Malignant melanoma is an aggressive form of skin cancer with poor prognosis. Despite improvements in awareness and prevention of this disease, its incidence is rapidly increasing. MicroRNAs (miRNAs) are a class of small RNA molecules that regulate cellular processes by repressing messenger RNAs (mRNAs) with partially complementary target sites. Several miRNAs have already been shown to attenuate cancer phenotypes, by limiting proliferation, invasiveness, tumor angiogenesis, and stemness. Here, we employed a genome-scale lentiviral human miRNA expression library to systematically survey which miRNAs are able to decrease A375 melanoma cell viability. We highlight the strongest inhibitors of melanoma cell proliferation, including the miR-15/16, miR-141/200a and miR-96/182 families of miRNAs and miR-203. Ectopic expression of these miRNAs resulted in long-term inhibition of melanoma cell expansion, both in vitro and in vivo. We show specifically miR-16, miR-497, miR-96 and miR-182 are efficient effectors when introduced as synthetic miRNAs in several melanoma cell lines. Our study provides a comprehensive interrogation of miRNAs that interfere with melanoma cell proliferation and viability, and offers a selection of miRNAs that are especially promising candidates for application in melanoma therapy.

During embryonic development, axons extend over long distances to establish functional connections. In contrast, axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing CNS regeneration. Here, we performed one of the first miRNome-wide functional miRNA screens to identify miRNAs with robust effects on axon growth. High-content screening identified miR-135a and miR-135b as potent stimulators of axon growth and cortical neuron migration and in male and female mice. Intriguingly, both of these developmental effects of miR-135s relied in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon growth and regeneration. These results prompted us to test the effect of miR-135s on axon regeneration after injury. Our results show that intravitreal application of miR-135s facilitates retinal ganglion cell (RGC) axon regeneration after optic nerve injury in adult mice in part by repressing KLF4. In contrast, depletion of miR-135s further reduced RGC axon regeneration. Together, these data identify a novel neuronal role for miR-135s and the miR-135-KLF4 pathway and highlight the potential of miRNAs as tools for enhancing CNS axon regeneration. Axon regeneration in the adult mammalian CNS is limited in part by a reduced intrinsic capacity for axon growth. Therefore, insight into the intrinsic control of axon growth may provide new avenues for enhancing regeneration. By performing an miRNome-wide functional screen, our studies identify miR-135s as stimulators of axon growth and neuron migration and show that intravitreal application of these miRNAs facilitates CNS axon regeneration after nerve injury in adult mice. Intriguingly, these developmental and regeneration-promoting effects rely in part on silencing of Krüppel-like factor 4 (KLF4), a well known intrinsic inhibitor of axon regeneration. Our data identify a novel neuronal role for the miR-135-KLF4 pathway and support the idea that miRNAs can be used for enhancing CNS axon regeneration.

Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.

MicroRNAs (miRNAs) are a family of small, non-coding RNAs and serve as small snippets of genetic material that regulate gene expression. As a result, miRNAs modulate a wide range of biological processes including; cell cycle control and apoptosis, cell signaling and differentiation, cell adhesion and motility. Thus, it is not surprising that their misregulation is linked to cancer initiation, treatment response, and metastasis. Due to the inherent ability of miRNAs to concurrently target multiple pathways, their therapeutic potential to be used as anti-cancer drugs is attractive. In this study, we are investigating the mode of action and development of a novel synthetic miRNA mimic (INT-1B3) as a therapeutic candidate for substitution and functional restoration of a depleted miRNA in variety of cancers. To identify miRNAs that control the survival of cancer cells, we performed a functional miRNA screening in which a wide range of relevant miRNAs were selected and miR-193a-3p was chosen as the potential candidate for further preclinical characterization. Compelling evidence demonstrate a tumor suppressor function for miR-193a-3p in many cancer types and link its reduced expression with cancer, metastasis and therapy resistance. Consistent with the described role for miR-193a-3p in literature, our cell-based assays conducted in various experimental tumor cell systems demonstrated efficient target engagement leading to reduced cell proliferation/survival, cell cycle arrest and induction of apoptosis, inhibition of cell motility as well as a potential immune-modulatory role (via adenosine-mediated tumor immune evasion). Furthermore, development of a novel lipid nanoparticle-based formulation as an efficient delivery system demonstrated a marked anti-tumor activity of INT-1B3 as single agent following systemic administration in tumor-bearing mice. Robust tumor growth inhibition in these experimental tumor models is correlated and consistent with efficient target engagement in tumor cells and relevant organs. We are currently investigating the in vitro and in vivo effect of INT-1B3 in variety of cancer types including hepatocellular carcinoma, melanoma and triple negative breast cancer. Our data thus far supports an anti-tumor role for INT-1B3 and encourages further preclinical and clinical investigation. Citation Format: Sanaz Yahyanejad, Thijs de Gunst, Iman Schultz, Harm den Boer, Monica Raimo, Bryony Telford, Rogier Vos, Laurens van Pinxteren, Roel Schaapveld, Michel Janicot. Pharmacologic profile of INT-1B3: A novel synthetic microRNA 193a-3p mimic for therapeutic intervention in oncology [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 4405.

Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.

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TPS2666 Background: MicroRNAs (miR) are naturally-occurring small non-coding RNA molecules involved in the regulation of gene expression and their dysregulation plays a fundamental role in several pathological conditions including cancer. The miR-193a-3p acts as a tumor suppressor and is downregulated in many cancer types. INT-1B3 is a novel lipid-nanoparticle (LNP) formulated 1B3, a 22-nucleotide double stranded chemically-modified miR-193a-3p mimic. INT-1B3 showed significant tumor growth inhibition in a large panel of human and syngeneic tumor models. It directly targets tumor cells and the tumor microenvironment by specific modulation of multiple signaling pathway components. Furthermore, in syngeneic mice models for e.g. TNBC (4T1) and HCC (H22), INT-1B3 was able to modulate the immune tumor microenvironment by turning ‘cold’ tumors into ‘hot’ tumors via upregulation of cytokines (e.g., IL-2 and IFN-g), decreasing immunosuppressive cells/Treg (e.g., CD4+ /LAG3+ and CD3+ /FoxP3+) and triggering cytotoxic CD8+ T cell-mediated long-term memory immune protection against re-challenge with tumor cells. These preclinical results suggest potential clinical benefit in a broad range of cancer indications, and a reduced potential to develop drug resistance due to its multi-targeted mode of action. Methods: This is a 2-part, multi-center, open-label, multiple ascending dose, first-in-human, clinical study to evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of INT-1B3 in the treatment of patients with advanced solid tumors. The phase 1 part follows a ‘hybrid’ 3+3 study design in ‘all-comer’ cancer patients enrolled and treated in cohorts to define the recommended phase 2 dose (RP2D). Upon completion of the dose escalation phase of the study, 2 expansion cohorts are planned to further confirm the safety, tolerability, and preliminary efficacy of the RP2D of INT-1B3 in patients with hepatocellular carcinoma and triple negative breast cancer. Major eligibility criteria include evaluable disease according to RECIST 1.1 and no more than one prior line of anti-PD-1/PD-L1 therapy. Patients will receive INT-1B3 via 60-min i.v. infusions twice a week in 21-day cycles. The first patient was enrolled on January the 14th 2021. Clinical trial information: NCT04675996.

As cancer is a multifactorial disease, the multimodal action of microRNAs makes them an attractive tool for novel therapeutic approaches. The tumor suppressive miR-7-5p has been shown to act on many aspects of oncogenesis, including cell proliferation, migration and angiogenesis, by targeting a spectrum of key genes. We developed a synthetic chemically modified miR-7-5p mimic, 5A2, and performed a comprehensive functional characterization in a panel of human cancer cell lines. 5A2 reduced cell proliferation in most cell lines by inducing cell cycle arrest. To enable systemic delivery of 5A2 to tumors, it was formulated in a novel lipid nanoparticle (INT-5A2) and we demonstrated the anti-tumor activity of INT-5A2 in an experimental human liver tumor-bearing mouse model. Next, RNA-sequencing was used to gain more insight into the molecular mechanism of action of 5A2 and demonstrated a broad repression of target mRNAs. Interestingly, Ingenuity Pathway Analysis revealed a new role for 5A2 in metabolic pathways. Validation experiments in vitro showed that 5A2 reduced the expression of key glycolysis and glutaminolysis enzymes, leading to a decrease in glycolysis, lactate secretion and intracellular glutamate availability. Taken together, these data strongly suggest that miR-7-5p/5A2 is a potent tumor suppressor that targets various key cellular pathways across cancer types. Therefore, 5A2 may represent a promising novel treatment strategy in oncology.