Engineering an oncolytic HSV-1 for the treatment of Melanoma

Abstract

Over 50,000 people die from cutaneous melanoma each year, despite it being a readily detectable skin cancer. Whilst survival has significantly improved in recent times due to the development of new treatments, severe toxicity and resistance to these therapies are common, meaning safer and more effective treatments are still needed. Most melanomas appear to carry at least one mutation that causes activation of ERK, a signaling protein that causes high levels of cell proliferation. Small molecules that inhibit MEK1/2 and mutant BRAF, signaling proteins directly upstream of ERK, are a currently approved combination therapy for melanoma. However, resistance to these inhibitors regularly occurs, with ERK frequently reactivated. DUSP6 is a protein found in human cells whose function is to specifically deactivate ERK, thus overexpression of DUSP6 stands as an interesting avenue for inhibiting ERK signaling in melanoma. To evaluate this idea, we generated several melanoma cell lines with inducible expression of DUSP6, enabling assessment of the effect DUSP6 overexpression, alone or in combination with either a BRAF or ERK inhibitor, had on BRAF mutant melanoma cells. In treatment naïve cell lines, DUSP6 overexpression and inhibitor combination led to greater suppression of ERK signaling, reduced cell proliferation, a higher proportion of apoptotic cells and/or a reduced propensity to develop resistance, than either treatment alone. Interestingly, combination treatment of DUSP6 overexpression and ERK inhibitor remained somewhat effective in cell lines that had previously developed resistance to mutant BRAF and/or MEK1/2 inhibitors. The oncolytic herpes simplex virus (HSV) T-Vec is another recently approved melanoma treatment that whilst having proven its safety, has also had limited success. The gene deletions that render T-Vec cancer specific potentially also reduce its efficacy, thus an alternate mechanism of rendering an oncolytic virus melanoma specific could increase its effectiveness. To achieve this, we attempted to generate an oncolytic HSV by replacing the promoter of UL54 with regulatory elements that would limit expression of this essential HSV gene, and thus virus replication, to melanoma cells. Usage of a tyrosinase enhancer/promoter construct was expected to limit transcription of UL54 to melanocytic cells (which includes many melanoma cells), whilst a long, structured 5’ untranslated region should restrict translation of UL54 to cancer cells (like melanoma). Evaluation of the regulatory elements indicated expression of a target gene would only occur in cells where tyrosinase was present. However, due to difficulties encountered with replacing the UL54 promoter with these elements, no virus could be produced and tested. However, the final results obtained indicated that the last strategy attempted to generate this oncolytic HSV had succeeded in producing the desired HSV recombinant genome, although further analysis is necessary to be certain.