ALBERT

Description: 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 vitro and 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. Disclosures: No relevant conflicts of interest to declare.

Description: MicroRNAs (miRNAs), short non-coding RNAs which tune gene expression at post-transcriptional level, are recently emerging as key players in pathogenesis, progression and drug-resistance of multiple myeloma (MM). In this disease, they can act either with tumor-promoting or tumor-suppressing functions, depending on the nature of target mRNAs. Nowadays, effective strategies are available both to replace or to inhibit the expression of deregulated miRNAs, thus prompting the design of miRNA-based therapeutic strategies. We have recently demonstrated that miR-125b has tumor suppressor activity in MM and that enforced expression of synthetic miR-125b-5p mimics induces significant anti-MM activity in vitro and in vivo by targeting cell addiction to IRF4/cMyc pro-survival signaling. Moreover, we uncovered a functional feedback loop between cMyc and miR-125b in MM cells, whereas cMyc directly suppresses miR-125b transcription which, in turn, negatively regulates cMyc expression by targeting IRF4 mRNA. In the present study, we investigated the therapeutic potential of synthetic miR-125b-5p mimics combined with cMyc targeting agents, including the 10058-F4 small molecule inhibitor of cMyc-Max heterodimerization and the BET-bromodomain inhibitor JQ1, which is reported to inhibit cMyc transcription. At this aim, 3 MM cell lines (NCI-H929, SK-MM-1 and RPMI-8226) transfected with either miR-125b-5p mimics or scrambled oligonucleotides (miR-NC) were exposed to 10058-F4 (ranging from 10 to 100 μM) or JQ1 (ranging from 0,1 to 2μM) or DMSO. Effects on cell proliferation were then evaluated by CCK-8 assay at 24h, 48h and 72h time points, while the occurrence of apoptotic cell death was assessed by Annexin V flow-cytometry assay. Importantly, we found that enforced expression of miR-125b-5p mimics significantly and synergistically (synergistic index, SI 〉1) increases growth-inhibitory and pro-apoptotic activities of both 10058-F4 and JQ1. Similar results were observed in SK-MM-1 cells co-transfected with miR-125b-5p and cMyc siRNAs, while cMyc-defective U266 cells were not sensitized to either 10058-F4 nor JQ1 upon transfection with miR-125b-5p mimics. Furthermore, combinatorial treatments with JQ1 and miR-125b-5p mimics resulted in a stronger downregulation of cMyc protein, as compared to single molecules alone. Indeed, these results confirmed that impairment of cMyc activity/expression mediates the anti-MM synergistic effects between 10058-F4 or JQ1 and overexpression of miR-125b-5p by synthetic mimics. In conclusion, our data demonstrate a cMyc-mediated synergistic anti-MM activity of synthetic miR-125b-5p mimics with 10058-F4 or JQ1 cMyc targeting agents, providing the rationale for a more advanced preclinical investigations for the design of early clinical trials. Disclosures No relevant conflicts of interest to declare.

Description: miR-221/222 are highly homologous microRNAs (miRNAs) whose upregulation has been found in several malignancies, including multiple myeloma (MM). Both miRNAs 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 proved that silencing of both miRNAs results in significant anti-MM activity and in downregulation of canonical targets both in vitro and in vivo. In the aim to progress to clinical translation, we designed an original 13mer synthetic inhibitor, specific for systemic delivery, named LNA-i-miR-221, which took advantage from both locked nucleic acid (LNA) technology and phosphorothioate backbone, for increasing the seed sequence binding and nuclease resistance in vivo, respectively. Since no data are presently available, we evaluated the specificity of LNA-i-miR-221 to inhibit endogenous miR-221 and the pharmacokinetic properties (i.e. tissue distribution) in mice and Cynomolgus monkeys. We demonstrated that LNA-i-miR-221 inhibit growth and survival of MM cells bearing high miR-221 levels. Moreover, LNA-i-miR-221-mediated silencing of miR-221 triggered upregulation of p27Kip1 mRNA and protein, evaluated by q-RT-PCR and western blotting, respectively. In vivo, i.p.treatment with 25 mg/kg of LNA-i-miR-221 reduced tumor growth in SCID/NOD mice bearing MM xenografts. Tumors and vital organs (including liver, kidney, bone marrow and heart) were harvested from treated animals and evaluated for pharmacokinetics purposes using in situ hybridization (ISH) assay. After a single i.p. dose of 25 mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues. Interestingly, no toxicity was observed even after long-lasting presence of the inhibitors in vital organs. We also evaluated the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS, which confirmed the rapid uptake of LNA-i-miR-221 in tissues. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed using doses from 10 to 100 mg/kg delivered at days 1,4,8,15,22. All treatments were well tolerated. No changes in mice behavior or organ toxicity were observed in treated mice. Finally, a pilot toxicity study was performed in Cynomolgus monkeys. PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary escretion. In conclusion, our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a safety profile in mice and in monkeys, supporting the rationale for development of this novel miR-221 inhibitor in early clinical trials. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2941 The enforced expression of tumor suppressor microRNAs (miRNAs) is a promising strategy for cancer treatment. Among miRNAs frequently deregulated in cancer, miR-29b is of great interest since its overexpression leads to tumor growth inhibition in solid tumors and leukemias. On these bases, we first investigated miR-29b expression pattern in multiple myeloma (MM) cells. miRNA profiling of primary CD138+ MM patient cells (n=55) revealed deregulated expression of miR-29b with almost 60% of cases showing strongly reduced levels. Moreover, as assessed by quantitative real time PCR (qRT-PCR), miR-29b expression was significantly downregulated in a panel of 11 MM cell lines and even further decreased when RPMI-8226 MM cells were co-cultured with bone marrow stromal cells (BMSCs), indicating that the human bone marrow microenvironment (huBMM) modulates miR-29b levels. Either transient expression of synthetic miR-29b mimics through electroporation or stable lentivirus-enforced miR-29b expression induced growth inhibition and apoptosis of MM cells in vitro. In vivo intratumoral or systemic delivery of neutral lipidic formulated-miR-29b mimics in different clinically relevant murine models of human MM, including our recent SCID-synth hu model which recapitulates the huBMM, led to significant tumor growth inhibition together with increased survival of miR-29b-treated animals. By Western blot and qRT-PCR analysis, we observed dramatic reduction of predicted targets of miR-29b including CDK6 and MCL-1, which can thus explain antiproliferative and pro-apoptotic effects triggered by miR-29b expression. Most importantly, we identified Sp1, a transcription factor endowed with oncogenic activity in MM, as a negative regulator of miR-29b expression providing evidence of a novel regulatory loop in MM cells: in fact, enforced expression of Sp1 was able to reduce miR-29b-promoter activity and miR-29b levels, whereas miR-29b expression decreased Sp1 mRNA and protein levels via 3'UTR binding, as assessed in luciferase reporter assays. Moreover, treatment of MM cells with the proteasome inhibitor bortezomib led to Sp1 downregulation and miR-29b upregulation. Strikingly, miR-29b transfection significantly strengthened the in vitro anti-proliferative and apoptotic effects induced by bortezomib, thus highlighting its role in the mechanism of anti-MM activity of this drug. Additional miR-29b targets include de novo DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B). Microarray profiling revealed increased DNMT3A and DNMT3B mRNA levels in cancer cells as compared to normal plasma cells. The integrated analysis of miRNA/mRNA profiling in a panel of 18 MM cell lines highlighted an inverse correlation between miR-29b and DNMT3B levels. Finally, miR-29b was proven to target DNMT3A and DNMT3B and significantly reduced the global DNA methylation levels in MM cells, as assessed by performing an in vitro DNA methylation assay with genomic DNA extracted from MM cells after synthetic miR-29b electroporation. In conclusion, our findings indicate that miR-29b exerts anti-MM activity by targeting relevant oncogenic pathways and by restoring the aberrant methylation pattern of MM cells. These results support a potential therapeutic role for miR-29b mimics in MM as single agent or in combination with the proteasome inhibitor bortezomib and/or demethylating agents. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Description: MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression at the post-transcriptional level by inducing mRNA decay or translation repression. A subclass of miRNAs, named epi-miRNAs, is known to exert anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role for the tumor suppressor miR-29b in reducing the global DNA methylation of multiple myeloma (MM) cells through the targeting of DNA-methyltransferases. In silico search of additional miR-29b targets contributing to clarify its role as epi-miRNA unveiled the class II histone deacetylase HDAC4. Since histone deacetylases represent promising molecular targets for cancer treatment, we sought to characterize HDAC4 expression and function and its regulation by miR-29b in MM cells. HDAC4 protein levels and enzymatic activity were found up-regulated in a panel of 11 MM cell lines as compared to peripheral blood mononuclear cells from healthy donors. Moreover, the analysis of HDAC4 mRNA levels in a microarray dataset consisting of 4 healthy controls, 55 MM and 29 plasma cell leukemia patients indicated significant over-expression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. Notably, the analysis of miRNA/mRNA paired expression in the same microarray dataset revealed an inverse correlation between miR-29b and HDAC4 mRNA, strengthening the relevance of miR-29b as a key regulator of HDAC4. Synthetic miR-29b mimics transfected in MM cells (SKMM1 and NCI-H929) down-regulated HDAC4 mRNA and protein levels and inhibited the 3’UTR of HDAC4 cloned in a luciferase reporter vector, whereas failed to regulate a 3’UTR devoid of two predicted miR-29b target sites. On the other hand, lentiviral-mediated overexpression of HDAC4 strongly inhibited miR-29b expression. These results underscore a negative feedback loop occurring between miR-29b and its target HDAC4 in MM cells. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable silencing of HDAC4 by shRNAs induced growth inhibition, caspase 3/7-dependent apoptosis and autophagy in U266 and KMS11 cells, which occurred together to miR-29b up-regulation. Interestingly, the pan-HDAC inhibitor vorinostat also triggered apoptosis and autophagy in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and other miR-29b-canonical targets like CDK6, MCL-1 and Sp1. miR-29b itself was able to promote autophagy, as assessed by beclin-1 up-regulation and LC3A/B proteolytic cleavage in miR-29b mimics-transfected MM cells, which was abrogated by constitutive expression of HDAC4. Finally, we provided evidence that miR-29b over-expression potentiated, whereas its stable inhibition dampened, apoptosis and autophagy triggered by vorinostat. In conclusion, our findings shed light on the oncogenic role of HDAC4, which can be targeted through miR-29b-based therapeutic approaches, and identify miR-29b as a relevant effector of vorinostat activity in MM cells. Disclosures: No relevant conflicts of interest to declare.

Description: Gene transcription within the tumor cell and its microenvironment can be affected by epigenetic modulation in histones and inhibition of histone deacetylases is presently a promising therapeutic option in multiple myeloma (MM). MicroRNAs (miRNAs) are non-coding RNAs that control gene expression; a subclass of miRNAs, named epi-miRNAs, exerts anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role of tumor suppressor miR-29b as epi-miRNA able to target DNA-methyltransferases and to reduce aberrant hypermethylation in MM. In silico search of miR-29b targets further clarifying its epi-miRNA function, unveiled the histone deacetylase HDAC4. Here, we aimed at characterizing HDAC4 expression, function and its regulation by miR-29b in MM. HDAC4 mRNA, protein levels and activity were found increased in 11 out of 11 MM cell lines as compared to 3 healthy controls. The analysis of our microarray dataset consisting of plasmacells from 4 healthy donors, 55 MM and 29 plasma cell leukemia patients indicated HDAC4 overexpression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. In the same dataset, HDAC4 mRNA expression inversely correlated with miR-29b levels. To investigate the mechanism by which miR-29b could affect HDAC4 expression, we transfected SKMM1 cells with miR-29b mimics, which resulted in down-modulation of HDAC4 at mRNA and protein level, along with increased acetylation at lysine 8 of histone H4 as well as of tubulin acetylation. This effect was isoform-specific, since levels of other HDACs (namely HDAC1, 2, 6) remained unchanged after miR-29b overexpression. Moreover, miR-29b mimics inhibited the 3’UTR of HDAC4 cloned in a luciferase vector whereas failed to regulate a mutant devoid of a predicted miR-29b target site. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable shRNA-mediated silencing of HDAC4 phenocopied our previously reported miR-29b-triggered biological effects, since it led to growth inhibition, caspase 3/7-dependent apoptosis, inhibition of cell migration and induction of autophagy in KMS11 and NCI-H929 MM cells. HDAC4 depletion by shRNAs potentiated the in vitro anti-MM activity of dexamethasone, bortezomib and vorinostat. Notably, silencing of HDAC4 led to miR-29b upregulation, likely as consequence of histone H4 hyperacetylation at miR-29b promoter, as assessed by Chip experiments; conversely, HDAC4 overexpression strongly reduced miR-29b levels. These results underscore the presence of a negative feedback loop between miR-29b and its target. Adhesion to BM stromal cells reduced miR-29b levels while induced up-regulation of HDAC4 mRNA levels in NCI-H929 cells, thus suggesting that the huBMM might increase HDAC activity in MM cells via miR-29b down-modulation. Importantly, the pan-HDAC inhibitor vorinostat also triggered apoptosis, autophagy and anti-migratory effects in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and of other miR-29b targets like Sp1, CDK6 and MCL-1; these phenotypical changes were potentiated by miR-29b mimics, whereas were strongly reduced by antagomiR-29b transduction, thus demonstrating a functional involvement of miR-29b in the anti-MM activity of vorinostat. Finally, in vivo studies using a KMS11 xenograft murine model of MM demonstrated a significant potentiation of vorinostat-induced tumor growth inhibition by subcutaneously delivered neutral lipid emulsion-formulated miR-29b synthetic mimics. Collectively, our results demonstrate that HDAC4 is aberrantly expressed and represents a novel therapeutic target in MM; moreover, our investigation provides the preclinical rationale for using miR-29b mimics to potentiate HDAC inhibitors activity in MM. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2910 Multiple myeloma (MM) remains an incurable disease despite important therapeutic advances in the last few years. Small non-coding RNAs (miRNAs) synthetic mimics are a new class of biological agents which have recently demonstrated preclinical activity against a variety of human neoplasms. miRNA antitumor activity has been related to their capacity to interfere with mRNA stability and protein transducing activity. miR-34a has tumor suppressor activity and is transcriptionally regulated by p53. We investigated the in vitro and in vivo therapeutic potential of pre-miR-34a mimics against human MM cells. Transient expression of pre-miR-34a mimics, after electroporation of SKMM1 and RPMI-8226 MM cell lines which display low constitutive miR-34a levels, triggered antiproliferative effects, as demonstrated by MTT and long-term soft-agar colony assays. 48 hours after cell transfection, apoptotic events were detected in both cell lines exposed to miR-34a mimics. In parallel experiments, MM cells stably transduced with miR-34a gene cloned in a lentiviral vector showed significant growth reduction as compared to empty vector-transduced cell colonies, providing additional evidence of miR-34a tumor suppressor activity in MM cells. qRT-PCR analysis of treated MM cells showed that pre-miR-34a mimics induced down-regulation of mRNAs coding for Notch1 and the cell-cycle dependent kinase 6 (CDK6), validated miR-34a targets. Furthermore, decreased anti-apoptotic Bcl-2 and CDK6 proteins was detected after pre-miR-34a mimic expression, evidence by western blotting analysis. The anti-MM activity of pre-miR-34a mimics was also evaluated in vivo using xenografted SCID models of human MM. Intra-tumor delivery of pre-miR-34a was performed by a novel formulation with Neutral Lipid Emulsion (NLE). Following 4 injections (3 days apart) of pre-miR34a formulated in NLE particles, a highly significant inhibition of tumor growth was detected in SKMM1 xenografted SCID mice. At day 13 after the first treatment, tumors in mice treated with formulated pre-miR-34a were significantly smaller than tumors in mice treated with the formulated scrambled sequence (P=0.0002) or vehicle (P=0.0002) or PBS (P=0.0001). Interestingly, at day 21a three mice enrolled in the miR-34a treated group showed complete regression of tumors. Formulated synthetic pre-miR-34a also produced a significant increase of mice survival (P=0.01 versus formulated scrambled sequence). A similar in vivo tumor growth inhibition was observed in mice xenografted with SKMM1 MM cells stably transduced with a miR-34a lentiviral construct, as compared to cells transduced with the empty vector. We here provide the first proof-of-principle demonstrating that replacement of miR-34a produces therapeutic activity against MM cells with low constitutive miR-34a expression. Our findings provide a framework for development of miR-34a-based therapeutic strategies in MM. Supported by AIRC 5 per mille, Molecular Clinical Oncology Program No. 9980 Disclosures: Anderson: Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3960 Overwhelming osteoclast (OCL) activation plays a central role in multiple myeloma (MM)-related bone disease. It is well known that in MM, OCL differentiation and final maturation rely upon RANKL stimulation by bone marrow mesenchymal cells and osteoblasts (OBLs). The clinical relevance of this pathway has been recently underlined by the anti-resorptive activity mediated by the mAb Denosumab. Since the discovery of microRNAs (miRNAs), findings on their role on intracellular pathway control have undergone a tremendous progress suggesting their potential use as a therapeutic tool against specific targets. Based on these premises, we aimed to identify miRNAs that can be relevant for the management of MM-related bone disease. Among miRNAs deregulated in MM, miR-29 family has been implicated in bone pathophysiology. Indeed, miR-29 family promotes OBL generation, while miR-29b might interfere with OCL differentiation and function by targeting RANKL axis and Metalloproteinase II (MMP2), that confers the property to degradate type I collagens. Therefore, we studied whether miR-29b can have a detrimental effect for terminally differentiated OCLs. We generated OCLs in vitro upon RANKL/MCSF stimulation starting from CD14+ hematopoietic precursors and found that miR-29b levels decline along OCL differentiation, reaching statistical significance as compared with its precursors (p=0,013). These data suggested that terminally differentiated OCLs do not require miR-29b. In order to assess whether miR-29b reconstitution could affect OCL activity, we transduced OCLs with a miR-29b coding sequence- carrying lentiviral vector (29b OCLs) or an empty lentiviral vector (eOCLs) and evaluated them in morphological and functional assays. As we expected, 29b OCLs showed a faint and irregular expression of tartrate acid phosphatase (TRAcP), which is highly and uniformously distributed within WT/eOCLs. Furthermore, when 29b OCLs were seeded on dentin, generation of lacunae on dentin surface, which recapitulates bone resorption, was significantly reduced as compared with WT/eOCLs (see Figure). According to these results, we measured the release of type I collagen fragments by OCLs and found that degradation of type I collagens was significantly impaired in 29b OCLs, suggesting that constitutive expression of miR-29b strongly antagonizes OCL differentiation and bone lytic functions. In order to reproduce the features of MM related bone disease, we co-cultured OCLs with RPMI-8226 MM cells, which are able to stimulate WT/eOCLs to generate lacunae on dentin slices in the absence of exogenous RANKL/MCSF. We observed that 29b OCLs failed to generate comparable numbers and areas of the pits in presence of MM cells (p=0,035/p=0,04). To support these functional data and ascertain the effects of miR-29b on specific pathways, we evaluated the expression of SP1 and NFATc-1, which are relevant transcription factors for OCL differentiation and work through RANK-L axis. We found that both factors are down-modulated in 29b OCLs as compared to WT/eOCLs. Down modulation was observed also for MMP2, thus mirroring the reduced capability to lyse type I collagens. Overall, our data indicate that miR-29b impairs OCL differentiation and function even in presence of robust stimuli such as RANKL and MCSF. We provided molecular support to these functional data, showing that SP1 and NFATc are down modulated in presence of miR-29b as well as MMP2, which is involved in collagen degradation. Intriguingly, MM cells, which represent a strong pro-osteoclastic factor, were not able to revert OCL functional impairment. We believe that these relevant preclinical findings allow to propose miR-29b mimics as a suitable and attractive candidate to be developed as a novel and innovative treatment of MM-related bone disease. Disclosures: No relevant conflicts of interest to declare.