Long-term proliferation of immature hypoxia-dependent JMML cells supported by a 3D in vitro system.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal stem cell disorder that occurs in early childhood and is characterized by the hyperactivation of the RAS pathway in 95% of the patients. JMML is characterized by a hyperproliferation of granulocytes and monocytes, and little is known about the heterogeneous nature of leukemia-initiating cells, as well as of the cellular hierarchy of the JMML bone marrow. In this study, we report the generation and characterization of a novel patient-derived three-dimensional (3D) in vitro JMML model, called patient-derived JMML Atypical Organoid (pd-JAO), sustaining the long-term proliferation of JMML cells with stem cell features and patient-specific hallmarks. JMML cells brewed in a 3D model under different microenvironmental conditions acquired proliferative and survival advantages when placed under low oxygen tension. Transcriptomic and microscopic analyses revealed the activation of specific metabolic energy pathways and the inactivation of processes leading to cell death. Furthermore, we demonstrated the pd-JAO-derived cells' migratory, propagation, and self-renewal capacities. Our study contributes to the development of a robust JMML 3D in vitro model for studying and defining the impact of microenvironmental stimuli on JMML disease and the molecular mechanisms that regulate JMML initiating and propagating cells. Pd-JAO may become a promising model for compound tests focusing on new therapeutic interventions aimed at eradicating JMML progenitors and controlling JMML disease.

Verteporfin induces apoptosis and reduces the stem cell-like properties in Neuroblastoma tumour-initiating cells through inhibition of the YAP/TAZ pathway.

Neuroblastoma is an embryonal malignancy of early childhood arising from the embryonic sympatho-adrenal lineage of the neural crest. About half of all cases are currently classified as high-risk of disease recurrence, with an overall survival rate of less than 40% at 5 years despite intensive therapy. Recent studies on matched primary tumours and at the relapse revealed downregulation of genes transcriptionally silenced by YAP as significant association with neuroblastoma relapse. Here, we evaluated the pharmacological targeting of YAP/TAZ with the YAP/TAZ-TEAD inhibitor Verteporfin (VP) in Tumour Initiating Cells (TICs) derived from High-Risk Neuroblastoma patients. VP treatment suppresses YAP/TAZ expression, induces apoptosis and causes the re-organization of the cytoskeleton reducing cells migration and clonogenic ability. Moreover, VP reduces the percentage of side population cells and ABC transporters involved in drug resistance, and the percentage of stem cell subpopulations CD133+ and CD44+ of TICs. Finally, we demonstrated that VP sensitizes TICs to the standard drugs used for neuroblastoma therapy etoposide and cis-platin opening the way to use VP as drug repositioning candidate for recurrent neuroblastoma.

Rhabdomyosarcoma Cells Produce Their Own Extracellular Matrix With Minimal Involvement of Cancer-Associated Fibroblasts: A Preliminary Study.

BACKGROUND: The interplay between neoplastic cells and surrounding extracellular matrix (ECM) is one of the determinant elements for cancer growth. The remodeling of the ECM by cancer-associated fibroblasts (CAFs) shapes tumor microenvironment by depositing and digesting ECM proteins, hence promoting tumor growth and invasion. While for epithelial tumors CAFs are well characterized, little is known about the stroma composition of mesenchymal cancers, such as in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma during childhood and adolescence. The aim of this work is to identify the importance of CAFs in specifying RMS microenvironment and the role of these stromal cells in RMS growth. METHODS: We assessed in two dimensional (2D) and three dimensional (3D) systems the attraction between RMS cells and fibroblasts using epithelial colon cancer cell line as control. CAFs were studied in a xenogeneic mouse model of both tumor types and characterized in terms of fibroblast activation protein (FAP), mouse PDGFR expression, metalloproteases activation, and ECM gene and protein expression profiling. RESULTS: In 2D model, the rate of interaction between stromal and malignant cells was significantly lower in RMS with respect to colon cancer. Particularly, in 3D system, RMS spheroids tended to dismantle the compact aggregate when grown on the layer of stromal cells. In vivo, despite the well-formed tumor mass, murine CAFs were found in low percentage in RMS xenogeneic samples. CONCLUSIONS: Our findings support the evidence that, differently from epithelial cancers, RMS cells are directly involved in their own ECM remodeling, and less dependent on CAFs support for cancer cell growth.

Dissecting molecular mechanisms of resistance to NOTCH1-targeted therapy in T-cell acute lymphoblastic leukemia xenografts.

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors.

Correction to: LIN28B increases neural crest cell migration and leads to transformation of trunk sympathoadrenal precursors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LIN28B increases neural crest cell migration and leads to transformation of trunk sympathoadrenal precursors.

The RNA-binding protein LIN28B regulates developmental timing and determines stem cell identity by suppressing the let-7 family of microRNAs. Postembryonic reactivation of LIN28B impairs cell commitment to differentiation, prompting their transformation. In this study, we assessed the extent to which ectopic lin28b expression modulates the physiological behavior of neural crest cells (NCC) and governs their transformation in the trunk region of developing embryos. We provide evidence that the overexpression of lin28b inhibits sympathoadrenal cell differentiation and accelerates NCC migration in two vertebrate models, Xenopus leavis and Danio rerio. Our results highlight the relevance of ITGA5 and ITGA6 in the LIN28B-dependent regulation of the invasive motility of tumor cells. The results also establish that LIN28B overexpression supports neuroblastoma onset and the metastatic potential of malignant cells through let-7a-dependent and let-7a-independent mechanisms.

Circular RNA differential expression in blood cell populations and exploration of circRNA deregulation in pediatric acute lymphoblastic leukemia.

Circular RNAs (circRNAs) are abundantly expressed in the haematopoietic compartment, but knowledge on their diversity among blood cell types is still limited. Nevertheless, emerging data indicate an array of circRNA functions exerted through interactions with other RNAs and proteins, by translation into peptides, and circRNA involvement as regulatory molecules in many biological processes and cancer mechanisms. Interestingly, the role of specific circRNAs in leukemogenesis has been disclosed by a few studies, mostly in acute myeloid leukemia. In this study, circRNA expression in B-cells, T-cells and monocytes of healthy subjects is described, including putative new circRNA genes. Expression comparison considered 6,228 circRNAs and highlighted cell population-specific expression and exon usage patterns. Differential expression has been confirmed by qRT-PCR for circRNAs specific of B-cells (circPAX5, circAFF3, circIL4R, and circSETBP1) or T-cells (circIKZF1, circTNIK, circTXK, and circFBXW7), and for circRNAs from intronic (circBCL2) and intergenic regions that were overexpressed in lymphocytes. Starting from this resource of circRNA expression in mature blood cell populations, targeted examination identified striking and generalized upregulated expression of circPAX5, circPVT1 and circHIPK3 in pediatric B-precursor acute lymphoblastic leukemia, and disclosed circRNAs with variable expression across cytogenetic subtypes.

Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

In Chronic Hepatitis C Infection, Myeloid-Derived Suppressor Cell Accumulation and T Cell Dysfunctions Revert Partially and Late After Successful Direct-Acting Antiviral Treatment.

Chronic HCV infection is characterized by several immunological alterations, such as the accumulation of suppressor cells and of hyperactivated T lymphocytes. However, it is unclear whether direct-acting antiviral (DAA)-mediated HCV clearance restores immune dysfunctions. We performed a phenotypic characterization by flow cytometry of different immune cell subsets, including monocytic myeloid-derived suppressor cells (M-MDSCs) and T lymphocytes in 168 patients with persistent HCV infection not treated, under DAA therapies and sustained virological responders. Chronic HCV infection prompted the accumulation of M-MDSCs independently of patient and clinical characteristics, and altered their metabolic properties. HCV RNA was undetectable in the majority of patients just after few weeks of DAA therapy, whereas M-MDSC levels normalized only 6 months after therapy. In addition, HCV infection deeply perturbed the T cell compartment since a re-distribution of memory CD4+ and CD8+ T cells was observed at the expenses of naïve cells, and memory T lymphocytes displayed increased activation. Notably, these features were only partially restored by DAA therapies in the CD4, but not in the CD8, compartment as high immune activation levels persisted in the terminally differentiated memory CD8+ T cells even more than 1 year after sustained virological response. Together, these results suggest that successful DAA therapies do not lead to full immunological reconstitution as fast as viral clearance.

BMP9 counteracts the tumorigenic and pro-angiogenic potential of glioblastoma.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor, with a strong ability to disseminate and invade the surrounding parenchyma. In addition, a subpopulation of GBM stem cells has been reported to possess the ability to transdifferentiate into tumor-derived endothelial cells (TDECs), supporting the resistance to anti-angiogenic treatments of newly formed blood vessels. Bone Morphogenetic Protein 9 (BMP9) is critically involved in the processes of cancer cell differentiation, invasion and metastasis, representing a potential tool in order to impair the intrinsic GBM aggressiveness. Here we demonstrate that BMP9 is able to trigger the activation of SMADs in patient-derived GBM cells, and to strongly inhibit proliferation and invasion by reducing the activation of PI3K/AKT/MAPK and RhoA/Cofilin pathways, respectively. Intriguingly, BMP9 treatment is sufficient to induce a strong differentiation of GBM stem-like cells and to significantly counteract the already reported process of GBM cell transdifferentiation into TDECs not only in in vitro mimicked TDEC models, but also in vivo in orthotopic xenografts in mice. Additionally, we describe a strong BMP9-mediated inhibition of the whole angiogenic process engaged during GBM tumor formation. Based on these results, we believe that BMP9, by acting at multiple levels against GBM cell aggressiveness, can be considered a promising candidate, to be further developed, for the future therapeutic management of GBM.

Dual inhibition of PI3K/mTOR signaling in chemoresistant AML primary cells.

A main cause of treatment failure for AML patients is resistance to chemotherapy. Survival of AML cells may depend on mechanisms that elude conventional drugs action and/or on the presence of leukemia initiating cells at diagnosis, and their persistence after therapy. MDR1 gene is an ATP-dependent drug efflux pump known to be a risk factor for the emergence of resistance, when combined to unstable cytogenetic profile of AML patients. In the present study, we analyzed the sensitivity to conventional chemotherapeutic drugs of 26 samples of primary blasts collected from AML patients at diagnosis. Detection of cell viability and apoptosis allowed to identify two group of samples, one resistant and one sensitive to in vitro treatment. The cells were then analyzed for the presence and the activity of P-glycoprotein. A comparative analysis showed that resistant samples exhibited a high level of MDR1 mRNA as well as of P-glycoprotein content and activity. Moreover, they also displayed high PI3K signaling. Therefore, we checked whether the association with signaling inhibitors might resensitize resistant samples to chemo-drugs. The combination showed a very potent cytotoxic effect, possibly through down modulation of MDR1, which was maintained also when primary blasts were co-cultured with human stromal cells. Remarkably, dual PI3K/mTOR inactivation was cytotoxic also to leukemia initiating cells. All together, our findings indicate that signaling activation profiling associated to gene expression can be very useful to stratify patients and improve therapy.

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2.

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

Sex-dependent differences in inflammatory responses during liver regeneration in a murine model of acute liver injury.

A sexual dimorphism in liver inflammation and repair was previously demonstrated. Its cellular dissection in the course of acute liver injury (ALI) was explored. BALB/c mice were treated with carbon tetrachloride (CCl4) by intraperitoneal injection and killed after 3, 5, and 8 days. Histological and hepatic cell population analyses were performed. The correlation between androgen receptor (AR) expression and liver recruited inflammatory cells was investigated by treatment with the AR antagonist flutamide. Additionally, patients with a diagnosis of drug induced liver injury (DILI) were included in the study, with a particular focus on gender dimorphism in circulating monocytes. A delayed resolution of necrotic damage and a higher expression of proinflammatory cytokines were apparent in male mice along with a slower recruitment of inflammatory monocytes. F4/80+CD11b+ macrophages and CD11bhighGr-1high monocytes expressed AR and were recruited later in male compared with female livers after CCl4 treatment. Moreover, CD11bhighAR+Gr-1high recruitment was negatively modulated by flutamide in males. Analysis of DILI patients showed overall a significant reduction in circulating mature monocytes compared with healthy subjects. More interestingly, male patients had higher numbers of immature monocytes compared with female patients.A stronger cytotoxic tissue response was correlated with an impaired recruitment of CD11bhighAR+Gr-1high cells and F4/80+CD11b+ macrophages in the early inflammatory phase under AR signaling. During DILI, a dimorphic immune response was apparent, characterized by a massive recruitment of monocytes to the liver both in males and females, but only in males was this recruitment sustained by a turnover of immature monocytes.

Antitumor Activity of Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor, in ETV6-NTRK3-Positive Acute Myeloid Leukemia.

Activation of tropomyosin receptor kinase (TRK) family tyrosine kinases by chromosomal rearrangement has been shown to drive a wide range of solid tumors and hematologic malignancies. TRK fusions are actionable targets as evidenced by recent clinical trial results in solid tumors. Entrectinib (RXDX-101) is an investigational, orally available, CNS-active, highly potent, and selective kinase inhibitor against TRKA/B/C, ROS1, and ALK kinase activities. Here, we demonstrate that TRK kinase inhibition by entrectinib selectively targets preclinical models of TRK fusion-driven hematologic malignancies. In acute myelogenous leukemia (AML) cell lines with endogenous expression of the ETV6-NTRK3 fusion gene, entrectinib treatment blocked cell proliferation and induced apoptotic cell death in vitro with subnanomolar IC50 values. Phosphorylation of the ETV6-TRKC fusion protein and its downstream signaling effectors was inhibited by entrectinib treatment in a dose-dependent manner. In animal models, entrectinib treatment at clinically relevant doses resulted in tumor regression that was accompanied by elimination of residual cancer cells from the bone marrow. Our preclinical data demonstrate the potential of entrectinib as an effective treatment for patients with TRK fusion-driven AML and other hematologic malignancies. Mol Cancer Ther; 17(2); 455-63. ©2017 AACR.

Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia.

Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.

PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis.

Tumor genetics and escape from immune surveillance concur in the poor prognosis of PDAC. In this study an experimental model was set up to verify whether SMAD4, deleted in about 55% PDAC and associated with poor prognosis, is involved in determining immunosuppression through Exosomes (Exo). Potential mechanisms and mediators underlying SMAD4-dependent immunosuppression were evaluated by studying intracellular calcium (Fluo-4), Exo-miRNAs (microarray) and Exo-proteins (SILAC). Two PDAC cell lines expressing (BxPC3-SMAD4+) or not-expressing (BxPC3) SMAD4 were used to prepare Exo-enriched conditioned media, employed in experiments with blood donors PBMCs. Exo expanded myeloid derived suppressor cells (gMDSC and mMDSC, flow cytometry) and altered intracellular calcium fluxes in an SMAD4 dependent manner. BxPC3-SMAD4+, but mainly BxPC3 Exo, increased calcium fluxes of PBMCs (p = 0.007) and this increased intracellular calcium trafficking characterized mMDSCs. The analysis of de-regulated Exo-miRNAs and transfection experiments revealed hsa-miR-494-3p and has-miR-1260a as potential mediators of SMAD4-associated de-regulated calcium fluxes. Eleven main biological processes were identified by the analysis of SMAD4-associated de-regulated Exo-proteins, including translation, cell adhesion, cell signaling and glycolysis. A reverse Warburg effect was observed by treating PBMCs with PDAC-derived Exo: BxPC3 Exo induced a higher glucose consumption and lactate production than BxPC3-SMAD4+ Exo. CONCLUSION: PDAC-derived Exo from cells with, but mainly from those without SMAD4 expression, create an immunosuppressive myeloid cell background by increasing calcium fluxes and glycolysis through the transfer of SMAD4-related differentially expressed miRNAs and proteins.

ZNF521 sustains the differentiation block in MLL-rearranged acute myeloid leukemia.

Zinc finger protein 521 (ZNF521) is a multiple zinc finger transcription factor and a strong candidate as regulator of hematopoietic stem cell homeostasis. Recently, independent gene expression profile studies have evidenced a positive correlation between ZNF521 mRNA overexpression and MLL-rearranged acute myeloid leukemia (AML), leaving open the question on the role of ZNF521 in this subtype of leukemia. In this study, we sought to analyze the effect of ZNF521 depletion on MLL-rearranged AML cell lines and MLL-AF9 xenograft primary cells. Knockdown of ZNF521 with short-hairpin RNA (shRNA) led to decreased leukemia proliferation, reduced colony formation and caused cell cycle arrest in MLL-rearranged AML cell lines. Importantly, we showed that loss of ZNF521 substantially caused differentiation of both MLL-rearranged cell lines and primary cells. Moreover, gene profile analysis in ZNF521-silenced THP-1 cells revealed a loss of MLL-AF9-directed leukemic signature and an increase of the differentiation program. Finally, we determined that both MLL-AF9 and MLL-ENL fusion proteins directly interacted with ZNF521 promoter activating its transcription. In conclusion, our findings identify ZNF521 as a critical effector of MLL fusion proteins in blocking myeloid differentiation and highlight ZNF521 as a potential therapeutic target for this subtype of leukemia.

Expression Profiling of Circulating Microvesicles Reveals Intercellular Transmission of Oncogenic Pathways.

Circulating microvesicles have been described as important players in cell-to-cell communication carrying biological information under normal or pathologic condition. Microvesicles released by cancer cells may incorporate diverse biomolecules (e.g., active lipids, proteins, and RNA), which can be delivered and internalized by recipient cells, potentially altering the gene expression of recipient cells and eventually impacting disease progression. Leukemia in vitro model systems were used to investigate microvesicles as vehicles of protein-coding messages. Several leukemic cells (K562, LAMA-87, TOM-1, REH, and SHI-1), each carrying a specific chromosomal translocation, were analyzed. In the leukemic cells, these chromosomal translocations are transcribed into oncogenic fusion transcripts and the transfer of these transcripts was monitored from leukemic cells to microvesicles for each of the cell lines. Microarray gene expression profiling was performed to compare transcriptomes of K562-derived microvesicles and parental K562 cells. The data show that oncogenic BCR-ABL1 transcripts and mRNAs related to basic functions of leukemic cells were included in microvesicles. Further analysis of microvesicles cargo revealed a remarkable enrichment of transcripts related to cell membrane activity, cell surface receptors, and extracellular communication when compared with parental K562 cells. Finally, coculturing of healthy mesenchymal stem cells (MSC) with K562-derived microvesicles displayed the transfer of the oncogenic message, and confirmed the increase of target cell proliferation as a function of microvesicle dosage.Implications: This study provides novel insight into tumor-derived microvesicles as carriers of oncogenic protein-coding messages that can potentially jeopardize cell-directed therapy, and spread to other compartments of the body. Mol Cancer Res; 15(6); 683-95. ©2017 AACR.

Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.

The ability to induce autologous tissue-specific stem cells in culture could have a variety of applications in regenerative medicine and disease modeling. Here we show that transient expression of exogenous YAP or its closely related paralogue TAZ in primary differentiated mouse cells can induce conversion to a tissue-specific stem/progenitor cell state. Differentiated mammary gland, neuronal, and pancreatic exocrine cells, identified using a combination of cell sorting and lineage tracing approaches, efficiently convert to proliferating cells with properties of stem/progenitor cells of their respective tissues after YAP induction. YAP-induced mammary stem/progenitor cells show molecular and functional properties similar to endogenous MaSCs, including organoid formation and mammary gland reconstitution after transplantation. Because YAP/TAZ function is also important for self-renewal of endogenous stem cells in culture, our findings have implications for understanding the molecular determinants of the somatic stem cell state.

Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.