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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

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.

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.

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