Approximately 80,000 Americans are diagnosed with melanoma annually, leading to nearly 10,000 deaths. Despite recent progress in targeted therapies and immunotherapies, melanoma remains one of the deadliest human cancers and the vast majority of patients with advanced melanoma will die of their disease. Abnormal DNA methylation is nearly universal in melanoma, but the functional consequences and the role of DNA methylation in melanoma development is largely unknown. Our preliminary data from a genetically engineered mouse model of melanoma and human melanoma lines suggest important functional roles for DNA methylation in melanoma development and establishment of intratumor heterogeneity. Based on these observations, we hypothesize that DNA methylation is required for melanomagenesis and regulates melanoma sub-populations.
Specific aims : This proposal is comprised of two specific aims.
The first aim i s focused on elucidating the role of the de-novo methyltransferase Dnmt3b in melanoma formation and growth. We will use a recently developed genetically engineered mouse model of melanoma (Braf/Pten) and tetracycline inducible Dnmt3b allele to study the effect on melanoma growth in vivo. We will also use RNAi to functionally evaluate the role of Dnmt3b in proliferation of human melanoma lines. To elucidate the mechanism of growth alterations that occurs with Dnmt3b loss, we will investigate the changes in PI3K/mTOR signaling. Furthermore, we will determine the mechanism of Dnmt3b upregulation in melanoma using a luciferase reporter assay and determine the value of Dnmt3b a prognostic biomarker, using quantitative immunofluorescence.
The second aim will focus on the role of Dnmt3b and DNA methylation in melanoma tumor heterogeneity. Intratumor heterogeneity poses a significant challenge to cancer treatment in general, and may be an important factor that leads to primary drug resistance. The cellular mechanisms that give rise to and regulate intratumor heterogeneity are poorly understood, but epigenetic regulation including DNA methylation may play a central role in this process. Based on our preliminary data showing that treatment with 5-aza-dC, a DNA demethylating agent, dramatically alters the distribution of intratumor subsets, we hypothesize that DNA methylation by Dnmt3b plays key roles in defining and regulating tumor subsets in melanoma. We propose to investigate this hypothesis using flow cytometry, bisulfite sequencing, and quantitative immunofluorescence. Relevance: This project will evaluate the role of DNA methylation in melanoma formation, progression, establishment and maintenance of tumor heterogeneity. Results of proposed studies will advance our understanding of melanoma biology and could define DNA methyltransferases as therapeutic targets in melanoma.

Public Health Relevance

Melanoma is the deadliest form of skin cancer, claiming nearly 10,000 lives in the United States annually. Abnormal DNA methylation is a genetic change that occurs in most melanomas, but its functional role is poorly understood. In this proposal, we examine the functional role of DNA methylation in melanoma formation and growth with the aim of advancing our understanding of melanoma biology which may lead to development of improved therapies for melanoma.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30CA196089-03
Application #
9217594
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Damico, Mark W
Project Start
2015-03-16
Project End
2018-03-15
Budget Start
2017-03-16
Budget End
2018-03-15
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Graduate Schools
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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