The incidence of melanoma has been increasing at an alarming rate over the past 20 years. Approximately 70,230 new cases were expected in 2011 with nearly 8,790 resulting in death. Melanoma is the most rapidly increasing malignancy among young people in the United States and is the most common form of cancer in young adults 25-29 years old. Over half of melanoma patients are younger than 60 years old. Melanoma accounts for the majority of skin cancer deaths, and prognosis is poor for advanced stages of the disease. The five-year survival rate for patients with metastatic melanoma is less than 15%.
Current FDA-approved drugs for advanced melanoma include interleukin-2 (IL-2), dacarbazine, ipilimumab (anti-CTLA-4), and vemurafenib (mutant BRAF inhibitor). While treatment with IL-2 or ipilimumab can produce a durable response, only a small percentage of patients ultimately respond and side effects can be severe. Results from clinical studies with vemurafenib have been very encouraging in the treatment of tumors with mutant BRAF. Initial responses, however, are not durable and resistance occurs in the majority of patients. Further advances in the management of this disease require model systems that aid in understanding the behavior of melanoma and assist in identifying mechanisms of resistance.
A major initiative in the Holmen Lab's research has been to develop a novel high-throughput mouse model of melanoma based on retroviral-mediated gene delivery to melanocytes. We have successfully developed this model and further employed the use of this model to better understand melanoma biology and response to therapy. We have identified genes and proteins with differential roles in melanoma initiation and progression as well as intrinsic resistance to mitogen-activated protein kinase (MAPK) inhibition. Our group has also extended the utility of this mouse model system by engineering the viruses to be responsive to doxycycline in the presence of Tet-off or Tet-on proteins. This allows us to regulate the expression of the delivered genes post-infection in vivo, define the role of specific genes in tumor maintenance, and develop models of resistance. We hope to use these tools to design rational combination therapies to improve outcome in patients with advanced melanoma.
In the melanoma model we developed, tumors evolve from gene mutations in developmentally normal somatic cells in the context of an unaltered microenvironment and, therefore, more closely mimic the human disease. Tumor development is a dynamic process that depends on the interactions between the tumor and its microenvironment. In our model, only a small number of cells are modified and therefore the cells surrounding the tumor are normal. Using this system, newly identified genes can be rapidly validated for their role(s) in tumor formation, progression, maintenance, and resistance to therapy. This method is based on the RCAS/TVA retroviral vector system that allows for tissue- and cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. This system utilizes a viral vector, RCASBP(A), derived from the avian leukosis virus (ALV). The receptor for RCASBP(A) is encoded by the TVA gene and is normally expressed in avian cells but not in mammalian cells. In mammalian cells engineered to express TVA, the viral vector is capable of stably integrating into the DNA and expressing the inserted experimental gene, but the virus is replication-defective, which allows for multiple rounds of infection. The ability of TVA-expressing mammalian cells to be infected by multiple ALV-derived viruses allows efficient modeling of human melanoma because multiple genetic alterations can be introduced into the same animal without the expense or time associated with creating new strains of mice. In addition, by restricting the expression of the viral receptor to melanocytes and by delivering the virus through subcutaneous injection, two levels of targeting are achieved.
The dopachrome tautomerase (DCT) promoter, also known as tyrosinase-related protein 2 (TRP2), was chosen to drive expression of the viral receptor TVA specifically in melanocytes since this gene is expressed early in melanocyte development when the cells are mitotically active. Because a significant percentage of both familial and sporadic melanomas have mutations that functionally inactivate both INK4a and ARF, DCT-TVA mice were crossed to Ink4a/Arf lox/lox mice to generate DCT-TVA;Ink4a/Arf lox/lox mice. As proof-of-principle, newborn mice were injected subcutaneously with RCAS viruses containing Cre-recombinase and NRASQ61R. Whereas no tumors were detected in TVA-negative mice, melanomas were visible in DCT-TVA;Ink4a/Arf lox/lox mice as early as three weeks of age. Within 12 weeks, more than one-third of DCT-TVA;Ink4a/Arf lox/lox mice developed melanoma that was histologically similar to the human disease. Delivery of a virus in which NRASQ61R and Cre expression was linked by an internal ribosomal entry site (IRES) resulted in tumor formation in nearly two-thirds of TVA-positive mice.