Pharmacokinetics and pharmacodynamics of a 13-mer LNA-inhibitor-miR-221 in mice and non-human primates

Locked nucleic acid (LNA) oligonucleotides have been successfully used to efficiently inhibit endogenous small noncoding RNAs in vitro and in vivo. We previously demonstrated that the direct miR-221 inhibition by the novel 13-mer LNA-i-miR-221 induces significant antimyeloma activity and upregulates canonical miR-221 targets in vitro and in vivo. To evaluate the LNA-i-miR-221 pharmacokinetics and pharmacodynamics, novel assays for oligonucleotides quantification in NOD.SCID mice and Cynomolgus monkeys (Macaca fascicularis) plasma, urine and tissues were developed. To this aim, a liquid chromatography/mass spectrometry method, after solid-phase extraction, was used for the detection of LNA-i-miR-221 in plasma and urine, while a specific in situ hybridization assay for tissue uptake analysis was designed. Our analysis revealed short half-life, optimal tissue biovailability and minimal urine excretion of LNA-i-miR-221 in mice and monkeys. Up to 3 weeks, LNA-i-miR-221 was still detectable in mice vital organs and in xenografted tumors, together with p27 target upregulation. Importantly, no toxicity in the pilot monkey study was observed. Overall, our findings indicate the suitability of LNA-i-miR-221 for clinical use and we provide here pilot data for safety analysis and further development of LNA-miRNA-based therapeutics for human cancer

Replacement of miR-155 elicits tumor suppressive activity and antagonizes bortezomib resistance in multiple myeloma

Aberrant expression of microRNAs (miRNAs) has been associated to the pathogenesis of multiple myeloma (MM). While miR-155 is considered a therapeutic target in several malignancies, its role in MM is still unclear. The analysis of miR-155 expression indicates its down-regulation in MM patient-derived as compared to healthy plasma cells, thus pointing to a tumor suppressor role in this malignancy. On this finding, we investigated miR-155 replacement as a potential anti-tumor strategy in MM. The miR-155 enforced expression triggered anti-proliferative and pro-apoptotic effects in vitro. Given the lower miR-155 levels in bortezomib-resistant as compared to sensitive MM cells, we analyzed the possible involvement of miR-155 in bortezomib resistance. Importantly, miR-155 replacement enhanced bortezomib anti-tumor activity both in vitro and in vivo in a xenograft model of human MM. In primary MM cells, we observed an inverse correlation between miR-155 and the mRNA encoding the proteasome subunit gene PSMβ5, whose dysregulation has been largely implicated in bortezomib resistance, and we validated PSMβ5 30UTR mRNA targeting, along with reduced proteasome activity, by miR-155. Collectively, our findings demonstrate that miR-155 elicits anti-MM activity, likely via proteasome inhibition, providing the framework for miR-155-based anti-MM therapeutic strategies

Influence of the fabrication accuracy of hot-embossed PCL scaffolds on cell growths

Polycaprolactone (PCL) is a biocompatible and biodegradable polymer widely used for the realization of 3D scaffold for tissue engineering applications. The hot embossing technique (HE) allows the obtainment of PCL scaffolds with a regular array of micro pillars on their surface. The main drawback affecting this kind of micro fabrication process is that such structural superficial details can be damaged when detaching the replica from the mold. Therefore, the present study has focused on the optimization of the HE processes through the development of an analytical model for the prediction of the demolding force as a function of temperature. This model allowed calculating the minimum demolding force to obtain regular micropillars without defects. We demonstrated that the results obtained by the analytical model agree with the experimental data. To address the importance of controlling accurately the fabricated microstructures, we seeded on the PCL scaffolds human stromal cell line (HS-5) and monocytic leukemia cell line (THP-1) to evaluate how the presence of regular or deformed pillars affect cells viability. In vitro viability results, scanning electron and fluorescence microscope imaging analysis show that the HS-5 preferentially grows on regular microstructured surfaces, while the THP-1 on irregular microstructured ones

Trabectedin triggers direct and NK-mediated cytotoxicity in multiple myeloma

Background: Genomic instability is a feature of multiple myeloma (MM), and impairment in DNA damaging response (DDR) has an established role in disease pathobiology. Indeed, a deregulation of DNA repair pathways may contribute to genomic instability, to the establishment of drug resistance to genotoxic agents, and to the escape from immune surveillance. On these bases, we evaluated the role of different DDR pathways in MM and investigated, for the first time, the direct and immune-mediated anti-MM activity of the nucleotide excision repair (NER)-dependent agent trabectedin. Methods: Gene-expression profiling (GEP) was carried out with HTA2.0 Affymetrix array. Evaluation of apoptosis, cell cycle, and changes in cytokine production and release have been performed in 2D and 3D Matrigel-spheroid models through flow cytometry on MM cell lines and patients-derived primary MM cells exposed to increasing nanomolar concentrations of trabectedin. DNA-damage response has been evaluated through Western blot, immunofluorescence, and DNA fragmentation assay. Trabectedin-induced activation of NK has been assessed by CD107a degranulation. miRNAs quantification has been done through RT-PCR. Results: By comparing GEP meta-analysis of normal and MM plasma cells (PCs), we observed an enrichment in DNA NER genes in poor prognosis MM. Trabectedin triggered apoptosis in primary MM cells and MM cell lines in both 2D and 3D in vitro assays. Moreover, trabectedin induced DDR activation, cellular stress with ROS production, and cell cycle arrest. Additionally, a significant reduction of MCP1 cytokine and VEGF-A in U266-monocytes co-cultures was observed, confirming the impairment of MM-promoting milieu. Drug-induced cell stress in MM cells led to upregulation of NK activating receptors ligands (i.e., NKG2D), which translated into increased NK activation and degranulation. Mechanistically, this effect was linked to trabectedin-induced inhibition of NKG2D-ligands negative regulators IRF4 and IKZF1, as well as to miR-17 family downregulation in MM cells. Conclusions: Taken together, our findings indicate a pleiotropic activity of NER-targeting agent trabectedin, which appears a promising candidate for novel anti-MM therapeutic strategies

Therapeutic vulnerability of multiple myeloma to MIR17PTi, a first-in-class inhibitor of pri-mir-17-92

The microRNA cluster miR-17-92 is oncogenic and represents a valuable therapeutic target in c-MYC (MYC)-driven malignancies. Here, we developed novel LNA gapmeR antisense oligonucleotides (ASOs) to induce RNase H-mediated degradation of MIR17HG primary transcripts and, consequently, to prevent biogenesis of miR-17-92 microRNAs (miR-17-92s). The leading LNA-ASO, named MIR17PTi, impaired proliferation of several cancer cell lines (n=48) established from both solid and hematologic tumors by on-target antisense activity, and more effectively as compared to miR-17-92s inhibitors. By focusing on multiple myeloma (MM), we found that MIR17PTi triggers apoptosis via impairment of homeostatic MYC/miR-17-92 feed-forward loops (FFLs) in patient-derived MM cells; and induced MYC-dependent synthetic lethality. We show that alteration of a BIM-centered FFL is instrumental for MIR17PTi to induce cytotoxicity in MM cells. MIR17PTi exerts strong in vivo anti-tumor activity in NOD-SCID mice bearing clinically relevant models of MM, with advantageous safety and pharmacokinetics profiles in non-human primates. Altogether, MIR17PTi is a novel pharmacological tool to be tested in early-phase clinical trials against MM and other MYC-driven malignancies

Replacement of miR-155 Elicits Tumor Suppressive Activity and Antagonizes Bortezomib Resistance in Multiple Myeloma

Aberrant expression of microRNAs (miRNAs) has been associated to the pathogenesis of multiple myeloma (MM). While miR-155 is considered a therapeutic target in several malignancies, its role in MM is still unclear. The analysis of miR-155 expression indicates its down-regulation in MM patient-derived as compared to healthy plasma cells, thus pointing to a tumor suppressor role in this malignancy. On this finding, we investigated miR-155 replacement as a potential anti-tumor strategy in MM. The miR-155 enforced expression triggered anti-proliferative and pro-apoptotic effects in vitro. Given the lower miR-155 levels in bortezomib-resistant as compared to sensitive MM cells, we analyzed the possible involvement of miR-155 in bortezomib resistance. Importantly, miR-155 replacement enhanced bortezomib anti-tumor activity both in vitro and in vivo in a xenograft model of human MM. In primary MM cells, we observed an inverse correlation between miR-155 and the mRNA encoding the proteasome subunit gene PSM\u3b25, whose dysregulation has been largely implicated in bortezomib resistance, and we validated PSM\u3b25 3'UTR mRNA targeting, along with reduced proteasome activity, by miR-155. Collectively, our findings demonstrate that miR-155 elicits anti-MM activity, likely via proteasome inhibition, providing the framework for miR-155-based anti-MM therapeutic strategies

Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo

Interferon regulatory factor 4 (IRF4) is an attractive therapeutic target in multiple myeloma (MM). We here report that expression of IRF4 mRNA inversely correlates with microRNA (miR)-125b in MM patients. Moreover, we provide evidence that miR-125b is downregulated in TC2/3 molecular MM subgroups and in established cell lines. Importantly, constitutive expression of miR-125b-5p by lentiviral vectors or transfection with synthetic mimics impaired growth and survival of MM cells and overcame the protective role of bone marrow stromal cells (BMSCs) in vitro. Apoptotic and autophagy-associated cell death were triggered in MM cells upon miR-125b-5p ectopic expression. Importantly, we found that the anti-MM activity of miR-125b-5p was mediated via direct downregulation of IRF4 and its downstream effector BLIMP-1. Moreover, inhibition of IRF4 translated into downregulation of c-Myc, caspase-10 and cFlip, relevant IRF4-downstream effectors. Finally, in vivo intra-tumor or systemic delivery of formulated miR-125b-5p mimics against human MM xenografts in SCID/NOD mice induced significant anti-tumor activity and prolonged survival. Taken together, our findings provide evidence that miR-125b, differently from other hematologic malignancies, has tumor suppressor activity in MM. Furthermore, our data provide proof-of-concept that synthetic miR-125b-5p mimics are promising anti-MM agents to be validated in early clinical trials

Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM