Holmen Lab Research - Melanoma

The incidence of melanoma has been increasing at an alarming rate over the past thirty years. Approximately 90,000 new cases occur each year with nearly 10,000 resulting in death. Melanoma accounts for the majority of skin cancer deaths due to its propensity to metastasize to distant organs, including the lung and brain, and brain metastases are responsible for up to half of all melanoma deaths. Among all cancers that frequently metastasize to the brain, including breast, lung, colon, and renal cancer, melanomas have the highest frequency for colonizing this organ. As many as 43% of stage IV melanoma patients present with brain metastases at diagnosis and nearly 75% of autopsy reports identify central nervous system (CNS) involvement. Melanoma patients with brain metastases have a poor prognosis and overall survival for these patients ranges between 4 and 9 months from the time of diagnosis. Since 2011, several new therapies have been FDA approved for this disease but brain metastases are often a major component of treatment failure. Given the poor prognosis for these patients, improved prognostic biomarkers are desperately needed to identify those patients at highest risk for disease progression and development of brain metastases as early as possible. To date, multiple clinical, pathologic, and gene expression features associated with higher risk of development of metastases have been published. However, many individual published metastatic prognostic factors have failed validation in independent studies, and no detailed, integrated clinical and molecular predictor for brain metastases has been published. Development of more sensitive and specific predictors of disease spread to the brain would enable the implementation of clinical trials testing the effectiveness of directed surveillance and/or therapeutic prevention strategies in those patients at highest risk of developing future brain metastases. While earlier detection may have been of little consequence in the era prior to approval of effective systemic therapies, this is likely not the case today where multiple treatment options exist.

Further advances in the management of this disease require model systems that aid in the understanding of the behavior of melanoma and assist in the identification of mechanisms of resistance. A major initiative in our research program 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 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 twelve 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. However, these tumors were not metastatic.

Metastasis is responsible for over 90% of all cancer-related deaths and unfortunately melanoma has a propensity to metastasize early in disease progression. In melanoma patients, the most common sites of metastases are skin, lung, brain, liver, bone, and intestine. Brain metastases are associated with extremely poor prognosis and are often responsible for treatment failure. Studies have suggested that as many as half of all melanoma deaths are due to brain metastases. Therefore, it is critical that we further our understanding of the metastatic process such that improved therapies can be developed for these patients. To this end, we have used our novel mouse model of melanoma to identify genes that promote metastasis. We have induced melanoma in this model by expression of mutant NRAS or BRAF in the context of Ink4a/Arf loss, Pten loss or AKT activation. Interestingly, tumors induced with mutant NRAS in the context of Ink4a/Arf loss are not metastatic tumors induced with mutant BRAF in the context of Ink4a/Arf loss and activated AKT1 metastasize to the lungs and brain similar to the human disease. We are currently evaluating the contribution of both Pten loss and activation of different AKT isoforms to melanoma metastasis in vivo with the goal of identifying more effective ways to target this pathway in tumor cells while limiting toxicity to normal cells.