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Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability. Here we provide evidence that hyper-activation of DNA ligase III (LIG3) is crucial for genomic instability and survival of MM cells. LIG3 mRNA expression in MM patients correlates with shorter survival and even increases with more advanced stage of disease. Knockdown of LIG3 impairs MM cells viability in vitro and in vivo, suggesting that neoplastic plasmacells are dependent on LIG3-driven repair. To investigate the mechanisms involved in LIG3 expression, we investigated the post-transcriptional regulation. We identified miR-22-3p as effective negative regulator of LIG3 in MM. Enforced expression of miR-22 in MM cells downregulated LIG3 protein, which in turn increased DNA damage inhibiting in vitro and in vivo cell growth. Taken together, our findings demonstrate that myeloma cells are addicted to LIG3, which can be effectively inhibited by miR-22, promoting a novel axis of genome stability regulation.

Bone disease (BD) represents a hallmark of multiple myeloma (MM), characterized by classical osteolytic lesions and/or osteoporosis, that can be life threatening and contribute to poor outcomes. The complex interplay among MM conditioned bone effectors such as MM cells, osteoclasts (OCLs) and osteoblasts within BM microenvironment (BMM) supports MMBD. Several studies have shown that the extent of BD is tightly correlated to Th17 cells development and maintenance. Th17 secrete pro OCL cytokines such as RANKL, TNF, and IL-17, contributing to MMBD development. Recent findings showed that microRNAs (miRNAs) are involved in MMBD. MiRNAs are short non-coding RNAs that regulate gene expression at a post transcriptional level. Among others, miR-21 is involved in Th17 differentiation by targeting SMAD-7, a negative regulator of TGF-β signaling, promoting the activation of SMAD-2/3, suppressing IL-2 expression, and ultimately enhancing the activity of the TGF-β signaling pathway. Taken together, these data support a therapeutic strategy of miR-21 targeting to ameliorate MMBD through Th17 inhibition. We have previously demonstrated that miR-21 activity within BMM plays a crucial role in bone resorption/apposition balance through PIAS3 suppression and STAT3 dependent RANKL transcription in BM stromal cells. In the present study, we sought to evaluate the role of miR-21 in Th17 mediated BD. We enriched Th17 from BM CD4+CCR6+T cells and evaluated miR-21 levels. We found increased miR-21 in MM derived Th17 (MMTh17) as compared to controls, supporting the relevance of miR-21 in MMTh17. To further explore the mechanisms of miR-21 activity in Th17 function, freshly isolated naïve CD4+T cells were differentiated from healthy donors into Th17 along 18 days of culture in the presence of polarizing cytokines. At the end of culture, T cells expressed the Th17 full maturation markers RORC and IL17. As a model of MMBD in vitro, OCL resorptive activity was evaluated on dentine discs with/without Th17 freshly isolated from MM patients or differentiated in vitro. In both cases, Th17 promoted potent OCL mediated dentin pits generation as compared to controls. To counteract miR-21 activity, we electroporated naïve CD4+T cells with miR-21 inhibitor (miR21i) or scramble control mirna before starting Th17 differentiation culture. MiR-21 expression levels were assessed by rt pcr before and at the end of differentiation culture to ascertain its effective inhibition. To map signaling pathways differences elicited by miR21i-Th17, we performed a mass spectrometry-based quantitative phosphoproteomics analysis. 1134 phosphorylated proteins were identified, with 386 significant changes as compared to controls. The global-pairwise-normalized data underwent to Ingenuity Pathway Analysis (IPA). Interestingly, analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition included PI3K/AKT and 14-3-3 Signaling. Among Th17 potential regulators, enhanced activity of PDCD4, STAT1, MINK1 and FOXO1 were observed, while ITGAL showed reduced activity. On these bases, we performed WB to validate IPA analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of RORC were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated in the presence of miR-21 antagonism. STAT3 is a master transcription factor for Th17 upon IL-6 stimulation. As PIAS3 and PDCA4 negatively regulate STAT3, we checked phospho-STAT3 expression and found it downregulated as expected. Overall, these data indicated that Th17 differentiation was impaired by miR-21i. In the second set of experiments, we aimed to evaluate Th17 functional impairment by miR-21 antagonism in MMBD. OCLs were plated with dentine discs and exposed to either Th17 alone, miR21i-Th17, or RANKL/MCSF cytokine alone as positive control. Dentine pits and areas were significantly lower in the presence of miR21i-Th17 as compared to Th17 alone and positive control (Fig.1). These data indicated that miR-21 antagonism negatively regulates Th17 mediated OCL activity and support the role of miR-21 inhibition as an innovative treatment tool in MMBD.

Dendritic cells (DCs) are potent antigen presenting cells that regulate the development of both innate and adaptive immune responses. According to their maturation status, DCs may ignite immune response or induce immune tolerance. Indeed, immature DCs (iDCs) present low levels of costimulatory molecules such as CD80 and CD86, and high levels of tolerogenic molecules such as B7H3. Upon exposure to maturation stimuli, DCs upregulate CD83 on their surface and gain the competence of stimulating T cell response. An efficient maturation is crucial for the generation of a specific cytotoxic T lymphocyte response, specially against cancer. However, recent reports have shown that Multiple Myeloma (MM) milieucan recruit DCs and reprogram them to sustain growth and survival of MM cells and protect them against immune response. Therapeutic approaches to restore DC functions rely on the identification of the pathways that are directly involved in induction of tolerance. Emerging evidence supports the role of microRNAs (miRNA) in the regulation of immune response and DC function. Among others, the miR-29 family seems to be involved in the modulation of NK activity, of Th1/Th2 phenotype switch and of DC differentiation from monocyte precursors. Besides, miR-29b targets and inhibits different and crucial immune-modulatory molecules such as B7H3, VEGF and IL-4. These findings suggest that miR-29 may play an important role in the multifaceted interplay between tumor cells and host's immune system. To address this hypothesis, we generated iDCs from CD14+ monocytes of healthy donors and co-cultured them with: i) allogeneic (allo-) lymphocytes; ii) VEGF and IL-6 producing MM cells (RPMI8226 and U266); iii) allo-lymphocyte and MM cells. We found a consistent increase of miR-29b expression by RT-PCR during differentiation and maturation of DCs induced by allo-lymphocytes. However, when immature DCs were co-cultured with MM cells +/- allo-lymphocytes, a significant 3-fold reduction of miR-29b levels (p= 0.02) was observed (fig 1). This event occurred together with the absence of maturation markers, the persistence of high levels of B7H3 on the cell surface and with a raise in VEGF, IL-10, and IFN-gamma levels in the supernatant, confirming the MM-dependent impairment of the physiological DC maturation process. This latter concept was supported by the finding of increased number of CD4+CD25+Foxp3+T-regs in the DC/MM cell/allo-lymphocyte co-cultures as compared to the DC/allo-lymphocyte co-cultures (p= 0.05). To promote the recovery from the MM related immune-bias, we transiently transfected iDCs with miR-29b (29b-DCs) mimics or with a negative control (NC-DCs). We observed improved DC maturation (82.46% versus39.89% of CD11c+/CD83+/CD86+ cells), reduced expression of B7H3 (33% reduction in MFI) and reduction of the T-reg number in 29b-DC/MM cell/allo-lymphocyte co-cultures as compared to NC-DC/MM cell/allo-lymphocyte co-culture. To investigate whether 29b-DCs were able to promote a specific CTL response against MM cells in vivo, we engrafted NOD/SCID γ chain-null mice with peripheral blood mononuclear cells (PBMCs) from HLA-A2+ healthy donors. DCs from the same donor were differentiated, transfected with either miR-29b or NC and then co-cultured with U266 for 48h. Mice were then vaccinated twice with either 29b-DCs or NC-DCs. Two weeks following the first injection, CD3+ human lymphocytes were recovered from mouse spleens (CD3 hu-splenocytes). We found an increased CD8/CD4 ratio in the CD3 hu-splenocytes collected from the 29b-DCs treated mouse as compared to control. To assess the capability of CD3+ hu-splenocytes to selectively kill U266 cells, we kept CD3 lymphocytes in culture in the presence of IL-15 for 48h. Then, we carried a cytotoxicity assay against U266 cell target. The highest specific lysis was attained with miR-29b DC primed CD3 hu-splenocytes (fig.2, p=0.03).

miR-221/222 are two highly homologous microRNAs (miRNAs), encoded in tandem on the chromosome X, whose up-regulation has been found in several malignancies and are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression. We demonstrated up-regulation of both miRNAs in malignant plasma cells (PCs) from multiple myeloma (MM) patients belonging to distinct TC (translocation/Cyclin) groups, including TC2 and TC4. A rising body of evidence suggests that silencing miRNAs with oncogenic potential could represent a novel approach for human cancer therapy. We previously demonstrated that silencing miR-221/222 exerts significant anti-MM activity and triggers canonical targets in vitroand in vivo. Here, in the aim to progress to clinical translation of our proof-of-principle findings, we investigated the anti-tumor activity and the appropriateness for systemic delivery of a novel and originally designed LNA-miR-221, a 13-mer antisense miR-221 inhibitor, which took advantage of locked nucleic acid (LNA) technology and phosphorothioate backbone chemistry for increasing affinity for miR-221 and nuclease resistance. We found that enforced ectopic expression of LNA-miR-221 in t(4;14) MM cells significantly inhibited growth and survival of MM cells in vitro. In treated cells, we detected knock down of miR-221/222 together and increased levels of both p27Kip1 mRNA and protein. Specific activity of this LNA-miR-221 inhibitor was confirmed by the use of a 3'UTR reporter (luciferase renilla/firefly) constructs containing, miR-221 target site. This construct was co-transfected either with miR-221/222 mimics or LNA-miR-221 inhibitor into MM cells. As predicted, a reduced luciferase activity was detected in miR-221/222 mimics co-transfected cells with each 3'UTR reporter plasmid, while increase luciferase activity was measured in MM cells co-transfected LNA-miR-221 inhibitors indicating an efficient and stable binding to the miRNA target sequence. Importantly, we evaluated the systemic delivery of the LNA-miR-221 inhibitor with saline solution vehicle alone by intraperitoneal or intravenous injection route against MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved after 2 weeks of treatment at similar extent by both injection routes. Retrieved tumors from treated animals showed efficient inhibition of miR-221/222, as demonstrated by increased levels of p27Kip1 protein in vivo. H&E staining and immunohistochemical analysis showed wide necrosis areas, reduced Ki67 and a significant increase of p27Kip1 cytoplasmic expression in retrieved tumors from LNA-miR-221 inhibitor-treated mice. No changes in mice behavior or organ toxicity were observed in treated mice. Taken together these findings support the rationale for development of this novel and highly efficient LNA-miR-221 inhibitor as a promising anti-MM drug in subsequent primate toxicology studies. Supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n. 9980, 2010/15.