The role of PIP5K1α/pAKT and targeted inhibition of growth of subtypes of breast cancer using PIP5K1α inhibitor

Despite recent improvement in adjuvant therapies, triple-negative, and ER+ subtypes of breast cancer (BC) with metastatic potentials remain the leading cause of BC-related deaths. We investigated the role of phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), a key upstream factor of PI3K/AKT, and the therapeutic effect of PIP5Kα inhibitor on subtypes of BC. The clinical importance of PIP5K1α and its association with survivals were analyzed using three BC cohorts from Nottingham (n = 913), KM plotter (n = 112) and TCGA (n = 817). Targeted overexpression or knockdown of PIP5K1α were introduced into BC cell lines. The effects of PIP5K1α and its inhibitor on growth and invasion of BC were confirmed by using in vitro assays including proliferation, migration, apoptosis and luciferase reporter assays and in vivo xenograft mouse models. All statistical tests were two-sided. PIP5K1α was associated with poor patient outcome in triple-negative BC (for PIP5K1α protein, p = 0.011 and for mRNA expression, p = 0.028, log-rank test). 29% of triple-negative BC had PIP5K1A gene amplification. Elevated level of PIP5K1α increased expression of pSer-473 AKT (p < 0.001) and invasiveness of triple-negative MDA-MB-231 cells (p < 0.001). Conversely, inhibition of PIP5K1α using its inhibitor ISA-2011B, or via knockdown suppressed growth and invasiveness of MDA-MB-231 xenografts (mean vehicle-treated controls = 2160 mm3, and mean ISA-2011B-treated = 600 mm3, p < 0.001). ISA-2011B-treatment reduced expression of pSer-473 AKT (p < 0.001) and its downstream effectors including cyclin D1, VEGF and its receptors, VEGFR1 and VEGFR2 (p < 0.001) in xenograft tumors. In ER+ cancer cells, PIP5K1α acted on pSer-473 AKT, and was in complexes with VEGFR2, serving as co-factor of ER-alpha to regulate activities of target genes including cyclin D1 and CDK1. Our study suggests that our developed PIP5K1α inhibitor has a great potential on refining targeted therapeutics for treatment of triple-negative and ER+ BC with abnormal PI3K/AKT pathways

Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma

Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.National Institutes of Health (U.S.) (U24 CA180922

oHSV Genome Editing by Means of galK Recombineering

open8noThis work was supported by European Research Council (ERC) Advanced Grant number 340060, VII framework program to G. C.-F., by RFO (University of Bologna) to L.M. and T.G, and by Fondi Pallotti to T.G.Since the cloning of the herpes simplex virus (HSV) genome as BAC (bacterial artificial chromosome), the genetic engineering of the viral genome has become readily feasible. The advantage is that the modification of the animal virus genome is carried out in bacteria, with no replication or production of viral progeny, and is separated from the reconstitution or regeneration of the recombinant virus in mammalian cells. This allows an easy engineering of essential genes, as well. Many technologies have been developed for herpesvirus BAC engineering. In our hands the most powerful is galK recombineering that exploits a single marker (galK) for positive and negative selection and PCR amplicons for seamless modification in the desired genome locus. Here we describe the engineering of the HSV recombinant BAC 115 by the insertion of a heterologous cassette for the expression of murine interleukin 12 (mIL12) in the intergenic sequence between US1 and US2 ORFs.embargoed_20201017Laura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-FiumeLaura Menotti, Valerio Leoni, Valentina Gatta, Biljana Petrovic, Andrea Vannini, Simona Pepe, Tatiana Gianni, Gabriella Campadelli-Fium

Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68

<p>Abstract</p> <p>Background</p> <p>Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo.</p> <p>Methods</p> <p>In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection.</p> <p>Results</p> <p>We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection.</p> <p>Conclusions</p> <p>Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection.</p

Cancer Screening by Systemic Administration of a Gene Delivery Vector Encoding Tumor-Selective Secretable Biomarker Expression

Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a “theragnostic” agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world

Intravenously Administered Alphavirus Vector VA7 Eradicates Orthotopic Human Glioma Xenografts in Nude Mice

VA7 is a neurotropic alphavirus vector based on an attenuated strain of Semliki Forest virus. We have previously shown that VA7 exhibits oncolytic activity against human melanoma xenografts in immunodeficient mice. The purpose of this study was to determine if intravenously administered VA7 would be effective against human glioma.In vitro, U87, U251, and A172 human glioma cells were infected and killed by VA7-EGFP. In vivo, antiglioma activity of VA7 was tested in Balb/c nude mice using U87 cells stably expressing firefly luciferase in subcutaneous and orthotopic tumor models. Intravenously administered VA7-EGFP completely eradicated 100% of small and 50% of large subcutaneous U87Fluc tumors. A single intravenous injection of either VA7-EGFP or VA7 expressing Renilla luciferase (VA7-Rluc) into mice bearing orthotopic U87Fluc tumors caused a complete quenching of intracranial firefly bioluminescence and long-term survival in total 16 of 17 animals. In tumor-bearing mice injected with VA7-Rluc, transient intracranial and peripheral Renilla bioluminescence was observed. Virus was well tolerated and no damage to heart, liver, spleen, or brain was observed upon pathological assessment at three and ninety days post injection, despite detectable virus titers in these organs during the earlier time point.VA7 vector is apathogenic and can enter and destroy brain tumors in nude mice when administered systemically. This study warrants further elucidation of the mechanism of tumor destruction and attenuation of the VA7 virus

Activation of Cytotoxic and Regulatory Functions of NK Cells by Sindbis Viral Vectors

Oncolytic viruses (OVs) represent a relatively novel anti-cancer modality. Like other new cancer treatments, effective OV therapy will likely require combination with conventional treatments. In order to design combinatorial treatments that work well together, a greater scrutiny of the mechanisms behind the individual treatments is needed. Sindbis virus (SV) based vectors have previously been shown to target and kill tumors in xenograft, syngeneic, and spontaneous mouse models. However, the effect of SV treatment on the immune system has not yet been studied. Here we used a variety of methods, including FACS analysis, cytotoxicity assays, cell depletion, imaging of tumor growth, cytokine blockade, and survival experiments, to study how SV therapy affects Natural Killer (NK) cell function in SCID mice bearing human ovarian carcinoma tumors. Surprisingly, we found that SV anti-cancer efficacy is largely NK cell-dependent. Furthermore, the enhanced therapeutic effect previously observed from Sin/IL12 vectors, which carry the gene for interleukin 12, is also NK cell dependent, but works through a separate IFNγ-dependent mechanism, which also induces the activation of peritoneal macrophages. These results demonstrate the multimodular nature of SV therapy, and open up new possibilities for potential synergistic or additive combinatorial therapies with other treatments

Identification of diagnostic serum protein profiles of glioblastoma patients

Diagnosis of a glioblastoma (GBM) is triggered by the onset of symptoms and is based on cerebral imaging and histological examination. Serum-based biomarkers may support detection of GBM. Here, we explored serum protein concentrations of GBM patients and used data mining to explore profiles of biomarkers and determine whether these are associated with the clinical status of the patients. Gene and protein expression data for astrocytoma and GBM were used to identify secreted proteins differently expressed in tumors and in normal brain tissues. Tumor expression and serum concentrations of 14 candidate proteins were analyzed for 23 GBM patients and nine healthy subjects. Data-mining methods involving all 14 proteins were used as an initial evaluation step to find clinically informative profiles. Data mining identified a serum protein profile formed by BMP2, HSP70, and CXCL10 that enabled correct assignment to the GBM group with specificity and sensitivity of 89 and 96%, respectively (p < 0.0001, Fischer’s exact test). Survival for more than 15 months after tumor resection was associated with a profile formed by TSP1, HSP70, and IGFBP3, enabling correct assignment in all cases (p < 0.0001, Fischer’s exact test). No correlation was found with tumor size or age of the patient. This study shows that robust serum profiles for GBM may be identified by data mining on the basis of a relatively small study cohort. Profiles of more than one biomarker enable more specific assignment to the GBM and survival group than those based on single proteins, confirming earlier attempts to correlate single markers with cancer. These conceptual findings will be a basis for validation in a larger sample size