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Supplementary Table 1, Figures 1-4 from Oncogenic Role of miR-483-3p at the IGF2/483 Locus

Simple Summary For the first time, neuronal features are described in blastic plasmacytoid dendritic cell neoplasm (BPDCN) by a complex array of molecular techniques, including microRNA and gene expression profiling, RNA and Chromatin immunoprecipitation sequencing, and immunohistochemistry. The discovery of unexpected neural features in BPDCN may change our vision of this disease, leading to the designing of a new BPDCN cell model and to re-thinking the relations occurring between BPDCN and nervous system. The observed findings contribute to explaining the extreme tumor aggressiveness and also to propose novel therapeutic targets. In view of this, the identification, in this work of new potential neural metastatic inducers might open the way to therapeutic approaches for BPDCN patients based on the use of anti-neurogenic agents. Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Burkitt lymphoma (BL) is an aggressive neoplasm characterized by consistent morphology and phenotype, typical clinical behavior and distinctive molecular profile. The latter is mostly driven by the MYC over-expression associated with the characteristic translocation (8;14) (q24; q32) or with variant lesions. Additional genetic events can contribute to Burkitt Lymphoma pathobiology and retain clinical significance. A pathogenetic role for Epstein-Barr virus infection in Burkitt lymphomagenesis has been suggested; however, the exact function of the virus is largely unknown. In this study, we investigated the molecular profiles (genes and microRNAs) of Epstein-Barr virus-positive and -negative BL, to identify specific patterns relying on the differential expression and role of Epstein-Barr virus-encoded microRNAs. First, we found significant differences in the expression of viral microRNAs and in selected target genes. Among others, we identified LIN28B, CGNL1, GCET2, MRAS, PLCD4, SEL1L, SXX1, and the tyrosine kinases encoding STK10/STK33, all provided with potential pathogenetic significance. GCET2, also validated by immunohistochemistry, appeared to be a useful marker for distinguishing EBV-positive and EBV-negative cases. Further, we provided solid evidences that the EBV-encoded microRNAs (e.g. BART6) significantly mold the transcriptional landscape of Burkitt Lymphoma clones. In conclusion, our data indicated significant differences in the transcriptional profiles of EBV-positive and EBV-negative BL and highlight the role of virus encoded miRNA.

The regulatory protein nucleolin controls the expression of a subset of miRNAs involved in breast cancer progression and can be targeted to inhibit breast cancer growth in vivo., Numerous studies have described the altered expression and the causal role of microRNAs (miRNAs) in human cancer. However, to date, efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here we find that nucleolin (NCL), a major nucleolar protein, posttranscriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, that are causally involved in breast cancer initiation, progression, and drug resistance. We also show that NCL is commonly overexpressed in human breast tumors and that its expression correlates with that of NCL-dependent miRNAs. Finally, inhibition of NCL using guanosine-rich aptamers reduces the levels of NCL-dependent miRNAs and their target genes, thus reducing breast cancer cell aggressiveness both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer.

Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.

Vol. 210, No. 5, May 2013. Pages [951–968][1]. The authors regret that in their original paper, the RNU6 loading control panels shown in Fig. 1 (E and F) were incorrect as a result of an error in figure presentation during the final production process. The conclusions of the experiments shown in

hsa-mir-483 is located within intron 2 of the IGF2 gene. We have previously shown oncogenic features of miR-483-3p through cooperation with IGF2 or by independently targeting the proapoptotic gene BBC3/PUMA. Here we demonstrate that expression of miR-483 can be induced independently of IGF2 by the oncoprotein β-catenin through an interaction with the basic helix–loop–helix protein upstream stimulatory transcription factor 1. We also show that β-catenin itself is a target of miR-483-3p, triggering a negative regulatory loop that becomes ineffective in cells harboring an activating mutation of β-catenin. These results provide insights into the complex regulation of the IGF2/miR-483 locus, revealing players in the β-catenin pathway.

hsa-mir-483 is located within intron 2 of the IGF2 locus. We found that the mature microRNA (miRNA) miR-483-3p is overexpressed in 100% of Wilms' tumors. In addition, colon, breast, and liver cancers exhibit high or even extremely high levels of miR-483-3p in ∼30% of the cases. A coregulation with IGF2 mRNA was detected, although some tumors exhibited high expression of miR-483-3p without a concomitant increase of IGF2. These findings suggested that miR-483-3p could cooperate with IGF2 or act as an autonomous oncogene. Indeed, here we prove that an anti-miRNA oligonucleotide against miR-483-3p could inhibit the miRNAs without affecting IGF2 mRNA and it could suppress tumorigenicity of HepG2 cells, a cell line that overexpresses miR-483-3p and IGF2. Conversely, no antitumor effect was elicited by inhibition of IGF2. The oncogenic mechanism of miR-483-3p was at least partially clarified by the finding that it could modulate the proapoptotic protein BBC3/PUMA and miR-483-3p enforced expression could protect cells from apoptosis. Our results indicate that miR-483-3p could function as an antiapoptotic oncogene in various human cancers and reveal a new, potentially important target for anticancer therapy. Cancer Res; 70(8); 3140–9. ©2010 AACR.

Introduction: The first FDA-approved deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA, Vorinostat), was shown to be effective in vitro by a number of anti-neoplastic mechanisms. Despite minimal single-agent activity in multiple myeloma (MM), phase 1b studies combining HDACi’s with bortezomib salvaged some relapsed patients and prolonged progression free survival (PFS) from days (Vorinostat) to months (Panobinostat), albeit at the cost of significant side effects including fatigue, nausea, and vomiting. Phase 1/2 studies in combination with lenalidomide have demonstrated tolerability and activity in lenalidomide-refractory patients, but randomized trials are lacking. In MM, the anti-neoplastic mechanism of action for HDACi’s is unknown, but at biologically achievable concentrations, it has been theorized that they sensitize MM cells to other drugs by interfering with cell adhesion mediated drug resistance (CAM-DR), primarily involving the integrins CD44, CD49d (VLA-4), CD54 (ICAM-1), and/or CD184 (CXCR4). AR-42 (ARNO Therapeutics) is a novel orally bioavailable phenylbutyrate-based class I/II HDAC inhibitor that has greater anti-proliferative effects compared to Vorinostat in vitro and in vivo. It has been previously shown that in MM cell lines, AR-42 down-regulates the expression of gp130, and inhibits IL-6 induced activation of STAT3 and downstream targets including BCL-XL and Cyclin-D1, with minimal effects on the PI3K/AKT and MAPK pathways. CD44 is a type I transmembrane glycoprotein, which is directly transcribed by β-catenin, and its role in cell adhesion-mediated drug resistance (CAM-DR) for MM as well as other cancers has been largely investigated. Recent published data have shown that CD44 forms a complex with STAT3 and p300 (acetyltransferase) causing STAT3 activation in a cytokine- and growth factor-independent manner, and that CD44 over-expression is one of the main molecular mechanisms that contributes to Lenalidomide resistance in MM cells. Hypothesis: We hypothesize that CD44 down-regulation, both surface expression on MM cells as well as the soluble form in the blood, is the primary effect of AR-42 at concentrations achievable in humans, explaining its weak single agent effect and its improvement when combined with other therapeutic agents in the clinic. Methods: As part of a single center, dose-escalating, first-in-man phase 1 trial of single agent oral AR-42 administered orally three times weekly in 28-day cycles (3 weeks of treatment followed by a 7-day off treatment period), patients were accrued at 20-70 mg TIW in a standard 3+3 cohort design. Using peripheral blood obtained during cycle 1 of therapy, nCounter® GX Human Immunology assays and nCounter miRNA expression profile was performed to assess differentially expressed genes after AR-42 treatment. Enzyme-linked immunoabsorbent assay (ELISA), qRT-PCR and luciferase assays were also performed.To examine whether AR-42 treatment could sensitize the cells to Lenalidomide in vivo, we used GFP+/Luc+ MM.1S cells engrafted in NOD-SCID mice. Results: AR-42 in relapsed MM showed no confirmed partial responses, but did result in marginal responses in 3 out of 13 MM patients. We found that AR-42 in MM cells modulates the expression of many genes coding for surface receptors including CD44, CD48, CD46 and TRAF5 and affects the expression of several miRNAs. Our data show a decrease of CD44 mRNA expression in the CD138+ MM plasma cells and of the soluble CD-44 in the serum of AR-42 treated patients. We also show that in MM cells CD44 down-regulation upon AR-42 treatment is associated with impairment of STAT-3 signaling pathways and direct targeting of its regulatory RNA binding protein, Insulin grow factor 3 binding protein 3 (IGF2BP3), by miR-9-5p. We found that miR-9-5p is up-regulated in vitro and in the cancer cells of MM patients after AR-42 treatment. Moreover we show that AR-42 in combination with Lenalidomide show synergistic apoptotic effect on MM cells, enhancing Lenalidomide anti-myeloma activity invivo. Conclusions: These findings show that CD44 is a therapeutic target for the HDACi AR-42 in MM patients, providing the rationale to support further clinical investigation of AR-42 in combination with IMiDs in patient cohorts with high pretreatment CD44 expression in the serum and on the surface of MM cells. Disclosures Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO Therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.

MicroRNAs (miRNAs) are noncoding single-stranded RNA molecules of ∼22 nucleotides in length. They have a critical role in regulating gene expression by targeting messenger RNAs (mRNAs) in a sequence-specific manner. Active mature miRNAs are produced from primary miRNA transcripts (pri-miRNAs) through sequential cleavages by the microprocessor complex, which includes DROSHA, DGCR8, and DICER proteins. Several miRNAs are specifically up-regulated in various types of tumors, and a wide range of studies have demonstrated how their down-regulation could potentially affect tumorigenesis, metastasis formation and drug resistance. However, most of these studies have indicated the possibility of modulating miRNA expression at the transcriptional level, while only a few focused on post-transcriptional control. Nucleolin (NCL) is a highly conserved multifunctional protein involved in ribosomal RNA (rRNA) biogenesis and in the stabilization of different mRNAs, in the nucleus and in the cytoplasm of normal and cancer cells. NCL was also found on the surface of different types of cancer cells, but not on their normal counterparts, shuttling between the inner and the outer part of the cell membrane. These observations suggested that NCL might be considered a cancer cell specific receptor, able to mediate tumor-selective uptake of specific ligands such as RNA and DNA G-rich aptamers. Here we show that NCL binds the terminal loop and promotes the maturation of a specific set of miRNAs, including miR-21, miR-103, miR-221 and miR-222, whose over-expression is causally associated with greater aggressiveness and resistance to anti-neoplastic therapies of several kind of tumors, such as breast cancer. Accordingly, a direct correlation between the expression levels of NCL and NCL-dependent miRNAs in human breast cancer samples was also observed. Conversely, NCL impairment down-regulated NCL-dependent miRNAs and up-regulates their target in vitro, affecting cancer cell proliferation, migration and anti-neoplastic drug resistance. Finally, we used AS1411, the first NCL-targeting G-rich aptamer that has reached phase II clinical trials for cancer therapy, to inhibit NCL activity in orthotopc xenograft models of breast cancer. Our in vivo data demonstrate that AS1411 down-regulates NCL-dependent miRNAs, hindering breast cancer metastasis. These findings provide insights into the molecular function of NCL in miRNA biology, as well as one of the first realistic strategies for miRNA regulation in cancer therapy. Citation Format: Dario Palmieri, Luciana De Luca, Jessica Consiglio, Alberto Rocci, Tiffany Talabere, Claudia Piovan, Alessandro Lagana, Luciano Cascione, Jingwen Guan, Pierluigi Gasparini, Veronica Balatti, Vincenzo Coppola, Craig Hofmeister, Guido Marcucci, John Byrd, Stefano Volinia, Charles Shapiro, Michael Freitas, Carlo Croce, Flavia Pichiorri. In vivo NCL-targeting affects breast cancer aggressiveness through miRNA regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1122. doi:10.1158/1538-7445.AM2013-1122

Abstract 1304 Recently, a class of noncoding RNAs called microRNA (miRNAs) has emerged as critical gene regulators in cell growth, differentiation, disease and development. MiRNAs are 18–24 nucleotide long noncoding RNAs, which regulate gene expression by pairing with 3′ untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. Deregulated miRNA expression is reported in various human diseases including lymphomas, suggesting an important role in their pathogenesis. According to WHO classification, Burkitt lymphoma (BL) is a rare, highly aggressive NHL composed of monomorphic medium-sized B cells with multiple nucleoli and numerous mitotic figures and is more common in children than in adults. The molecular feature of BL is the translocation that places MYC under the control of immunoglobulin gene regulatory elements. High levels of c-MYC have been clearly shown to have a tumour-promoting effect. However, there is recent evidence that infrequent cases may lack an identifiable MYC translocation, the explanation for which is still uncertain, though suggesting the existence of pathogenetic mechanisms alternative to genetic alterations. Over the past years miRNA signatures have been described to characterize and classify different types of BL or to investigate the expression of miRNAs possibly regulated by c-Myc in BL cases positive or negative for Myc translocation. However, it remained unclear the functional role of differentially expressed miRNAs and no further studies have been conducted. We performed miRNA expression profile to gain further insights into the molecular pathology of BL. We conducted array analysis on a set of 5 sporadic BL patients, 3 endemic BL patients, 9 reactive tissues and 11 cases of mononucleosis. Our profile is the first one that shows the different expression between BL cases and normal B cells whereas recent miRNA profiles have been conducted in BL compared to other B-NHL (B-CLL, MCL & FL). A common trend of miRNAs altered expression was also observed by NanoString analysis in 10 BL cell lines compared to 5 normal CD-19+ B cells. Among several miRNAs previously described be deregulated in BL we identified a severe down-regulation of miR-221, miR-222 in all classes of comparisons we analyzed. The down-regulation of miR-221 and miR-222 associated to BL has been also confirmed by q-RT-PCR method in a different cohort of BL patients (20) compared to the healthy controls (6). We found that interesting considering the up-regulation of miR-221 and miR-222 previously confirmed in a lot of solid tumors by multiple studies. We are investigating a different role of the cluster miR-222 and miR-221 in lymphomas that have a different process in carcinogenesis than solid tumors. In vivo models to study the lymphomagenesis of BL have been created but until now no one studied the importance of the miRNAs in vivo. We analyzed the expression of miR-221 and miR-222 in a Myc transgenic mouse model. The transgene construct consists of the Myc oncogene (c-myc) in association with the Emu immunoglobulin heavy chain enhancer and Myc promoter. Expression of the mouse Myc transgene is restricted to the B cell lineage. Previously it has been shown an increase of pre-B cells in the bone marrow throughout life of hemizygotes and a transient increase in large pre-B cells in the blood at 3–4 weeks of age; moreover spontaneous pre-B and B cell lymphomas reach an incidence of 50% at 15–20 weeks in hemizygous progeny of a wildtype female mated with a hemizygous male. We observed the development of Burkitt lymphoma within 10 weeks of birth in 14 out of 25 Eu-Myc transgenic mice and a premature death in 5 out or 25 transgenic mice within 6–8 weeks of birth without showing any enlarged lymph nodes. Transgenic mice with masses showed the same phenotype characterized by enlarged spleen (3 fold), lymphosarcomas associated with BL and enlarged lymph nodes around the neck area. B-cells have been negatively selected from enlarged lymph nodes and enlarged spleen. A qRT-PCR has been conducted to evaluate the miR-221 and miR-222 expression. The miRNA levels showed a down-regulation in B cells collected from the masses when compared to normal B cells derived from the spleen of WT mice. In conclusion, our study reveals new insights into the functional significance in loss of miR-221 and miR-222 expression in BL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

The hsa-mir-483 locus is located within intron 2 of the IGF2 locus. Here, we show that the mature microRNA (miRNA) miR-483-3p is over-expressed and co-regulated with IGF2 in 100% of Wilms’ tumors. Additionally, several types of common adult cancers exhibit high or even extremely high levels of miR-483-3p. We show that the mutation status of the oncoprotein β-catenin is correlated with the expression of the miR-483-3p in hepatocarcinoma and colorectal cancer. These findings suggested an autonomous oncogenic function for this miRNA. Indeed, anti-microRNA oligonucleotide (AMO) against miR-483-3p could inhibit tumorigenicity of HepG2 cells. At the same time, no anti-tumor effect was elicited by inhibition of IGF2. The oncogenic properties of miR-483-3p were further supported by the finding that its ectopic expression could protect cells from apoptosis through the regulation of one important pro-apoptotic protein, Puma. Our results indicate that miR-483-3p can function as an anti-apoptotic oncogene in various human cancers and reveal a new potentially important target for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2087.