Development of a New Class of Microrna Controlled OHSV Vectors for Treatment of Glioblastoma

Glioblastoma Multiforme (GBM) is a highly aggressive brain tumor for which there is no treatment. Oncolytic Viruses (OVs) are attenuated tumor-selective viruses designed to replicate in and kill tumor cells without affecting normal tissue. Herpes Simplex Virus type-1 derived oncolytic vectors (oHSV-1) are a promising and safe alternative to current GBM therapies, but the deletions that render these oHSV-1 incapable of replicating in normal tissue also compromise their lytic activity in tumor cells. Consequently, these viruses fail to effectively eradicate the tumor. In this study, I developed a new oHSV-1, named ICP4-miR124t, whose selective replication in GBM cells does not rely on defective genes, but rather is mediated by micro (mi)RNAs differentially expressed between normal tissue and GBM. MiRNAs recognize complementary target sequences in mRNAs resulting in repression of the mRNA. MiR124 is one of the most abundant miRNAs in developing and mature neurons, but is absent in GBM since its presence is incompatible with the tumor phenotype and viability. After introducing four tandem copies of the miR124 target sequence in the 3’UTR of the HSV-1 essential ICP4 gene, I observed robust replication of the resulting ICP4-miR124t oHSV-1 in primary glioblastoma cells, while viral growth was highly impaired in the same cells upon induction of mir124. Toxicity tests involving intracranial injection of immunocompetent and immunodeficient mice showed a dramatic reduction in vector toxicity compared to unregulated control virus. Moreover, ICP4-miR124t-injected animals showed a loss of viral DNA in the brain over time, whereas an increase was observed in control virus injected animals. In conclusion the ICP4-miR124t oHSV-1 developed in this study is promising for GBM treatment because it is capable of killing miR124-negative tumor cells as efficiently as wild-type HSV-1, while lacking toxicity for normal brain. These results are of major Public Health importance not only as a potentially more effective alternative to current inefficient GBM treatments, but also as a new paradigm to treat other kinds of tumors by taking advantage of the differential miRNA expression between tumor and normal tissue

Structure-function studies of the bHLH phosphorylation domain of TWIST1 in prostate cancer cells

The TWIST1 gene has diverse roles in development and pathologic diseases such as cancer. TWIST1 is a dimeric basic helix-loop-helix (bHLH) transcription factor existing as TWIST1-TWIST1 or TWIST1-E12/47. TWIST1 partner choice and DNA binding can be influenced during development by phosphorylation of Thr125 and Ser127 of the Thr-Gln-Ser (TQS) motif within the bHLH of TWIST1. The significance of these TWIST1 phosphorylation sites for metastasis is unknown. We created stable isogenic prostate cancer cell lines overexpressing TWIST1 wild-type, phospho-mutants, and tethered versions. We assessed these isogenic lines using assays that mimic stages of cancer metastasis. In vitro assays suggested the phospho-mimetic Twist1-DQD mutation could confer cellular properties associated with pro-metastatic behavior. The hypo-phosphorylation mimic Twist1-AQA mutation displayed reduced pro-metastatic activity compared to wild-type TWIST1 in vitro, suggesting that phosphorylation of the TWIST1 TQS motif was necessary for pro-metastatic functions. In vivo analysis demonstrates that the Twist1-AQA mutation exhibits reduced capacity to contribute to metastasis, whereas the expression of the Twist1-DQD mutation exhibits proficient metastatic potential. Tethered TWIST1-E12 heterodimers phenocopied the Twist1-DQD mutation for many in vitro assays, suggesting that TWIST1 phosphorylation may result in heterodimerization in prostate cancer cells. Lastly, the dual phosphatidylinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor BEZ235 strongly attenuated TWIST1-induced migration that was dependent on the TQS motif. TWIST1 TQS phosphorylation state determines the intensity of TWIST1-induced pro-metastatic ability in prostate cancer cells, which may be partly explained mechanistically by TWIST1 dimeric partner choice

K13 blocks KSHV lytic replication and deregulates vIL6 nad hIL6 expression: A model of lytic replication induced clonal selection in viral oncogenesis

Background. Accumulating evidence suggests that dysregulated expression of lytic genes plays an important role in KSHV (Kaposi's sarcoma associated herpesvirus) tumorigenesis. However, the molecular events leading to the dysregulation of KSHV lytic gene expression program are incompletely understood. Methodoloxy/Principal Findings. We have studied the effect of KSHV-encoded latent protein vFLIP K13, a potent activator of the NF-κB pathway, on lytic reactivation of the virus. We demonstrate that K13 antagonizes RTA, the KSHV lytic-regulator, and effectively blocks the expression of lytic proteins, production of infectious virions and death of the infected cells. Induction of lytic replication selects for clones with increased K13 expression and NF-κB activity, while siRNA-mediated silencing of K13 induces the expression of lytic genes. However, the suppressive effect of K13 on RTA-induced lytic genes is not uniform and it falls to block RTA-induced viral IL6 secretion and cooperates with RTA to enhance cellular IL-6 production, thereby dysregulating the lytic gene expression program. Conclusions/Significance. Our results support a model in which ongoing KSHV, lytic replication selects for clones with progressively higher levels of K13 expression and NF-κB activity, which in turn drive KSHV tumorigenesis by not only directly stimulating cellular survival and proliferation, but also indirectly by dysregulating the viral lytic gene program and allowing non-lytic production of growth-promoting viral and cellular genes. Lytic Replication-Induced Clonal Selection (LyRICS) may represent a general mechanism in viral oncogenesis. 2007 Zhao et al

Inhibition of Indoleamine-2,3-dioxygenase (IDO) in Glioblastoma Cells by Oncolytic Herpes Simplex Virus

Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO) is expressed in response to interferon γ (IFNγ) and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγ treatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G

BRCA1 Interaction of Centrosomal Protein Nlp Is Required for Successful Mitotic Progression*♦

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability