the affected cardiomyocytes of FD females. Western blot analysis confirmed an increase in FD cardiomyocyte protein nitration compared with controls. 8-OHdG was expressed in 25% of cardiomyocyte nuclei from FD patients, whereas it was absent in controls. The intensity of immunostaining for iNOS/nitrotyrosine correlated with 8-OHdG expression in cardiomyocyte nuclei. Apoptosis of FD cardiomyocytes was 187-fold higher than in controls, and apoptotic nuclei were positive for 8-OHdG. Cardiac dysfunction of FD reflects increased myocardial nitric oxide production with oxidative damage of cardiomyocyte myofilaments and DNA, causing cell dysfunction and death.

Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor recognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I–related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM) cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is upregulated upon chemotherapeutic treatment both in patient-derived CD138+/CD38+ plasma cells and in several MM cell lines, and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs and metalloproteinase inhibitors enhances NK cell–mediated recognition of MM cells, preserving MIC molecules on the cell surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell–based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune responses.

Background 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. Methods 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0362-2) contains supplementary material, which is available to authorized users.