Charles Kore Kaufman - US grants

DESCRIPTION (provided by applicant): Melanocytes are pigment-producing cells in the skin that arise from multipotent neural crest cells. The specification of melanocytes requires the activity of a transcription factor called micropthalmia-associated transcription factor (Mitf). In addition to directing formation of melanocytes, the normal functions of Mitf are also linked as a lineage oncogene to the abnormal properties of melanoma. This proposal aims to better understand how to alter melanocyte lineage character and will test the hypothesis that mechanisms that disfavor formation and survival of melanocytes may also inhibit melanoma formation. The vertebrate model organism, Danio rerio (zebrafish), will be used to 1) identify chemicals that inhibit expression of mitf and thus disfavor production of melanocytes and 2) use misexpression of neural crest transcription factors to skew neural crest cells away from the melanocyte lineage or reprogram committed melanocytes to a different but related neural crest lineage (i.e. glial/Schwann cell). In the first approach, a high-throughput chemical genetic screen using ~2280 bioactive compounds is being completed to identify chemicals that specifically decrease mitf expression in developing zebrafish embryos. Compounds that reproducibly decrease mitf expression will be assayed for their effects on neural crest stem cells, precursors, and committed descendants (e.g. glial, neural, cartilage) to determine where in neural crest development each acts. Based on the likely mechanism of action of mitf-inhibitory compounds, standard gain- and loss-of-function approaches in the zebrafish will be used to establish the regulatory mechanism(s) affecting mitf expression. In the second approach, the lineage identity of developing and committed melanocytes will be altered in zebrafish by forcing expression of transcription factors linked to the formation of the adjacent Schwann cell neural crest lineage. Transcription factors shown to skew away from melanocytes during development or to reprogram committed melanocytes will be tested in combination with the identified mitf-inhibitory compounds in a zebrafish melanoma model for their ability to prevent formation and/or cause regression of de novo melanoma tumors. Dr. Kaufman is a clinical fellow in adult oncology at the Dana Farber Cancer Institute (DFCI) and will perform his research project in the laboratory of noted stem cell biologist, Dr. Leonard Zon, in the Division of Hematology/Oncology at the Children's Hospital Boston (CHB). Building on his experience studying mechanisms of transcriptional regulation in the epidermis in mice and humans, Dr. Kaufman will expand his scientific and technical training to studies of lineage regulation in melanocytes and the neural crest using the zebrafish model. Dr. Zon's proven mentorship coupled with the rigorous and nurturing scientific environment offered by the research community at CHB and affiliated institutions offer the maximal opportunity for Dr. Kaufman's success during the award period as a fellow and in his transition to an independent investigator.

Project Summary/Abstract Within a group of cancer-prone cells that share an oncogenic mutation, sometimes described as a ?cancerized field?, only select cells transition to a malignant state. Due to the rarity and transient nature of these events, cancer initiation has been difficult to study and remains incompletely understood. Identifying cells at the earliest stages of transformation and understanding the underlying mechanisms leading to their malignant conversion would allow for earlier detection and treatment of cancer, which should lead to better patient outcomes. Such is the case in the skin, where non-cancerous overgrowths of pigmented melanocytes, called nevi or moles, harbor a potentially cancer-causing mutation, termed BRAFV600E, which is also found in over half of melanoma cancers. Yet nevi rarely progress to invasive melanoma. To enable studies of melanoma initiation and the mechanisms driving this conversion to cancer, we developed a novel reporter of melanoma initiation in a zebrafish melanoma model (that relies on this same BRAFV600E mutation) that expresses a fluorescent protein (EGFP) in the first cell of melanoma and can be visualized in a live animal. Using this model, we found that embryonic neural crest identity reemerges during melanoma initiation, and that regulation of sox10 neural crest transcription factor levels in melanocytes is one key control point in this process. In this proposal, we will test the hypothesis that a specific subset of normally embryonic transcriptional inputs modulates sox10 activity during melanoma initiation and that upregulation of neural crest transcription factors -- in addition to and potentially upstream of sox10 -- directs this transition to malignancy. By defining the role of key transcriptional inputs in modulating sox10 in its native context, we will understand the molecular mechanisms modulating the melanocyte to melanoma transition. We further identify additional neural crest transcription factors that may modulate this process and determine their role and regulation in melanoma initiation. Finally, we use human melanoma patient-derived xenografts (PDX) to further clarify if ongoing expression of these factors is needed for melanoma viability or if their role is primarily during tumor initiation. Overall, this proposal describes innovative approaches to determine the precise regulation of altered transcriptional and epigenetic programs driving melanoma cancer initiation, which may define new and earlier- acting therapeutic targets for treating melanoma.