pDEST FG12-CMV DsRed Vector

Melanoma is the most rapidly increasing malignancy among young people in the United States. If detected early, the disease is easily treated; however, once the disease has metastasized it is largely refractory to conventional therapies and is associated with a high mortality rate. The development of human cancer from a pre-malignant primary tumor to a metastatic lesion that develops at secondary sites is thought to be a multi-step process, requiring many genetic and epigenetic events that provide a growth advantage to cells. It is still unclear which of the many genetic changes in human cancers are required for metastasis. Therefore, it is critical to evaluate each step in the metastatic process. To this end, we will generate novel lentiviral vectors containing fluorescent reporter genes to better understand the metastatic potential of melanoma cells. Vectors containing green fluorescent protein (GFP) have already been generated while vectors containing red fluorescent protein (RFP) and yellow fluorescent protein (YFP) will be cloned. Viruses will be generated and used to infect syngeneic explanted tumor cells. Since each vector will be marked with a reporter gene of a different color, we will be able to track the movement of these cells in vivo and determine the source of each metastatic tumor. Whole body fluorescence will be detected using the FluorVivo Imaging System (INDEC BioSystems, Santa Clara, CA). The experiments proposed will contribute to an increased understanding of the biology of melanoma, which has the potential to identify specific molecular targets and promote the development of more effective therapies for advanced stages of this disease

Characterizing and inhibiting two pathways activated in Glioblastoma Multiforme

Despite major improvements in imaging, radiation, and surgery, the prognosis for patients with Glioblastoma multiforme (GBM) remains clinically challenging. New treatment strategies are badly needed to reduce the mortality and morbidity associated with this disease. The resistance of these tumors to conventional treatments makes GBM patients ideal candidates for molecularly targeted therapies and several agents are currently being developed(1). Because GBM is genetically heterogeneous, combination therapies or the use of multikinase inhibitors are more likely to achieve the greatest therapeutic benefit(2,3). However, genes that can be productively targeted for effective therapies in patients remain to be identified. The overall objective of this project was to better understand the signaling pathways driving cell survival so that new targets can be identified in gliomas. These studies will lead to an increased understanding of the proteins that are altered in this disease and should provide promising opportunities to develop better treatment strategies based on specific molecular targets. Two parallel pathways, which are both activated in GBM, converge on downstream survival signaling cascades. Studies have demonstrated that blocking only one pathway often leads to a transient response (e.g., delayed time to progression), but tumors eventually progress(4). More effective therapies are likely to be those that inhibit more than one target or pathway(5). Targeting antiapoptotic Bcl-2 proteins in combination with RAS/MAPK or AKT/mTOR inhibition is a rationale approach. To determine if inhibiting both the RAS/MAPK and AKT/mTOR pathways in combination results in increased apoptosis in glioma cells, I compared the level of apoptosis in cells treated with each inhibitor alone and in combination. Treatment of glioma cells with a MEK inhibitor in combination with a PI(3)K inhibitor has not previously been reported and therefore represents a new approach in the field. We already know that just inhibiting RAS/MAPK or AKT/mTOR alone results in cell cycle arrest but not death. I tested the effect on cell death when combining the inhibitors of both pathways, and saw an increase in cell death. I determined the growth inhibitory and apoptotic sensitivity of several human glioma cell lines to inhibition of both RAS/MAPK and AKT/mTOR pathways. Due to the heterogeneous nature of GBM, I predicted and saw that these cell lines display varying levels of sensitivity to MEK/PI(3)K inhibition. These differences can then be used in the future to further define the mechanism(s) by which the AKT and MAPK pathways mediate survival signaling in glioma cells

Defining the role of NRAS in melanoma maintenance

The incidence of melanoma has increased 600 percent over the last four decades; it is the most rapidly increasing malignancy among young people in the United States and is currently the leading cause of cancer death in women aged 25- 29. If detected early, the disease is easily treated; however, once the disease has metastasized it is largely refractory to conventional therapies and is associated with a high mortality rate. The development of cancer from a pre-malignant primary tumor to a metastatic cancer that develops at secondary sites is a multi-step process, thought to require many genetic and epigenetic events that provide a growth advantage to cells. It is still unclear, however, which of the many genetic changes are required late in tumor progression. The increased incidence of melanoma, combined with the poor prognosis of patients with advanced disease, make it imperative that we increase our understanding of the underlying genetic causes of melanoma such that better targeted therapeutic strategies can be developed

Melanoma central nervous system metastases: current approaches, challenges, and opportunities

Melanoma central nervous system metastases are increasing, and the challenges presented by this patient population remain complex. In December 2015, the Melanoma Research Foundation and the Wistar Institute hosted the First Summit on Melanoma Central Nervous System (CNS) Metastases in Philadelphia, Pennsylvania. Here, we provide a review of the current status of the field of melanoma brain metastasis research; identify key challenges and opportunities for improving the outcomes in patients with melanoma brain metastases; and set a framework to optimize future research in this critical area

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma

We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids’ quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium.Medicin

The BRAF kinase domain promotes the development of gliomas in vivo

ABSTRACT In-frame BRAF fusions have been observed in over 80% of sporadic pilocytic astrocytomas. In each fusion, the N-terminal autoinhibitory domain of BRAF is lost, which results in constitutive activation via the retained C-terminal kinase domain (BRAF-KD). We set out to determine if the BRAF-KD is sufficient to induce gliomas alone or in combination with Ink4a/Arf loss. Syngeneic cell lines demonstrated the transforming ability of the BRAF-KD following Ink4a/Arf loss. In vivo, somatic cell gene transfer of the BRAF-KD did not cause tumors to develop; however, gliomas were detected in 21% of the mice following Ink4a/Arf loss. Interestingly, these mice demonstrated no obvious symptoms. Histologically the tumors were highly cellular and atypical, similar to BRAF V600E tumors reported previously, but with less invasive borders. They also lacked the necrosis and vascular proliferation seen in BRAF V600E -driven tumors. The BRAF-KD-expressing astrocytes showed elevated MAPK signaling, albeit at reduced levels compared to the BRAF V600E mutant. Pharmacologic inhibition of MEK and PI3K inhibited cell growth and induced apoptosis in astrocytes expressing BRAF-KD. Our findings demonstrate that the BRAF-KD can cooperate with Ink4a/Arf loss to drive the development of gliomas and suggest that glioma development is determined by the level of MAPK signaling

Melanoma Brain Metastasis: Mechanisms, Models, and Medicine

The development of brain metastases in patients with advanced stage melanoma is common, but the molecular mechanisms responsible for their development are poorly understood. Melanoma brain metastases cause significant morbidity and mortality and confer a poor prognosis; traditional therapies including whole brain radiation, stereotactic radiotherapy, or chemotherapy yield only modest increases in overall survival (OS) for these patients. While recently approved therapies have significantly improved OS in melanoma patients, only a small number of studies have investigated their efficacy in patients with brain metastases. Preliminary data suggest that some responses have been observed in intracranial lesions, which has sparked new clinical trials designed to evaluate the efficacy in melanoma patients with brain metastases. Simultaneously, recent advances in our understanding of the mechanisms of melanoma cell dissemination to the brain have revealed novel and potentially therapeutic targets. In this review, we provide an overview of newly discovered mechanisms of melanoma spread to the brain, discuss preclinical models that are being used to further our understanding of this deadly disease and provide an update of the current clinical trials for melanoma patients with brain metastases

Reduced Affinity to and Inhibition by DKK1 Form a Common Mechanism by Which High Bone Mass-Associated Missense Mutations in LRP5 Affect Canonical Wnt Signaling

The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1