Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.

Personalized oncology and BRAFK601N melanoma: model development, drug discovery, and clinical correlation.

PURPOSE: Mutations in BRAF are the most prominent activating mutations in melanoma and are increasingly recognized in other cancers. There is currently no accepted treatment regimen for patients with mutant BRAFK601N melanoma, and the study of melanoma driven by BRAF mutations at the 601 locus is lacking due to a paucity of cellular model systems. Therefore, we sought to better understand the treatment and clinical approach to patients with mutant BRAFK601N melanoma and subsequently develop a novel personalized oncology platform for rare or treatment-refractory cancers. METHODS: We developed and characterized the first patient-derived, naturally occurring BRAFK601N melanoma model, described herein as OHRI-MEL-13, and assessed efficacy using the Prestwick Chemical Library and select targeted therapeutics. RESULTS: OHRI-MEL-13 exhibits loss of heterozygosity of BRAF, closely mimics the original tumor's gene expression profile, is tumorigenic in immune-deficient murine models, and is available for public accession through American Type Culture Collection. We present in silico modeling data, which illustrates the therapeutic failure of BRAFV600E-targeted therapies in BRAFK601N mutants. Our platform elucidated a unique role for MEK inhibition with cobimetinib, which resulted in short-term clinical success by reducing the metastatic burden. CONCLUSION: Our model of BRAFK601N-activated melanoma was developed, thoroughly characterized, and made available for public accession. This model served to demonstrate the feasibility of a novel personalized oncology platform that could be optimized at an institutional level for rare variant or treatment-refractory cancers. We also demonstrate the clinical utility of monotherapy MEK inhibition in a case of BRAFK601N melanoma.

Generating Primary Models of Human Cancer to Aid in the Development of Clinically Relevant Oncolytic Viruses.

Oncolytic viruses (OVs) represent a rapidly advancing class of cancer immunotherapeutic that is helping to shift the treatment paradigm of cancer. Initially thought to only have direct oncolysis ability, the established anticancer mechanisms of action of OVs have rapidly expanded to incorporate a multitude of immune-activating properties. Specifically, the field is focused on harnessing OVs to train patients' immune systems to recognize and eradicate cancer. Recent scientific progress has enabled the shift in focus to clinical translation, and as such it has become increasingly important to utilize clinically relevant models in the development and testing of novel OVs. As a result of this need, some research groups have developed their own experimental models derived from clinical or surgical specimens at their home institutions. This chapter outlines methodology for the generation of three distinct patient-derived experimental models. This includes cell cultures derived from (1) malignant ascites of ovarian cancer patients, (2) direct excision of metastatic melanoma, and (3) core biopsies of pancreatic cancer. Each of these methodologies has been utilized to generate replicating cellular models and have been demonstrated as valuable tools in in vitro, in vivo, and ex vivo studies.

Lighting a Fire in the Tumor Microenvironment Using Oncolytic Immunotherapy.

Oncolytic virus (OV) therapy is potentially a game-changing cancer treatment that has garnered significant interest due to its versatility and multi-modal approaches towards tumor eradication. In the field of cancer immunotherapy, the immunological phenotype of the tumor microenvironment (TME) is an important determinant of disease prognosis and therapeutic success. There is accumulating data that OVs are capable of dramatically altering the TME immune landscape, leading to improved antitumor activity alone or in combination with assorted immune modulators. Herein, we review how OVs disrupt the immunosuppressive TME and can be used strategically to create a "pro-immune" microenvironment that enables and promotes potent, long-lasting host antitumor immune responses.

Diversity in yeast-mycelium dimorphism response of the Dutch elm disease pathogens: the inoculum size effect.

Dutch elm disease (DED) is caused by the dimorphic fungi Ophiostoma ulmi, Ophiostoma novo-ulmi, and Ophiostoma himal-ulmi. A cell population density-dependent phenomenon related to quorum sensing was previously shown to affect the reversible transition from yeast-like to mycelial growth in liquid shake cultures of O. novo-ulmi NRRL 6404. Since the response to external stimuli often varies among DED fungal strains, we evaluated the effect of inoculum size on 8 strains of the 3 species of DED agents by determining the proportion of yeast and mycelium produced at different spore inoculum concentrations in defined liquid shake medium. The results show that not all DED fungi strains respond similarly to inoculum size effect, since variations were observed among strains. It is thus possible that the different strains belonging to phylogenetically close species use different signalling molecules or molecular signalling pathways to regulate their growth mode via quorum-sensing mechanisms.