miR34a is a tumor-suppressor miRNA that functions within the p53 pathway to regulate cell-cycle progression and apoptosis. With apparent roles in metastasis and cancer stem cell development, miR34a provides an interesting opportunity for therapeutic development. A mimic of miR34a was complexed with an amphoteric liposomal formulation and tested in two different orthotopic models of liver cancer. Systemic dosing of the formulated miR34a mimic increased the levels of miR34a in tumors by approximately 1,000-fold and caused statistically significant decreases in the mRNA levels of several miR34a targets. The administration of the formulated miR34a mimic caused significant tumor growth inhibition in both models of liver cancer, and tumor regression was observed in more than one third of the animals. The antitumor activity was observed in the absence of any immunostimulatory effects or dose-limiting toxicities. Accumulation of the formulated miR34a mimic was also noted in the spleen, lung, and kidney, suggesting the potential for therapeutic use in other cancers. Mol Cancer Ther; 13(10); 2352-60. Ó2014 AACR.

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD 2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for highrisk NB patients.

Metabolic dysregulation is a hallmark of cancer. Many tumors exhibit auxotrophy for various amino acids, such as arginine, because they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase-1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase-1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct antitumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments antitumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Tumor-bearing mice (CT26, MC38, and MCA-205) receiving pegzilarginase in combination with anti–PD-L1 or agonist anti-OX40 experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust antitumor immunity characterized by increased intratumoral effector CD8+ T cells and M1 polarization of tumor-associated macrophages. Our data suggest potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both antigen-presenting cells and tumor cells, and increased presence of M1-like antitumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.

Tumor suppressor miRNAs function by regulating multiple cancer-related genes and pathways. The tumor suppressor miRNAs miR-34, let-7, miR-16, miR-26, and miR-145 have proven to be effective in inhibiting tumor growth in mouse models of cancer. Based upon the availability of systemic delivery technologies that provide significant accumulation of siRNAs in liver tumors, we chose to identify tumor suppressor miRNAs that might be used as therapies for hepatocellular carcinoma (HCC). Cell culture studies with mimics of eight tumor suppressor miRNAs revealed five with the capacity to significantly inhibit the proliferation of multiple HCC cell lines. The five miRNA mimics were complexed with several lipid formulations and injected into NOD/SCID mice with orthotopically grown Hep3B liver cancer xenografts. All five miRNAs significantly inhibited the growth of the liver tumors compared to animals treated with formulated negative control miRNAs. Intriguingly, mimics of three of the tumor suppressor miRNAs eliminated detectable tumors. Preliminary studies indicate that the formulated miRNAs are neither toxic nor immunostimulatory. The formulated miRNAs are currently being evaluated for therapeutic activity in mouse models of other cancers and a development program has been initiated in anticipation of creating candidates for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C142.

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MicroRNA (miRNA) mimics have emerged as a novel class of therapeutics with promising anti-oncogenic activity. These mimics are modeled after naturally occurring tumor suppressor miRNAs that are ubiquitously expressed in normal cells but frequently show a loss-of-function in human malignancies. The premise for the strong inhibitory activity is based on the observation these endogenous miRNAs control multiple oncogenic pathways commonly deregulated in cancer. Therefore, “miRNA replacement therapy” acts in accordance with our current understanding of cancer as a pathway disease that can only be successfully treated when intervening with multiple cancer pathways. We have identified a series of key tumor suppressor miRNAs, including miR-34, and validated the therapeutic potential in cultured cancer cells and mouse models of cancer. The translation of this potential into future medicines, however, was hampered by the lack of a robust clinically relevant delivery system. To facilitate a rapid route to the clinic, we have screened a panel of external delivery systems that are in pre-clinical development or have already reached the clinic featuring another oligonucleotide. Here, we present the pharmacologic and pharmacodynamic parameters of miRNA mimics complexed in ionizable NOV340 liposomes (SMARTICLEs, Marina Biotech, Bothell, WA) in an orthotopic tumor model of hepatocellular carcinoma. Treatment of mice carrying existing tumors mimics led to significant tumor regression, prolonged survival and lacked notable drug-related side effects. Some of the mice appeared to be tumor-free. The data demonstrate the therapeutic utility of the NOV340/miRNA formulation and support the initiation of IND-enabling studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5636. doi:1538-7445.AM2012-5636

The effective treatment of advanced Neuroblastoma (NB) is still a challenge in pediatric oncology, because the clinical use of most therapeutics is limited by insufficient drug delivery to the tumor and high systemic toxicity. The discovery of the RNAi has great promise for anti-cancer therapeutics but, as in high-grade solid tumors a single ‘oncogene addiction’ is rare, multi-'gene' target combinations are required and a targeted delivery system is mandatory to successfully translate RNAi-based therapeutics into the clinics. It is now ascertained the master role of ALK and related genes such as PHOX2B, able to promote its transcription, in NB growth and survival, opening new perspectives for RNAi-mediated therapies for NB. Nevertheless, even if the counterpart of tumor suppressor (TS) regulators in NB is not completely known, a number of miRNAs with TS functions have been found aberrantly under-expressed in NB and associated with aggressive phenotypes. To develop a new generation of targeted and multi-RNAi-directed therapeutics for NB, we are exploiting our well-established NB-specific delivery system, the GD2-Targeted Liposomes entrapping specific siRNAs, TL[siRNA], or miRNAs mimics, TL[miRNA]. The final purpose is to get simultaneously the silencing of the oncogenic functions of ALK and related genes and the replacement of miRNAs with TS functions in clinically relevant mouse models of NB. We have planned to test different combinations of individually entrapped TL[siRNA] or TL[miRNA] for in vivo therapeutic efficacy. In a pilot study, NB-bearing mice were treated with different TL[siRNA]. After 65 days from i.v. injection of HTLA-230 (WT ALK) cells, we observed: about 50% of TL[siALK]-treated mice still disease-free, marginal effects with TL[siPHOX2A] and moderate effects with TL[siPHOX2B] formulations alone and a significant increased mice survival with an equimolar combination of TL[siRNA] of each gene. The enhanced efficacy of ALK and PHOX2 genes knockdown combination highlights a powerful therapeutic potential. After pilot transfection studies of SH-SY5Y (F1174L mut ALK) and GI-LI-N (WT ALK) cell lines, TS miRNAs mimics were screened and selected. Cell proliferation was evaluated at different days post-transfection. miR-96, miR-34a and let7b replacement resulted the most effective in inhibiting NB cell growth. Liposomal preparation of miRNA-NC and the above mimics for in vivo studies was tuned up according to the number of negative charges of the stem-loop sequence with an entrapment efficiency of 80-90%. Currently, an ongoing in vivo trial indicates promising results after replacement of miR-34a and let7b. We believe that the use of tumor-targeted biocompatible carriers of RNAi molecules able to replace loss-of-functions and to knockdown gain-of-functions in cancer cells at the same time is likely to represent an innovative modality for a more effective gene therapy. Citation Format: Patrizia Perri, Daniela Di Paolo, Leslie Priddy, Annarita Di Fiore, Chiara Brignole, Fabio Pastorino, David Brown, Mirco Ponzoni. MicroRNA replacement and RNAi-mediated silencing of ALK as combined targeted therapies for neuroblastoma. [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 1453. doi:10.1158/1538-7445.AM2014-1453

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