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Alternative Non Homologous End Joining DNA repair as therapeutic target in Multiple MyelomaDaniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. UMG of Catanzaro, Catanzaro, ItalyBackground- Multiple Myeloma (MM) is a hematologic malignancy strongly characterized by genomic instability. We previously demonstrated that LIG3-dependent Alt-NHEJ repair is significantly involved in the genomic instability which promote survival and drug resistance of MM cells. Based on this rationale, we aimed to elucidate the efficacy of available Alt-NHEJ pathway inhibitors as new therapeutic agents in MM. Materials and methods- Cell proliferation and apoptosis were evaluated with CellTiter-Glo assay and Annexin V staining. Alt-NHEJ repair was evaluated using EJ2-GFP and EJ5-GFP plasmids. LIG3, LIG1, PARP1, Caspase 3 and p-H2AX, levels were analyzed by Western blot of whole protein extracts. In vivo anti-MM activity was evaluated in NOD-SCID mice bearing subcutaneous AMO-1 Bortezomib resistant (ABZB) xenografts, daily treated with Olaparib via oral gavage. Results- By interrogating public available datasets, we found that higher mRNA expression of Alt-NHEJ core components was correlated with shorter survival of MM patients. Indeed, plasmid-based reporter assays confirmed a prevalent activation of Alt-NHEJ repair in MM cells as compared to normal lymphoblastoid cell lines, except for U266 cells which instead displayed a prevalence of Canonical-NHEJ repair activity. Next, we attempted to evaluate the effects of available Alt-NHEJ inhibitors on MM cell survival. Consistently, L67 (LIG3-LIG1 inhibitor) or Olaparib (PARP-inhibitor) induced a significant reduction of proliferation and clonogenic cell growth of different MM cell lines at low micromolar concentrations, while no significant effects on survival were observed in U226 as well as in normal lymphoblastoid cells. Importantly L67 or Olaparib impaired viability of MM cell lines or primary plasmacells co-cultured on stromal cells, thus overcoming the supporting role played by MM-microenvironment on plasma-cells survival. As result of Alt-NHEJ repair inhibition, anti-proliferative effects were correlated to increase of DNA double-strand breaks (DSBs), cell cycle arrest and finally apoptosis. Notably, we observed that Bortezomib resistant AMO-1 derivative cells (ABZB) exhibited significantly increased sensitivity to a combination of L67 and Olaparib that increased the number of DSBs. Moreover, Alt-NHEJ inhibitors synergistically sensitized primary cells as well as MM cell lines to Bortezomib. Finally, to demonstrate the in vivo relevance of our findings, we showed that clinically available Parp-inhibitor Olaparib, exerted a significant anti-MM activity on ABZB cells injected in immunocompromised mice. Conclusion- Taken together, our findings indicate that MM cells are addicted to Alt-NHEJ repair parthway, which therefore represent a novel druggable target pathway in MM. Citation Format: Daniele Caracciolo, Martina Montesano, Emanuela Altomare, Grazia Consolo, Nicola Amodio, Maria Teresa Di Martino, Marco Rossi, Cirino Botta, Eugenio Morelli, Giada Juli, Pierosandro Tagliaferri, Pierfrancesco Tassone. Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma [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 2827.

Th17 cells play a critical role in Multiple Myeloma (MM) microenvironment by triggering osteoclastogenic activity, that leads to severe skeleton damage. We have recently found that Th17 cells directly promote osteoclast (OCL) differentiation and function via expression of RANKL. It has been demonstrated that miRNAs are involved in the differentiation of Th17 cells. Murugaiyan et al. demonstrated that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-β signaling. Indeed, we have been able to negatively modulate Th17 differentiation by a specific inhibitor of miR-21 (mir21i). The overall effect of mir-21 inhibition is the suppression of OCL resorptive activity, thus attenuating Th17 mediated multiple myeloma bone disease (MMBD). However, the molecular mechanisms underlying these biological effects have not been clarified. On these bases, we aimed to identify proteins involved in the effect of miR21i on Th17 cells. Quantitative phosphoproteomics technologies were applied to study the effects of miR21i in Th17 cells. To unveil the molecular targets responsible for this interaction, Ingenuity's IPA® software was interrogated. This approach allowed to identify several unknown miR-21 target proteins and the related signal transduction pathway.To perfom global phosphoproteome analysis, we performed a mass spectrometry study of phosphopeptides on protein extracts from miR21i- treated Th17 and control Th17 cells, enriched using SCX-IMAC/TiO2. High-resolution LC-Ms/MS data were processed using Proteome Discoverer software. Among analyses of more than 1134 phosphorylation sites, 386 have shown significant changes, as compared to controls. Interestingly, IPA analysis showed a generally higher level of phosphoserine sites and top canonical pathways affected by miR-21 inhibition including PI3K/AKT, 14-3-3 and GRANZYME A signaling. Moreover IPA analysis disclosed RANKL among the top upstream proteins. The most up regulated protein was CYTIP and the ones with enhanced activity were PDCD4, STAT1, MINK1, FOXO1, while ITGAL showed reduced activity. To validate these data, we performed WB analysis and explored additional widely acknowledged Th17 regulators. Upon miR-21i treatment, the expression of PDCD4 and FOXO1 were significantly increased while lower levels of the master Th17 transcription factor, RORC, were observed. Furthermore, PIAS3 and TBX21 were significantly upregulated by mir21i. As PIAS3 and PDCD4 negatively regulate STAT3, we checked phospho-STAT3 expression and found that it was indeed downregulated, as expected. Overall, this study defines a phosphoproteomic signature of miR-21i treated Th17 cells, providing new insights into molecular impairment of Th17 differentiation by miR21i and suggest innovative molecular targeted approaches to treat MMBD Citation Format: Emanuela Altomare, Marco Rossi, Daniele Caracciolo, Nicola Amodio, Marco Gaspari, Domenico Taverna, Domenica Scumaci, Cirino Botta, Annamaria Gullà, Eugenio Morelli, Paola Critelli, Mariamena Arbitrio, Pierosandro Tagliaferri, Pierfrancesco Tassone. Phosphoproteomic analysis of miR-21 modulation in Th17 cells: Potential implications for multiple myeloma bone disease therapy [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 4431.

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.

Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by deep genomic instability. Alternative non-homologous end-joining (A-NHEJ) DNA repair is up-regulated in a significant fraction of cancers and it is more error-prone than the major LIG4-dependent NHEJ repair pathway (classic-NHEJ). Principal components of A-NHEJ (PARP1, POLQ and LIG3) represent therefore a new class of cancer cell-specific therapeutic targets. There is Increasing evidence that microRNAs take actively part in the regulation of the DNA damage/repair network. Based on this rationale, we aimed to elucidate the role of miRNAs in the regulation of A-NHEJ-driven genomic instability in MM. We first examined the prognostic role of DNA Ligases involved in NHEJ pathways interrogating the public MM gene expression dataset GSE9782, which revealed higher expression of LIG3 in patients with shorter survival; conversely LIG4 expression was not associated with worse prognosis, thus supporting the idea that LIG3 has a pivotal role in genomic instability and progression of MM. Next, we aimed to identify LIG3-targeting miRNAs by an integrated approach that predicted miR-22 as negative regulator of LIG3 in MM patients. On this basis we evaluated miR-22 and LIG3 expression in a panel of MM cell lines and primary samples compared to plasma cells from healthy donors. We detected significant down-regulation of miR-22 and up-regulation of LIG3, strictly correlated to disease progression. Subsequently, we induced miR-22 overexpression (by transduction or transfection) in a panel of MM cell lines and we found cell growth inhibition, apoptosis induction and cell cycle arrest. Importantly, transfection of miR-22 mimics reduced the viability of primary MM cells but not of HD-PBMCs. We next examined the effects of enforced overexpression of miR-22 on DNA-damage response and we observed an increase of the DNA damage marker yH2AX, reduction of LIG3 at mRNA and protein level, and decrease of LIG3-3’UTR luciferase activity, indicating specific 3’ UTR targeting. Importantly, we observed inhibition of A-NHEJ repair by a functional assay based on the plasmid EJ2-GFP. Finally, miR-22 mimics potentiated cytotoxic effects of PARP-inhibitor Olaparib (Selleckchem), both in vitro and in vivo, confirming that co-inhibition of LIG3 and PARP-1 have a synergistic effect on A-NHEJ dependent MM cells. Taken together, our preliminary findings indicate that miR-22 inhibits A-NHEJ by specific targeting of LIG-3 and modulates sensitivity to PARP inhibition. Further investigations could shed new light on A-NHEJ in MM pathogenesis and define a role for miR-22 as therapeutic agent in this still incurable disease. Acknowledgement: This work has been supported by Italian Association for Cancer Research (AIRC). PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille” n.9980, 2010/15 Citation Format: Daniele Caracciolo, Eugenio Morelli, Maria Teresa Di Martino, Daniela Talarico, Cirino Botta, Nicola Amodio, Cinzia Federico, Emanuela Altomare, Lavinia Biamonte, Maria Angelica Stamato, Pierosandro Tagliaferri, Pierfrancesco Tassone. Synthetic miR-22 inhibits alternative non homologous end-joining DNA repair and increases sensitivity to PARP-inhibition in multiple myeloma cells. [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 1066.

The onset of resistance to melphalan (MF) in Multiple Myeloma (MM) patients adversely affects disease outcome. Presently there is increasing evidence on the pivotal role of microRNA (miRNAs) in mediating drug-resistance. We here investigated the effect of an original LNA inhibitor of microRNA-221 (LNA-i-miR-221), in restoring sensitivity to MF in MM cells. LNA-i-miR-221 has been previously demonstrated as anti-tumor agent in preclinical models of MM. We studied the MF-sensitizing effect of LNA-i-miR-221 on MM cells in vitro and in vivo. MiR-221/222 expression inversely correlated with MF-sensitivity in a panel of 6 MM cell lines. Noteworthy, LNA-i-miR-221 overcame MF-resistance in vitro significantly enhancing apoptotic cell death as demonstrated by activation/cleavage of caspase-8, caspase-9 and caspase-3, as well as by PARP cleavage and by histone H2AX (γ-H2AX) phosphorylation. Importantly, the synergistic activity of LNA-i-miR-221 with MF affected cell survival even in the presence of bone marrow stromal cells (BMSCs). Gene expression profiling (GEP) comparing treated and untreated MF-sensitive and -resistant MM cell lines was performed to investigate genes and pathways significantly affected by combinatory treatment. Based on the GEP findings, we confirmed by Western Blot that the growth impairment occurred together with up-regulation of pro-apoptotic BBC3/PUMA protein, a target of miR-221/222 which we validated by luciferase assay in our system. Moreover, GEP analysis showed modulation of drug-influx -efflux transporters such as L-type amino acid transporter 1 (LAT1) and multidrug resistance associated protein 1 (MRP1/ABCC1) which was confirmed by Western Blot analysis. Indeed, LNA-i-miR-222 led to a reduction of the Side Population in MF-resistant cells as assessed by Hoechst dye exclusion assay. Finally, treatment of human MM xenografts in SCID/NOD mice with LNA-i-miR-221 plus MF induced a marked antitumor effect, which again occurred together with BBC3/PUMA and MRP1/ABCC1 proteins modulation in retrieved tumors. Taken together, our findings provide proof-of-concept that LNA-i-miR-221 overcomes MF-resistance of MM cells, providing the rationale for clinical translation of LNA-i-miR-221/MF combination in MM patients. Citation Format: Annamaria Gulla, Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Eugenio Morelli, Nicola Amodio, Cirino Botta, Maria Rita Pitari, Santo Giovanni Lio, Pierosandro Tagliaferri, Pierfrancesco Tassone. A 13 mer LNA miR-221 inhibitor restores drug sensitivity in melphalan-refractory multiple myeloma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2015-4426

Upregulation of miR-221/222 has been found in several solid and hematologic malignancies, including multiple myeloma (MM), and is thought to have oncogenic potential and promote cell proliferation via down-regulation of p27 and/or p57, negative regulators of cell cycle progression. We previously demonstrated that upregulation of both miRNAs occurs in malignant plasma cells (PCs) from multiple myeloma (MM) patients belonging to TC2 and TC4 (traslocation/Cyclin) groups. 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 (Di Martino et al. Oncotarget, 2013). In the aim to progress to clinical translation of our proof-of-principle findings, we here investigated the anti-tumor activity and the appropriateness for systemic delivery of 10 different, originally designed, miR-221/222 inhibitors which took advantage from locked nucleic acid (LNA) technology and phosphorothioate backbone for increasing the seed sequence binding stability and nuclease resistance. We found that electroporation of t(4;14) MM cells with a novel 13-mer miR-221 inhibitor, named LNA-i-miR-221, significantly inhibited growth and survival of MM cells in vitro. In treated cells, we detected knocking down of miR-221 together with increased levels of p27 mRNA and protein. Specific activity of this LNA-i-miR-221 against mature miR-221 was confirmed by the use of two 3’UTR reporter (luciferase renilla/firefly) constructs containing miR-221 target sites. These constructs were separately co-transfected either with miR-221/222 mimics or LNA-i-miR-221 into MM cells. As predicted, a reduced luciferase activity was detected in miR-221/222 mimics co-trasfected cells, while increase luciferase activity was measured in LNA-i-miR-221 co-transfected MM cells indicating an efficient and stable binding to the miRNA target sequence. Importantly, we evaluated the systemic delivery of the naked LNA-miR-221 inhibitor alone by intraperitoneal or intravenous injection route against MM xenografts in NOD.SCID 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 and increased levels of p27Kip1 in vivo. H&E staining and immunohystochemical analysis showed wide necrosis areas, reduced Ki67 and a significant increase of p27 cytoplasmic expression in retrieved tumors from LNA-i-miR-221-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. Citation Format: Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Annamaria Gullà, Emanuela Altomare, Eugenio Morelli, Nicola Amodio, Emanuela Leone, Cirino Botta, Niels M. Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone. In vitro and vivo activity against multiple myeloma cells of a novel locked nucleic acid (LNA)-miR-221 inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2014-4789