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Fig. S1. MiR-221 and miR-222 expression levels in Melphalan resistant MM U266/LR7 and sensitive U266/S; Fig. S2. Enforced expression of miR-221/222 decreases melphalan sensitivity of MM cells in vitro; Fig. S3. MiR-221/222 expression levels in Melphalan resistant MM U266/LR7 adherent to human Hs-5; Fig. S4. Genome-wide expression patterns associated with miR-221/222 inhibitors plus melphalan treatment; Fig. S5. Validation by q-RT-PCR of gene expression results; Table S1. Synergistic index of combination treatment with miR-221/222 inhibitors plus Melphalan.

Purpose: The onset of drug resistance is a major cause of treatment failure in multiple myeloma. Although increasing evidence is defining the role of miRNAs in mediating drug resistance, their potential activity as drug-sensitizing agents has not yet been investigated in multiple myeloma.Experimental Design: Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory multiple myeloma cells to melphalan.Results: miR-221/222 expression inversely correlated with melphalan sensitivity of multiple myeloma cells. Inhibition of miR-221/222 overcame melphalan resistance and triggered apoptosis of multiple myeloma cells in vitro, in the presence or absence of human bone marrow (BM) stromal cells. Decreased multiple myeloma cell growth induced by inhibition of miR-221/222 plus melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx–efflux transporters SLC7A5/LAT1 and the ABC transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory multiple myeloma xenografts with systemic locked nucleic acid (LNA) inhibitors of miR-221 (LNA-i-miR-221) plus melphalan overcame drug resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice.Conclusions: Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan resistance in preclinical models of multiple myeloma, providing the framework for clinical trials to overcome drug resistance, and improve patient outcome in multiple myeloma. Clin Cancer Res; 22(5); 1222–33. ©2015 AACR.

Supplementary methods for pubblication only

Sample processing of formalin-fixed specimens constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature, dehydration, and embedding media affect downstream analysis, generating data dependent on technical processing rather than disease state. In this study, we investigated two different sample processing methods, including the use of the cytospin sample preparation and automated sample processing apparatuses for proteomic analysis of multiple myeloma (MM) cell lines using imaging mass spectrometry (IMS). In addition, two sample-embedding instruments using different reagents and processing times were considered. Three MM cell lines fixed in 4% paraformaldehyde were either directly centrifuged onto glass slides using cytospin preparation techniques or processed to create paraffin-embedded specimens with an automatic tissue processor, and further cut onto glass slides for IMS analysis. The number of peaks obtained from paraffin-embedded samples was comparable between the two different sample processing instruments. Interestingly, spectra profiles showed enhanced ion yield in cytospin compared to paraffin-embedded samples along with high reproducibility compared to the sample replicate.

Polyphenols from olive oil are endowed with several biological activities. Chemical modifications have been recently applied to these compounds to improve their therapeutic activity in different pathological settings, including cancer. Herein, we describe the in vitro effects on multiple myeloma (MM) cells of oleil hydroxytyrosol (HTOL), a synthetic fatty ester of natural hydroxytyrosol with oleic acid. HTOL reduced the viability of various human MM cell lines (HMCLs), even when co-cultured with bone marrow stromal cells, triggering ER stress, UPR and apoptosis, while it was not cytotoxic against healthy peripheral blood mononuclear cells or B lymphocytes. Whole-transcriptome profiling of HTOL-treated MM cells, coupled with protein expression analyses, indicate that HTOL antagonizes key survival pathways for malignant plasma cells, including the undruggable IRF4–c-MYC oncogenic axis. Accordingly, c-MYC gain- and loss-of-function strategies demonstrate that HTOL anti-tumor activity was, at least in part, due to c-MYC targeting. Taken together, these findings underscore the anti-MM potential of HTOL, providing the molecular framework for further investigation of HTOL-based treatments as novel anti-cancer agents.

Epigenetic mechanisms, such as alterations in histone acetylation based on histone deacetylases (HDACs) activity, have been linked not only to normal brain function but also to several brain disorders including epilepsy and the epileptogenic process. In WAG/Rij rats, a genetic model of absence epilepsy, epileptogenesis and mild-depression comorbidity, we investigated the effects of two HDAC inhibitors (HDACi), namely sodium butyrate (NaB), valproic acid (VPA) and their co-administration, on the development of absence seizures and related psychiatric/neurologic comorbidities following two different experimental paradigms. Treatment effects have been evaluated by EEG recordings (EEG) and behavioural tests at different time points. Prolonged and daily VPA and NaB treatment, started before absence seizure onset (P30), significantly reduced the development of absence epilepsy showing antiepileptogenic effects. These effects were enhanced by NaB/VPA co-administration. Furthermore, early-chronic HDACi treatment improved depressive-like behaviour and cognitive performance 1 month after treatment withdrawal. WAG/Rij rats of 7 months of age showed reduced acetylated levels of histone H3 and H4, analysed by Western Blotting of homogenized brain, in comparison to WAG/Rij before seizure onset (P30). The brain histone acetylation increased significantly during treatment with NaB or VPA alone and more markedly during co-administration. We also observed decreased expression of both HDAC1 and 3 following HDACi treatment compared to control group. Our results suggest that histone modifications may have a crucial role in the development of epilepsy and early treatment with HDACi might be a possible strategy for preventing epileptogenesis also affecting behavioural comorbidities.

The mitochondrial quality control network includes several epigenetically-regulated genes involved in mitochondrial dynamics, mitophagy, and mitochondrial biogenesis under physiologic conditions. Dysregulated expression of such genes has been reported in various disease contexts, including cancer. However, their expression pattern and the possible underlying epigenetic modifications remain to be defined within plasma cell (PC) dyscrasias. Herein, we compared the mRNA expression of mitochondrial quality control genes from multiple myeloma, plasma cell leukemia patients and human myeloma cell lines (HMCLs) with healthy plasma cells; moreover, by applying the Sequenom MassARRAY EpiTYPER technology, we performed a pilot investigation of their CpG methylation status in HMCLs. Overall, the results provided indicate dysregulated expression of several mitochondrial network’s genes, and alteration of the CpG methylation profile, underscoring novel potential myeloma biomarkers deserving in-depth functional investigation in the future.

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor cure rate for relapsed/resistant patients. Due to the lack of T-cell restricted targetable antigens, effective immune-therapeutics are not presently available and the treatment of chemo-refractory T-ALL is still an unmet clinical need. To develop novel immune-therapy for T-ALL, we generated an afucosylated monoclonal antibody (mAb) (ahuUMG1) and two different bispecific T-cell engagers (BTCEs) against UMG1, a unique CD43-epitope highly and selectively expressed by T-ALL cells from pediatric and adult patients.MethodsUMG1 expression was assessed by immunohistochemistry (IHC) on a wide panel of normal tissue microarrays (TMAs), and by flow cytometry on healthy peripheral blood/bone marrow-derived cells, on 10 different T-ALL cell lines, and on 110 T-ALL primary patient-derived cells. CD43-UMG1 binding site was defined through a peptide microarray scanning. ahuUMG1 was generated by Genetic Glyco-Engineering technology from a novel humanized mAb directed against UMG1 (huUMG1). BTCEs were generated as IgG1-(scFv)2 constructs with bivalent (2+2) or monovalent (2+1) CD3ε arms. Antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and redirected T-cell cytotoxicity assays were analysed by flow cytometry. In vivo antitumor activity of ahUMG1 and UMG1-BTCEs was investigated in NSG mice against subcutaneous and orthotopic xenografts of human T-ALL.ResultsAmong 110 T-ALL patient-derived samples, 53 (48.1%) stained positive (24% of TI/TII, 82% of TIII and 42.8% of TIV). Importantly, no expression of UMG1-epitope was found in normal tissues/cells, excluding cortical thymocytes and a minority (2 log lower as compared with ahuUMG1, with significant mice survival advantage in different T-ALL models in vivo.ConclusionAltogether our findings, including the safe UMG1-epitope expression profile, provide a framework for the clinical development of these innovative immune-therapeutics for this still orphan disease.

Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.

Polycaprolactone (PCL) is a biocompatible and biodegradable polymer widely used for the realization of 3D scaffold for tissue engineering applications. The hot embossing technique (HE) allows the obtainment of PCL scaffolds with a regular array of micro pillars on their surface. The main drawback affecting this kind of micro fabrication process is that such structural superficial details can be damaged when detaching the replica from the mold. Therefore, the present study has focused on the optimization of the HE processes through the development of an analytical model for the prediction of the demolding force as a function of temperature. This model allowed calculating the minimum demolding force to obtain regular micropillars without defects. We demonstrated that the results obtained by the analytical model agree with the experimental data. To address the importance of controlling accurately the fabricated microstructures, we seeded on the PCL scaffolds human stromal cell line (HS-5) and monocytic leukemia cell line (THP-1) to evaluate how the presence of regular or deformed pillars affect cells viability. In vitro viability results, scanning electron and fluorescence microscope imaging analysis show that the HS-5 preferentially grows on regular microstructured surfaces, while the THP-1 on irregular microstructured ones.

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.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Olive oil contains different biologically active polyphenols, among which oleacein, the most abundant secoiridoid, has recently emerged for its beneficial properties in various disease contexts. By using in vitro models of human multiple myeloma (MM), we here investigated the anti-tumor potential of oleacein and the underlying bio-molecular sequelae. Within a low micromolar range, oleacein reduced the viability of MM primary samples and cell lines even in the presence of bone marrow stromal cells (BMSCs), while sparing healthy peripheral blood mononuclear cells. We also demonstrated that oleacein inhibited MM cell clonogenicity, prompted cell cycle blockade and triggered apoptosis. We evaluated the epigenetic impact of oleacein on MM cells, and observed dose-dependent accumulation of both acetylated histones and &alpha, tubulin, along with down-regulation of several class I/II histone deacetylases (HDACs) both at the mRNA and protein level, providing evidence of the HDAC inhibitory activity of this compound, conversely, no effect on global DNA methylation was found. Mechanistically, HDACs inhibition by oleacein was associated with down-regulation of Sp1, the major transactivator of HDACs promoter, via Caspase 8 activation. Of potential translational significance, oleacein synergistically enhanced the in vitro anti-MM activity of the proteasome inhibitor carfilzomib. Altogether, these results indicate that oleacein is endowed with HDAC inhibitory properties, which associate with significant anti-MM activity both as single agent or in combination with carfilzomib. These findings may pave the way to novel potential anti-MM epi-therapeutic approaches based on natural agents.

Aberrant expression of microRNAs (miRNAs) has been associated to the pathogenesis of multiple myeloma (MM). While miR-155 is considered a therapeutic target in several malignancies, its role in MM is still unclear. The analysis of miR-155 expression indicates its down-regulation in MM patient-derived as compared to healthy plasma cells, thus pointing to a tumor suppressor role in this malignancy. On this finding, we investigated miR-155 replacement as a potential anti-tumor strategy in MM. The miR-155 enforced expression triggered anti-proliferative and pro-apoptotic effects in vitro. Given the lower miR-155 levels in bortezomib-resistant as compared to sensitive MM cells, we analyzed the possible involvement of miR-155 in bortezomib resistance. Importantly, miR-155 replacement enhanced bortezomib anti-tumor activity both in vitro and in vivo in a xenograft model of human MM. In primary MM cells, we observed an inverse correlation between miR-155 and the mRNA encoding the proteasome subunit gene PSM&beta, 5, whose dysregulation has been largely implicated in bortezomib resistance, and we validated PSM&beta, 5 3&prime, UTR mRNA targeting, along with reduced proteasome activity, by miR-155. Collectively, our findings demonstrate that miR-155 elicits anti-MM activity, likely via proteasome inhibition, providing the framework for miR-155-based anti-MM therapeutic strategies.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still open questions. Herein, we investigated the functional significance of the oncogenic lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in MM. Our study demonstrates that NEAT1 expression level is higher in MM than in the majority of hematological malignancies. NEAT1 silencing by novel LNA-gapmeR antisense oligonucleotide inhibits MM cell proliferation and triggers apoptosis in vitro and in vivo murine MM model as well. By transcriptome analyses, we found that NEAT1 targeting downregulates genes involved in DNA repair processes including the Homologous Recombination pathway, which in turn results in massive DNA damage. These findings may explain the synergistic impact on apoptosis observed in MM cell lines co-treated with inhibitors of both NEAT1 and PARP. The translational significance of NEAT1 targeting is further underlined by its synergistic effects with the most common drugs administered for MM treatment, including bortezomib, carfilzomib, and melphalan. Overall, NEAT1 silencing is associated with a chemo-sensitizing effect of both conventional and novel therapies, and its targeting could therefore represent a promising strategy for novel anti-MM therapeutic options.

Simple Summary Genomic instability (GI) plays an important role in the pathobiology of multiple myeloma (MM) by promoting the acquisition of several tumor hallmarks. Molecular determinants of GI in MM are continuously emerging and will be herein discussed, with specific regard to non-coding RNAs. Targeting non-coding RNA molecules known to be involved in GI indeed provides novel routes to dampen such oncogenic mechanisms in MM. Abstract Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal proliferation of malignant plasma cells (PCs) within a permissive bone marrow microenvironment. The pathogenesis of MM is unequivocally linked to the acquisition of genomic instability (GI), which indicates the tendency of tumor cells to accumulate a wide repertoire of genetic alterations. Such alterations can even be detected at the premalignant stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) and, overall, contribute to the acquisition of the malignant traits underlying disease progression. The molecular basis of GI remains unclear, with replication stress and deregulation of DNA damage repair pathways representing the most documented mechanisms. The discovery that non-coding RNA molecules are deeply dysregulated in MM and can target pivotal components of GI pathways has introduced a further layer of complexity to the GI scenario in this disease. In this review, we will summarize available information on the molecular determinants of GI in MM, focusing on the role of non-coding RNAs as novel means to tackle GI for therapeutic intervention.

The microRNA (miRNA) cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce ribonuclease H-mediated degradation of MIR17HG primary transcripts and consequently prevent biogenesis of miR-17-92 miRNAs (miR-17-92s). The leading LNA ASO, MIR17PTi, impaired proliferation of several cancer cell lines (n = 48) established from both solid and hematologic tumors by on-target antisense activity, more effectively as compared with miR-17-92 inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells and induces MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo antitumor activity in nonobese diabetic severe combined immunodeficient mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetic profiles in nonhuman primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies.

Integration of multi-omics data from different molecular levels with clinical data, as well as epidemiologic risk factors, represents an accurate and promising methodology to understand the complexity of biological systems of human diseases, including cancer. By the extensive use of novel technologic platforms, a large number of multidimensional data can be derived from analysis of health and disease systems. Comprehensive analysis of multi-omics data in an integrated framework, which includes cumulative effects in the context of biological pathways, is therefore eagerly awaited. This strategy could allow the identification of pathway-addiction of cancer cells that may be amenable to therapeutic intervention. However, translation into clinical settings requires an optimized integration of omics data with clinical vision to fully exploit precision cancer medicine. We will discuss the available technical approach and more recent developments in the specific field.

One of the major challenges in the epilepsy field is identifying disease-modifying drugs in order to prevent or delay spontaneous recurrent seizure onset or to cure already established epilepsy. It has been recently reported that fingolimod, currently approved for the treatment of relapsing-remitting multiple sclerosis, has demonstrated antiepileptogenic effects in 2 different preclinical models of acquired epilepsy. However, to date, no data exist regarding the role of fingolimod against genetic epilepsy. Therefore, we have addressed this issue by studying the effects of fingolimod in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, a well-established genetic model of absence epilepsy, epileptogenesis, and neuropsychiatric comorbidity. Our results have demonstrated that an early long-term treatment with fingolimod (1 mg/kg/day), started before absence seizure onset, has both antiepileptogenic and antidepressant-like effects in WAG/Rij rats. However, these effects were transitory, as 5 months after treatment discontinuation, both absence seizure and depressive like-behavior returned to control levels. Furthermore, a temporary reduction of mTOR signaling pathway activity, indicated by reduced phosphorylated mammalian target of rapamycin and phosphorylated p70S6k levels, and by increased phosphorylated Akt in WAG/Rij rats of 6 months of age accompanied the transitory antiepileptogenic effects of fingolimod. Surprisingly, fingolimod has demonstrated longer-lasting positive effects on cognitive decline in this strain. This effect was accompanied by an increased acetylation of lysine 8 of histone H4 (at both 6 and 10 months of age). In conclusion, our results support the antiepileptogenic effects of fingolimod. However, the antiepileptogenic effects were transitory. Moreover, fingolimod might also have a positive impact on animal behavior and particularly in protecting the development of memory decline.

Background Bone disease (BD) is a hallmark of multiple myeloma (MM) and is characterized by severe skeleton damage, reduced quality of life and overall survival (1-2). Several findings indicated that IL-17 producing CD4+ T cells (Th17) play a central role in triggering MMBD and support MM cell growth mainly by IL-17 production. There is compelling evidence that miR-21 is a central player in Th17 effector functions. Our preliminary data have shown that miR-21 is highly upregulated in MM-Th17 isolated from patients with active BD as compared to MM with no active BD and controls. We found that inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired differentiation towards Th17 in vitro, by reducing interleukin (IL)-17, IL-22, RANKL and RORC, leading to abrogation of osteoclast (OCL) bone resorption. Aims Based on these premises, we sought to explore miR-21 related underlying molecular networks that support pathogenic Th17 differentiation and function. As miRNAs may exert direct and indirect effects on gene expression and at post-transcriptional level, we performed a global head-to-head comparison by RNA-seq and proteomic -phosphoproteomic analysis on miR-21i-Th17. Then, we recapitulated and validated our findings in NOD/SCID gNULL mice, injected intratibially with miR-21i-T cells and MM cells. Methods RNAseq and proteomic/phosphoproteomic assays have been performed on in vitro differentiated Th17 cells originated from scramble control (SC) or miR-21i transfected naïve T cells (SC-Th17 and miR-21i-Th17 respectively) from 3 healthy donors through MARS-seq protocol adapted for bulk RNA and proteome/phosphoproteome analysis . Data have been analyzed through R by using different packages including limma, DESEQ2 and pheatmap. To perfom global proteome/phosphoproteome analysis, we conducted a mass spectrometry study of phosphopeptides protein extract from SC-Th17 and miR-21i-Th17, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software Results In the presence of miR-21i, we found 109 upregulated and 22 downregulated proteins in the global proteome analysis of Th17 cells, while 90 and 18 phosphoproteins were up and down modulated, respectively. Paired analysis showed that 46 proteins are modulated in expression but not in phosphorylation, 23 proteins are modulated in phosphorylation but not in expression, while 85 proteins are modulated in both conditions. These data suggest that selective miRNA modulation interferes with a specific and limited group of proteins/phosphoproteins according to cell type and despite predicted pleiotropic miRNA activity. To understand whether miR-21i-Th17 undergo a "molecular reprogramming", we evaluated gene expression by RNA seq Analysis of miR-21-related molecular pathways in Th17 cells and found upregulation of STAT-1/-5a-5b, downregulation of STAT-3 and redirection of Th17 to Th1/activated like cells as shown by a pair-to-pair RNAseq and proteome/phosphoproteome analysis. These data indicate that miR-21 plays a central role in driving Th17 differentiation and function in a proinflammatory milieu such as MM-Bone marrow microenvironment (BMM). However, when miR-21 activity is strongly counteracted, pathogenic Th17 can switch to a Th1 like phenotype (STAT 1 dependent gene/protein upregulation). This switch may partly explain the attenuation of MMBD observed in vitro. To confirm our observation in vivo, we injected intratibially miR-21i exposed- or scramble miR (SC) exposed-naïve CD4+ T cells together with MM cells into gamma null SCID mice. We observed that mice injected with SC CD4+ naïve T cells presented severe local skeleton damage, while bone structure was preserved in miR-21i naïve CD4+ T cells injected mice. Conclusions Our data highlight the relevance of miR-21 in supporting Th17 mediated MMBD onset and progression. The possibility to "reprogram" MM Th17 by miR-21 modulation opens a new avenue to develop miR-21 targeting therapeutic strategies to counteract BMM-dependent MM development and related-BD. Figure Disclosures Paiva: Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Locked nucleic acid (LNA) oligonucleotides have been successfully used to efficiently inhibit endogenous small noncoding RNAs in vitro and in vivo. We previously demonstrated that the direct miR-221 inhibition by the novel 13-mer LNA-i-miR-221 induces significant antimyeloma activity and upregulates canonical miR-221 targets in vitro and in vivo. To evaluate the LNA-i-miR-221 pharmacokinetics and pharmacodynamics, novel assays for oligonucleotides quantification in NOD.SCID mice and Cynomolgus monkeys (Macaca fascicularis) plasma, urine and tissues were developed. To this aim, a liquid chromatography/mass spectrometry method, after solid-phase extraction, was used for the detection of LNA-i-miR-221 in plasma and urine, while a specific in situ hybridization assay for tissue uptake analysis was designed. Our analysis revealed short half-life, optimal tissue biovailability and minimal urine excretion of LNA-i-miR-221 in mice and monkeys. Up to 3 weeks, LNA-i-miR-221 was still detectable in mice vital organs and in xenografted tumors, together with p27 target upregulation. Importantly, no toxicity in the pilot monkey study was observed. Overall, our findings indicate the suitability of LNA-i-miR-221 for clinical use and we provide here pilot data for safety analysis and further development of LNA-miRNA-based therapeutics for human cancer.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) accounts for about 20% of pediatric and adult ALL cases. Despite the use of intensive chemotherapy protocols, 25% of children and 50% of adult patients fail to respond or relapse. The 3-years prognosis for these patients is poor and novel treatment options are needed. The targeting of tumor-associated antigens by monoclonal antibodies (mAb) is among the most investigated immune-therapeutic strategies. Accordingly, we developed a new humanized mAb (hUMG1), directed against a heavy glycosylated epitope of CD43 which presents a high reactivity against T-ALL cells. Here we investigated the pre-clinical therapeutic activity and the mechanisms of action of hUMG1 in experimental models of T-ALL. Methods: The expression of hUMG1 target was assessed by flow cytometry on tumor cell lines and primary samples from either T-ALL patients (n=48) or healthy donors (n =6). Humanized mAbs were generated by combining the variable domains of the murine antibody to the corresponding human IgG1 constant domains. Through immunohistochemistry (IHC) we screened several tissue microarrays (TMA) including human, cynomolgusmonkey and macacus rhesus (according to FDA/CE guidelines), rat and mouse normal tissues. Complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) on T-ALL cells were evaluated by flow cytometry. Six different in vivo models on NSG mice (3 with administration of NK-92-CD16+ effectors) have been generated to evaluate mAbs activity in different disease settings: orthotopic, subcutaneous advanced disease (treatments started at 100 mmc) and subcutaneous limited disease (treatments started the day after injection). Results: By screening different cancer cell lines, we observed hUMG1 target to be highly expressed on malignant T-ALL cells. We then tested T-ALL patient-derived blasts from 48 samples (40 pediatric and 8 adults) collected at diagnosis. The antigen was expressed on 23 out of 48 (48%), and most of them belonged to the subset of cortical (EGIL TIII) T-ALL group. By contrast the target antigen was not expressed on normal bone marrow cells from healthy donors. The analysis of the TMA including human normal tissues revealed a specific binding for thymus cortical lymphocytes only, leading us to hypothesize an acceptable safety profile. A humanized mAb, named hUMG1, and an afucosylated version of this mAb (aUMG1) were then developed. By gene expression profiling, western blot and flow cytometry, we observed that target binding by hUMG does not exert any direct activity on neoplastic cells. Subsequently, to investigate CDC, ADCC or ADCP, T-ALL cells were cultured in the presence of complement, peripheral blood mononuclear cells or macrophages, at increasing concentrations of both antibodies. Neither hUMG1 nor aUMG1 were able to induce CDC on target cells. Conversely, both mAbs induced CD16 downregulation, IFN-g production and degranulation on NK cells (more evident with aUMG1) and significant cytotoxicity against both T-ALL cell lines and primary blasts. Additionally, both mAbs induced ADCP. Lastly we observed a mAb-dependent activation of monocytes in the presence of target cells, as demonstrated by the reduction of CD16+ "non-classical" monocytes. Furthermore, we demonstrated potent activity of both mAbs in different T-ALL in vivo models. In an orthotopic model we observed 5 out of 20 treated mice free of disease after 100 days from injection as compared to none of the control group. In both subcutaneous models, we observed a strong ability of our antibody to delay tumor growth and to increase mice survival. Of note, the addition of NK-92-CD16+ strongly improved the activity of aUMG1. In the attempt to bring this antibody from bench to bedside, we assessed, through IHC, the expression of the hUMG1 target in healthy tissues from cynomolgus monkey, macacus rhesus, rat and mouse. We did not observe any reactivity, suggesting that the mAb target is very specific for human cells. Conclusion: Here, we demonstrated that hUMG1 mAb recognizes an antigen specifically expressed on the majority of T-ALL, and its binding is able to mediate effective cytotoxicity against leukemia in vitro and in vivo, indicating that this antibody, in particular the aUMG1 version, may represent a novel promising immune-therapeutic tool for the treatment of T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Although estrogens have been shown to regulate normal B cell proliferation and differentiation, the impact of estrogen receptor (ER) signaling on B cell malignancies, including Waldenström Macroglobulinemia (WM), remains unexplored. To address this issue, we have analyzed, through preclinical WM models, the druggability either of the classical estrogen receptor ERβ, an emerging target for acute leukemias and certain types of lymphomas, and of the novel, more recently deorphanized G-protein coupled ER GPER, whose therapeutic potential in solid tumors has recently emerged. Both ERβ and GPER were found expressed at mRNA and protein level in a panel of WM and IgM-secreting lymphoma cell lines. However, while treatment with ERβ agonists did not elicit any effect, selective GPER activation significantly reduced WM cell viability. On this basis, we investigated GPER expression pattern and therapeutic potential in WM. By interrogating public microarray datasets, we found GPER transcript significantly upregulated in WM as compared to normal B cells; moreover, immunohistochemical analysis showed elevated GPER expression in lymph node biopsies from newly-diagnosed WM patients as compared to healthy lymph nodes. Treatment with the selective GPER agonist G-1 ( (±) -1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl) -3a,4,5,9b-tetrahydro-3H cyclopenta [c]quinolin-8-yl] ethanone) led to reduced viability, clonogenicity and migration of WM and IgM-secreting lymphoma cell lines (IC50 at 48h ranging between 2.5 and 5.0 mM), including MYD88 and CXCR4 mutated cell lines, and even in the presence of bone marrow-derived stromal cells, while it did not affect healthy CD19+ B cell viability; conversely, GPER antagonists G-36 and G-15 slightly enhanced cell proliferation. The growth inhibitory activity of G-1 was associated with accumulation in G2/M cell cycle phase and induction of apoptosis, the latter phenomenon assessed by Annexin V/7AAD staining, analysis of mitochondrial membrane depolarization and cleavage of caspase 3, 7 and 9; hybridization of an antibody-array also highlighted G-1-induced down-regulation of anti-apoptotic proteins, including survivin and Bcl2. A significant effect of G-1 was also observed in primary tumor cells from refractory WM patients. Moreover, G-1 synergistically enhanced bortezomib and ibrutinib cytotoxicity in vitro. Importantly, intraperitoneal injection of G-1 (2mg/kg) significantly reduced the growth of BCWM-1 xenografts in NOD/SCID mice. To gain further insight into the consequences of selective GPER activation, we performed gene expression profile and GSEA analysis of WM cell lines treated with G-1. Of note, the p53 signaling pathway was strongly induced upon GPER activation, and upregulation of p53 and of its target genes or microRNAs (p21CIP1, Bax, Bad, PUMA and miR-34a) was confirmed both in G-1 treated WM cell lines and primary WM cells; moreover, G-1 combination with bortezomib or ibrutinib led to stronger increase in p53 and p21CIP1 levels as compared to single agent treatment. Finally, p53 knock-down partially reversed G-1-dependent anti-WM effects, thus suggesting p53 as downstream mediator of GPER. In summary, we report a significant anti-tumor activity upon selective GPER activation in WM cells, with apoptosis induction and a potent synergistic anti-WM activity in combination with ibrutinib and bortezomib. Altogether, these preclinical data suggest GPER agonists as a potential therapeutic option in WM. Disclosures Munshi: OncoPep: Other: Board of director.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy accounting for about 25% of all acute leukemias. Due to the poor therapeutic scenario and severe prognosis of T-ALL, novel drugs are eagerly awaited. The targeting of tumor-associated antigens by monoclonal antibodies (mAb) for induction of immune-mediated cellular cytotoxicity is presently a promising immunotherapeutic strategy. We previously developed a novel mAb direct against a heavy glycosylated oncofetal epitope of CD43 (UN1) with known potential application as therapeutic and diagnostic tool. By screening different cancer cell lines, we observed that UN1 is highly and selectively expressed on malignant T-ALL cells. The expression of UN1 was then evaluated in 38 T-ALL patient-derived blasts and high correlation for a specific subset of patients (about 80%) belonging to the cortical T-ALL group (EGIL T3) was detected. Accordingly, we developed a humanized mAb, (UMG1) and an afucosylated enginereed version (a-UMG1). Therefore, to elucidate the mechanism of action of these mAbs, we investigated complement-mediated cytotoxicity (CDC), ADCC and antibody-dependent cellular phagocytosis (ADCP). To this aim, T-ALL cells (HPB-ALL and CCRF-CEM) have been cultured in the presence of complement, peripheral blood mononuclear cells (PBMCs), NK-92-CD16+ cells or macrophages, using increasing concentrations of both mAbs. Notably, we observed that mAbs treatment alone did not exert either cellular cytotoxicity or CDC on target cells. Conversely, both mAbs induced significant ADCC mediated by PBMCs and NK-92-CD16+ cell line in term of NK degranulation and cytotoxicity and macrophage-mediated ADCP. In vivo results demonstrated a powerful activity of UMG1 in 3 different models of T-ALL in NSG mice in the presence or absence of NK-92-CD16+ cells. Specifically, in two subcutaneous models, we observed a strong ability of both mAbs to delay tumor growth and increase survival of treated mice. As expected, the combinatory treatment with NK-92-CD16+ cell line strongly improved the activity of a-UMG1. Importantly, in the orthotopic disseminated model, which better reproduces the human T-ALL disease, we observed 5 out of 20 treated mice alive and free of disease 100 days after injection. Furthermore, to explore the possibility of combination therapy, the modulation of UN1 expression by chemotherapeutic agents in T-ALL was investigated. Interestingly, we observed that methotrexate and doxorubicin , alone or in combination, increased UN1 expression at low nanomolar concentrations, and improved ADCC in vitro. In conclusion, we demonstrated that UMG1 and a-UMG1 represent a novel promising immune-therapeutic tool for the treatment of T-ALL patients. Citation Format: Cirino Botta, Maria E. Gallo Cantafio, Chiara Buracchi, Maria A. Siciliano, Maria Cucè, Caterina Riillo, Franca M. Tuccillo, Daniele Caracciolo, Emanuela Altomare, Mariamena Arbitrio, Maria T. Di Martino, Marco Rossi, Andrea Biondi, Giuseppe Gaipa, Pierosandro Tagliaferri, Pierfrancesco Tassone. UMG1, a novel humanized monoclonal antibody against a glysosylated-CD43-related epitope, induces antibody-dependent cellular cytotoxicity (ADCC) on human T-cell acute lymphoblastic leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1783.

Purpose: The onset of drug resistance is a major cause of treatment failure in multiple myeloma. Although increasing evidence is defining the role of miRNAs in mediating drug resistance, their potential activity as drug-sensitizing agents has not yet been investigated in multiple myeloma. Experimental Design: Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory multiple myeloma cells to melphalan. Results: miR-221/222 expression inversely correlated with melphalan sensitivity of multiple myeloma cells. Inhibition of miR-221/222 overcame melphalan resistance and triggered apoptosis of multiple myeloma cells in vitro, in the presence or absence of human bone marrow (BM) stromal cells. Decreased multiple myeloma cell growth induced by inhibition of miR-221/222 plus melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx–efflux transporters SLC7A5/LAT1 and the ABC transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory multiple myeloma xenografts with systemic locked nucleic acid (LNA) inhibitors of miR-221 (LNA-i-miR-221) plus melphalan overcame drug resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice. Conclusions: Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan resistance in preclinical models of multiple myeloma, providing the framework for clinical trials to overcome drug resistance, and improve patient outcome in multiple myeloma. Clin Cancer Res; 22(5); 1222–33. ©2015 AACR.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p

Stable nucleic acid lipid vesicles (SNALPs) encapsulating miR-34a to treat multiple myeloma (MM) were developed. Wild type or completely 2′-O-methylated (OMet) MiR-34a was used in this study. Moreover, SNALPs were conjugated with transferrin (Tf) in order to target MM cells overexpressing transferrin receptors (TfRs). The type of miR-34a chemical backbone did not significantly affect the characteristics of SNALPs in terms of mean size, polydispersity index, and zeta potential, while the encapsulation of an OMet miR-34a resulted in a significant increase of miRNA encapsulation into the SNALPs. On the other hand, the chemical conjugation of SNALPs with Tf resulted in a significant decrease of the zeta potential, while size characteristics and miR-34a encapsulation into SNALPs were not significantly affected. In an experimental model of MM, all the animals treated with SNALPs encapsulating miR-34a showed a significant inhibition of the tumor growth. However, the use of SNALPs conjugated with Tf and encapsulating OMet miR-34a resulted in the highest increase of mice survival. These results may represent the proof of concept for the use of SNALPs encapsulating miR-34a for the treatment of MM.

Purpose: Primary plasma cell leukemia (pPCL) is a rare and very aggressive form of plasma cell dyscrasia. To date, no information on microRNA (miRNA) expression in pPCL has been reported. This study aimed at investigating the involvement of miRNAs in pPCL and their possible relationship with higher tumor aggressiveness. Experimental design: Global miRNA expression profiles were analyzed in highly purified malignant plasma cells from 18 pPCL untreated patients included in a prospective clinical trial. MiRNA expression patterns were evaluated in comparison with a representative series of multiple myeloma patients, in relation to the most recurrent chromosomal abnormalities (as assessed by fluorescence in situ hybridization and single-nucleotide polymorphism-array analysis), and in association with clinical outcome. MiRNA expression was also integrated with gene expression profiles in pPCL and multiple myeloma samples. Results: We identified a series of deregulated miRNAs in pPCL (42 upregulated and 41 downregulated) in comparison with multiple myeloma. Some of them, on the basis of their reported functions and putative target genes computed by integrative analysis, might have a role in the pathobiology of pPCL. As regards chromosomal aberrations, the expression of some miRNAs mapped to hotspot altered regions was associated with DNA copy number of the corresponding loci. Finally, 4 miRNA (miR-497, miR-106b, miR-181a*, and miR-181b) were identified as having expression levels that correlated with treatment response, and 4 (miR-92a, miR-330-3p, miR-22, and miR-146a) with clinical outcome. Conclusions: Overall, our study provides insights into the possible contribution of miRNAs in the pathogenesis of pPCL and suggests targets for future therapeutic investigations. Clin Cancer Res; 19(12); 3130–42. ©2013 AACR.

miRNAs therapeutics is based on the use of oligonucleotide engineered to efficiently replace or sequester endogenous miRNAs. miR-221 is upregulated in several tumors, including multiple myeloma (MM), where it promotes cancer cell proliferation mainly via targeting the negative regulators of G1/S cell cycle progression p27 and/or p57. We previously demonstrated that direct miR-221 inhibition by the use of an original LNA inhibitor, LNA-i-miR-221, induces significant anti-MM activity and upregulates canonical targets in vitro and in vivo.To assess pharmacokinetics (PK)/pharmacodynamics of LNA-i-miR-221, we set-up, and report here, novel assays for oligo quantification in plasma, urine and tissues of NOD.SCID mice and Cynomolgus monkeys. We investigated the LNA-i-miR-221 PKs by UHPLC-MS/MS, following solid-phase extraction, in plasma and urine, and tissue uptake by in-situ hybridization (ISH). In particular, PK profile was calculated by UHPLC-MS/MS after single dose i.p injection of LNA-i-miR-221 (25 mg/kg) in mouse and the Tmax was identified at 1.5 hours with Cmax of 1110 ng/mL. Three hours after bolus injection, the plasma concentration was about 60% reduced and the molecule was not detectable at later time-points. The plasma exposure of the oligo showed an AUClast of 2330 h*ng/mL. Similarly, in monkeys the LNA-i-miR-221 plasma concentration following a single i.v. bolus injection (8.75 mg/kg) peaked at a Tmax of ½ hour, with a Cmax of 23600 ng/mL. The plasma exposure of the oligo showed an AUClast of 49300h*ng/mL, C0 of 61400 ng/mL and the t1/2 was 12.8 hours. Urine PK evaluation was performed in monkey on samples collected from 2 up to 48 hours. The excreted LNA-i-miR-221 was quantified equal to 0.68 mg with a total urinary recovery of approximately 2.68% of the amount injected (25,375 mg). Specifically, the urinary concentration/time-points plot shows the excretion of 9394,5 ng/mL (1.63% of recovery) in the interval of 2-6 hours. In addition, murine tissues analyzed by ISH revealed strong signals and long-lasting accumulation of the oligo in mice vital organs together with upregulation of the main canonical miR-221 target p27, as assessed by IHC. In addition, ISH analysis excluded that the LNA-i-miR-221 crosses the blood-brain barrier. All together PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary excretion of the intact molecule, with a very long tissue half-life and biological activity. Moreover, we report preliminary toxicity analysis in non-human primates. No changes in the monkeys’ behavior, body weight or food consumption were observed in treated animals.In conclusion our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a favorable PK, long-lasting tissue bioavailability and good safety profile in mice and in monkeys, providing the rationale for translation of this novel miR-221 inhibitor in early clinical investigation Citation Format: Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Boye S. Nielsen, Mariamena Arbitrio, Annamaria Gullà, Chiara Mignogna, Massimo Breda, Niels M. Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone. Pharmacokinetics and biodistribution of LNA-i-miR-221 in NOD.SCID mice and Cynomolgus monkeys. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1079.

There is emerging evidence that miR-17-92 plays a crucial role in c-Myc driven tumorigenesis of multiple myeloma (MM). We attempted to antagonize its full-oncogenic activity by targeting primary transcripts (pri-miR-17-92) with RNase H-triggering antisense oligonucleotides (LNA gapmeRs). Specifically, 7 different molecules were generated and screened for their ability to inhibit pri-miR-17-92 expression. The most effective molecule, henceforth named miR17-92-i-PT, was selected for furher investigation. As assessed by qRT-PCR, transfection of MM cells with miR17-92-i-PT resulted in downregulation of both pri-miR-17-92 and all 6 mature miRNA transcripts. Importantly, miR17-92-i-PT inhibited MM cell proliferation more effectively than inhibitors targeting each cluster's miRNA. Lack of relevant off-target effects was demonstrated by specifically-designed negative (LNA mixmeRs) and positive (not-phosphorothioated LNA gapmeRs) control oligos, and a dominant-negative genome-edited cell line by CRISPR/CAS9 technology is currently under development. Importantly, treatment of MM cells with naked miR17-92-i-PT resulted in miR-17-92 downregulation. Low micromolar concentrations of miR17-92-i-PT significantly affected survival of MM patient plasma cells (n = 14) and MM cell lines (n = 15), while the viability of CD138+ cells from MGUS patients (n = 3) or PBMCs from healthy donors (n = 3) was not impaired. Further, a synergistic in vitro activity with Bortezomib,Carfilzomib or Melphalan was demonstrated. Molecular perturbations in MM cells exposed to miR17-92-i-PT were firstly investigated by gene expression profiling (HTA 2.0, Affimetrix). Ingenuity pathway analysis (IPA) of differentially expressed genes highlighted alteration of relevant molecular functions, including “cell cycle”, “DNA replication, recombination, and repair”, and “cell death and survival”, which were validated by functional assays. Moreover, impairment of BRD4 activity was indicated by upstream regulator analysis (Zscore Citation Format: Eugenio Morelli, Lavinia Biamonte, Cinzia Federico, Maria Teresa Di Martino, Nicola Amodio, Maria Eugenia Gallo Cantafio, Niels M. Frandsen, Maria Rita Pitari, Daniele Caracciolo, Annamaria Gullà, Marco Rossi, Pierosandro Tagliaferri, Pierfrancesco Tassone. Targeting oncogenic miR-17-92 primary transcripts by LNA gapmeRs in multiple myeloma: Molecular findings and therapeutic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1077.

Purpose: Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. Experimental Design: Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. Results: Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. Conclusions: Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a–based treatment strategies in MM patients. Clin Cancer Res; 18(22); 6260–70. ©2012 AACR.

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.

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.