Mouse models of multiple myeloma: Technologic platforms and perspectives

Murine models of human multiple myeloma (MM) are key tools for the study of disease biology as well as for investigation and selection of novel candidate therapeutics for clinical translation. In the last years, a variety of pre-clinical models have been generated to recapitulate a wide spectrum of biological features of MM. These systems range from spontaneous or transgenic models of murine MM, to subcutaneous or orthothopic xenografts of human MM cell lines in immune compromised animals, to platform allowing the engraftment of primary/bone marrowdependent MM cells within a human bone marrow milieu to fully recapitulate human disease. Selecting the right model for specific pre-clinical research is essential for the successful completion of investigation. We here review recent and most known pre-clinical murine, transgenic and humanized models of MM, focusing on major advantages and/or weaknesses in the light of different research aims

OSAnalyzer: A Bioinformatics Tool for the Analysis of Gene Polymorphisms Enriched with Clinical Outcomes

Background: The identification of biomarkers for the estimation of cancer patients\u2019 survival is a crucial problem in modern oncology. Recently, the Affymetrix DMET (Drug Metabolizing Enzymes and Transporters) microarray platform has offered the possibility to determine the ADME (absorption, distribution, metabolism, and excretion) gene variants of a patient and to correlate them with drug-dependent adverse events. Therefore, the analysis of survival distribution of patients starting from their profile obtained using DMET data may reveal important information to clinicians about possible correlations among drug response, survival rate, and gene variants. Methods: In order to provide support to this analysis we developed OSAnalyzer, a software tool able to compute the overall survival (OS) and progression-free survival (PFS) of cancer patients and evaluate their association with ADME gene variants. Results: The tool is able to perform an automatic analysis of DMET data enriched with survival events. Moreover, results are ranked according to statistical significance obtained by comparing the area under the curves that is computed by using the log-rank test, allowing a quick and easy analysis and visualization of high-throughput data. Conclusions: Finally, we present a case study to highlight the usefulness of OSAnalyzer when analyzing a large cohort of patient

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

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

A reappraisal of the role of the various opioid receptor subtypes in cell-mediated immunity.

Opioid peptides have been shown by several studies to modulate various parameters of the immune response, but scant experimental findings exist on the role played by specific opioid receptor subtypes in the control of immune mechanisms. This study focuses on the in vitro influences of [Trp4,Asn7]dermorphin, a mu-selective agonist, [D-Ala2]deltorphin I, a delta-selective agonist and U50,488, a kappa-selective agonist, on the proliferative response of splenocytes to concanavalin A (Con A). [Trp4,Asn7]dermorphin at low concentrations (10(-11P) and 10(-12) M) enhanced the proliferative response to Con A, whereas higher concentrations (10(-6) to 10(-7) M) inhibited it. Both effects were antagonized by naloxone. [D-Ala2]deltorphin I at very low concentrations (10(-12) to 10(-13) M) also produced a significant increase in the proliferative response of splenocytes to Con A. This effect was significantly antagonized by natrindole, a specific delta-receptor antagonist. Finally U50,488 at concentrations ranging from 10(-8) to 10(-9) M inhibited the proliferative response to Con A. The effects of U50,488 were mediated by the stimulation of the kappa-opioid receptors, since a preincubation of splenocytes with the selective antagonist norbinaltorphimine significantly reduced or abolished the U50,488-induced suppression of the mitotic response. In conclusion, our results clearly indicate that the different opioid receptor subtypes play a different role in the control of immune mechanisms and suggest that immunoenhancing effects of opioid peptides are very likely due to the stimulation of mu- and delta-receptors, whereas the immunosuppressive effects are mediated through the stimulation of kappa-opioid receptors

Use of hematopoietic cytokines to accelerate the recovery of the immune system in irradiated mice

In our previous studies aimed at designing appropriate strategies to accelerate recovery of the immune system after irradiation, we found that the hematopoietic cytokine recombinant murine (rmu) interleukin (IL)-3 was able to induce differentiation and growth of thymocytes and splenic T and B lymphocytes in mice exposed to x-rays (200-500 cGy). The recovery, however, was complete at 7 days only after a dose of 200 cGy, whereas 2, 3, and 4 weeks were necessary to achieve full recovery after 300, 400, and 500 cGy, respectively. These studies were extended to investigate the effects of another hematopoietic cytokine, recombinant human (rhu) IL-11, a bone marrow stromal-derived cytokine, administered together with IL-3 to irradiated mice. The synergistic effect of the two cytokines was evident when relatively small doses of rhu IL-11 were injected with an optimal dose of rmu IL-3

Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts

miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3'UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-2, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/ apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD