Marco Rossi

Role of nucleophosmin in embryonic development and tumorigenesis

Nucleophosmin (also known as NPM, B23, NO38) is a nucleolar protein directly implicated in cancer pathogenesis, as the NPM1 gene is found mutated and rearranged in a number of haematological disorders. Furthermore, the region of chromosome 5 to which NPM1 maps is deleted in a proportion of de novo human myelodysplastic syndromes (MDS), and loss of chromosome 5 is extremely frequent in therapy-related MDS. NPM is a multifunctional protein, and its role in oncogenesis is controversial as NPM has been attributed with both oncogenic and tumour suppressive functions. To study the function of Npm in vivo, we generated a hypomorphic Npm1 mutant series (Npm1+/- < Npm1hy/hy < Npm1-/-) in mouse. Here we report that Npm is essential for embryonic development and the maintenance of genomic stability. Npm1-/- and Npm1hy/hy mutants have aberrant organogenesis and die between embryonic day E11.5 and E16.5 owing to severe anaemia resulting from defects in primitive haematopoiesis. We show that Npm1 inactivation leads to unrestricted centrosome duplication and genomic instability. We demonstrate that Npm is haploinsufficient in the control of genetic stability and that Npm1 heterozygosity accelerates oncogenesis both in vitro and in vivo. Notably, Npm1+/- mice develop a haematological syndrome with features of human MDS. Our findings uncover an essential developmental role for Npm and implicate its functional loss in tumorigenesis and MDS pathogenesis.

Indoleamine 2,3-dioxygenase–expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells

A comprehensive understanding of the complex, autologous cellular interactions and regulatory mechanisms that occur during normal dendritic cell (DC)-stimulated immune responses is critical to optimizing DC-based immunotherapy. We have found that mature, immunogenic human monocyte-derived DCs (moDCs) up-regulate the immune-inhibitory enzyme, indoleamine 2,3-dioxygenase (IDO). Under stringent autologous culture conditions without exogenous cytokines, mature moDCs expand regulatory T cells (Tregs) by an IDO-dependent mechanism. The priming of resting T cells with autologous, IDO-expressing, mature moDCs results in up to 10-fold expansion of CD4(+)CD25(bright)Foxp3(+)CD127(neg) Tregs. Treg expansion requires moDC contact, CD80/CD86 ligation, and endogenous interleukin-2. Cytofluorographically sorted CD4(+) CD25(bright)Foxp3(+) Tregs inhibit as much as 80% to 90% of DC-stimulated autologous and allogeneic T-cell proliferation, in a dose-dependent manner at Treg:T-cell ratios of 1:1, 1:5, and as low as 1:25. CD4(+)CD25(bright)Foxp3(+) Tregs also suppress the generation of cytotoxic T lymphocytes specific for the Wilms tumor antigen 1, resulting in more than an 80% decrease in specific target cell lysis. Suppression by Tregs is both contact-dependent and transforming growth factor-beta-mediated. Although mature moDCs can generate Tregs by this IDO-dependent mechanism to limit otherwise unrestrained immune responses, inhibition of this counter-regulatory pathway should also prove useful in sustaining responses stimulated by DC-based immunotherapy.

Synthetic miR-34a Mimics as a Novel Therapeutic Agent for Multiple Myeloma: In Vitro and In Vivo Evidence

Purpose: Deregulated expression of miRNAs has been shown in multiple myeloma (MM). A promising strategy to achieve a therapeutic effect by targeting the miRNA regulatory network is to enforce the expression of miRNAs that act as tumor suppressor genes, such as miR-34a. Experimental Design: Here, we investigated the therapeutic potential of synthetic miR-34a against human MM cells in vitro and in vivo. Results: Either transient expression of miR-34a synthetic mimics or lentivirus-based miR-34a-stable enforced expression triggered growth inhibition and apoptosis in MM cells in vitro. Synthetic miR-34a downregulated canonic targets BCL2, CDK6, and NOTCH1 at both the mRNA and protein level. Lentiviral vector-transduced MM xenografts with constitutive miR-34a expression showed high growth inhibition in severe combined immunodeficient (SCID) mice. The anti-MM activity of lipidic-formulated miR-34a was further shown in vivo in two different experimental settings: (i) SCID mice bearing nontransduced MM xenografts; and (ii) SCID-synth-hu mice implanted with synthetic 3-dimensional scaffolds reconstituted with human bone marrow stromal cells and then engrafted with human MM cells. Relevant tumor growth inhibition and survival improvement were observed in mice bearing TP53-mutated MM xenografts treated with miR-34a mimics in the absence of systemic toxicity. Conclusions: Our findings provide a proof-of-principle that formulated synthetic miR-34a has therapeutic activity in preclinical models and support a framework for development of miR-34a–based treatment strategies in MM patients. Clin Cancer Res; 18(22); 6260–70. ©2012 AACR.

miR-29b negatively regulates human osteoclastic cell differentiation and function: Implications for the treatment of multiple myeloma-related bone disease†‡

Skeletal homeostasis relies upon a fine tuning of osteoclast (OCL)‐mediated bone resorption and osteoblast (OBL)‐dependent bone formation. This balance is unsettled by multiple myeloma (MM) cells, which impair OBL function and stimulate OCLs to generate lytic lesions. Emerging experimental evidence is disclosing a key regulatory role of microRNAs (miRNAs) in the regulation of bone homeostasis suggesting the miRNA network as potential novel target for the treatment of MM‐related bone disease (BD). Here, we report that miR‐29b expression decreases progressively during human OCL differentiation in vitro. We found that lentiviral transduction of miR‐29b into OCLs, even in the presence of MM cells, significantly impairs tartrate acid phosphatase (TRAcP) expression, lacunae generation, and collagen degradation, which are relevant hallmarks of OCL activity. Accordingly, expression of cathepsin K and metalloproteinase 9 (MMP9) as well as actin ring rearrangement were impaired in the presence of miR‐29b. Moreover, we found that canonical targets C‐FOS and metalloproteinase 2 are suppressed by constitutive miR‐29b expression which also downregulated the master OCL transcription factor, NAFTc‐1. Overall, these data indicate that enforced expression of miR‐29b impairs OCL differentiation and overcomes OCL activation triggered by MM cells, providing a rationale for miR‐29b‐based treatment of MM‐related BD. J. Cell. Physiol. 228: 1506–1515, 2013.

Targeting miR-21 Inhibits In Vitro and In Vivo Multiple Myeloma Cell Growth

Purpose: Deregulated expression of miRNAs plays a role in the pathogenesis and progression of multiple myeloma. Among upregulated miRNAs, miR-21 has oncogenic potential and therefore represents an attractive target for the treatment of multiple myeloma. Experimental Design: Here, we investigated the in vitro and in vivo anti-multiple myeloma activity of miR-21 inhibitors. Results: Either transient-enforced expression or lentivirus-based constitutive expression of miR-21 inhibitors triggered significant growth inhibition of primary patient multiple myeloma cells or interleukin-6–dependent/independent multiple myeloma cell lines and overcame the protective activity of human bone marrow stromal cells. Conversely, transfection of miR-21 mimics significantly increased proliferation of multiple myeloma cells, showing its tumor-promoting potential in multiple myeloma. Importantly, upregulation of miR-21 canonical validated targets (PTEN, Rho-B, and BTG2), together with functional impairment of both AKT and extracellular signal–regulated kinase signaling, were achieved by transfection of miR-21 inhibitors into multiple myeloma cells. In vivo delivery of miR-21 inhibitors in severe combined immunodeficient mice bearing human multiple myeloma xenografts expressing miR-21induced significant antitumor activity. Upregulation of PTEN and downregulation of p-AKT were observed in retrieved xenografts following treatment with miR-21 inhibitors. Conclusion: Our findings show the first evidence that in vivo antagonism of miR-21 exerts anti-multiple myeloma activity, providing the rationale for clinical development of miR-21 inhibitors in this still incurable disease. Clin Cancer Res; 19(8); 2096–106. ©2013 AACR.

Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma.

The Hedgehog (Hh) pathway is required for cell-fate determination during the embryonic life, as well as cell growth and differentiation in the adult organism, where the inappropriate activation has been implicated in several cancers. Here we demonstrate that Hh signaling plays a significant role in growth and survival of multiple myeloma (MM) cells. We observed that CD138(+) MM cells express Hh genes and confirmed Smoothened (Smo)-dependent Hh signaling in MM using a novel synthetic Smo inhibitor, NVP-LDE225 (Novartis), which decreased MM cell viability by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of Hh activity. In addition, we detected a nuclear localization of Gli1 in MM cells, which is completely abrogated by Forskolin, a Gli1-modulating compound, confirming Smo-independent mechanisms leading to Hh activation in MM. Finally, we identified that bone marrow stromal cells are a source of the Shh ligand, although they are resistant to the Hh inhibitor because of defective Smo expression and Ptch1 up-regulation. Further in vitro as well as in vivo studies showed antitumor efficacy of NVP-LDE225 in combination with bortezomib. Altogether, our data demonstrate activation of both canonical and noncanonical Hh pathway in MM, thus providing the rationale for testing Hh inhibitors in clinical trials to improve MM patient outcome.

miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells

Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.

Role of gemcitabine-based combination therapy in the management of advanced pancreatic cancer: A meta-analysis of randomised trials

BACKGROUNDnPancreatic cancer is the fourth leading cause of cancer-related death worldwide. Gemcitabine is the mainstay treatment for advanced disease. However, almost all up-to-date trials, that evaluated the benefit of gemcitabine-combination schedules, failed to demonstrate an improvement in overall survival (OS). In this study, we performed a systematic review and a meta-analysis of randomised clinical trials (RCTs) to investigate the efficacy and safety of gemcitabine-based combination regimens as compared to gemcitabine alone in the management of pancreatic cancer.nnnMETHODSnClinical trials were collected by searching different databases (PubMed, Embase and the Central Registry of Controlled Trials of the Cochrane Library) and abstracts from major cancer meetings. We considered period ranging from January 1997 to January 2012. Primary end-point was OS, secondary end-points were response rate (RR), disease control rate (DCR) and safety. Hazard ratios (HRs) of OS, odds-ratios (ORs) of RR, DCR and risk ratios of grade 3-4 toxicity rates (TRs), were extracted as presented in retrieved studies and used for statistical analysis. Meta-analytic estimates were derived using random-effects model.nnnFINDINGSnThirty-four trials for a total of 10,660 patients were selected and included in the final analysis. The analysis showed that combination chemotherapy confers benefit in terms of OS (HR: 0.93; 95% confidence interval (CI): 0.89-0.97; p=0.001). ORs for both RR and DCR demonstrated a significant advantage for combination therapy (OR for RR: 0.60, 95%CI: 0.47-0.76, p<0.001; OR for DCR: 0.79; 95%CI: 0.66-0.93; p=0.006). Toxicities were more frequent with the combination treatment and significance in terms of risk ratio was reached for diarrhoea (0.53, 95%CI: 0.36-0.79), nausea (0.74, 95%CI: 0.56-0.96), neutropenia (0.71, 95%CI: 0.59-0.85) and thrombocytopenia (0.57, 95%CI: 0.43-0.75).nnnINTERPRETATIONnThe combination chemotherapy as compared to gemcitabine alone significantly improves OS in advanced pancreatic cancer (APC). However, this advantage is marginal whereas the treatment-related toxicity is increased, suggesting the use of gemcitabine-based combination regimens only in selected patient populations. New prospective trials, based on translational approaches and innovative validated biomarkers, are eagerly awaited on this topic.

In vivo anti-myeloma activity and modulation of gene expression profile induced by valproic acid, a histone deacetylase inhibitor.

Valproic acid (VPA) is a well‐tolerated anticonvulsant that exerts anti‐tumour activity as a histone deacetylase inhibitor. This study investigated the in vitro and in vivo activity of VPA against multiple myeloma (MM) cells. In vitro exposure of interleukin‐6‐dependent or ‐independent MM cells to VPA inhibited cell proliferation in a time‐ and dose‐dependent manner and induced apoptosis. In a cohort of severe combined immunodeficiency mice bearing human MM xenografts, VPA induced tumour growth inhibition and survival advantage in treated animals versus controls. Flow cytometric analysis performed on MM cells from excised tumours showed increase of G0–G1 and a decreased G2/M‐ and S‐phase following VPA treatment, indicating in vivo effects of VPA on cell cycle regulation. Gene expression profiling of MM cells exposed to VPA showed downregulation of genes involved in cell cycle progression, DNA replication and transcription, as well as upregulation of genes implicated in apoptosis and chemokine pathways. Pathfinder analysis of gene array data identified cell growth, cell cycle, cell death, as well as DNA replication and repair as the most important signalling networks modulated by VPA. Taken together, our data provide the preclinical rationale for VPA clinical evaluation as a single agent or in combination, to improve patient outcome in MM.

Peptide-Loaded Langerhans Cells, Despite Increased IL15 Secretion and T-Cell Activation In Vitro, Elicit Antitumor T-Cell Responses Comparable to Peptide-Loaded Monocyte-Derived Dendritic Cells In Vivo

Purpose: We compared the efficacy of human Langerhans cells (LC) as tumor immunogens in vivo with monocyte-derived dendritic cells (moDC) and investigated how interleukin 15 (IL15) supports optimal DC-stimulated antitumor immunity. Experimental Design: American Joint Committee on Cancer stage III/IV melanoma patients participated in this first clinical trial comparing melanoma peptide-pulsed LC with moDC vaccines (NCT00700167, www.ClinicalTrials.gov). Correlative studies evaluated mechanisms mediating IL15 support of DC-stimulated antitumor immunity. Results: Both DC vaccines were safe and immunogenic for melanoma antigens. LC-based vaccines stimulated significantly greater tyrosinase–HLA-A*0201 tetramer reactivity than the moDC-based vaccines. The two DC subtypes were otherwise statistically comparable, in contrast to extensive prior data in vitro showing LC superiority. LCs synthesize much more IL15 than moDCs and stimulate significantly more antigen-specific lymphocytes with a cytolytic IFN-γ profile even without exogenous IL15. When supplemented by low-dose IL15, instead of IL2, moDCs stimulate 5 to 6 logs more tumor antigen–specific effector memory T cells (TEMRA) over 3 to 4 weeks in vitro. IL2 and IL15 can be synergistic in moDC stimulation of cytolytic T cells. IL15 promotes T-cell expression of the antiapoptotic bcl-2 and inhibits candidate regulatory T-cell (Treg) expansion after DC stimulation, countering two effects of IL2 that do not foster tumor immunity. Conclusions: MoDC-based vaccines will require exogenous IL15 to achieve clinical efficacy. Alternatively, LCs can couple the endogenous production of IL15 with potent T-cell stimulatory activity. Optimization of full-length tumor antigen expression for processing into multiple immunogenic peptides for presentation by both class I and II MHC therefore merits emphasis to support more effective antitumor immunity stimulated by LCs. Clin Cancer Res; 17(7); 1984–97. ©2011 AACR.