Abstract A26: Integrated epigenomic profiling reveals widespread demethylation in melanoma, and reveals aberrant CSF-1 receptor expression as a regulator of malignant growth and invasion inhibited by PLX3397
Authors: Mo Yongkai , Giricz Orsolya , Hu Caroline , Dahlman Kimberly , Bhattacharyya Sanchari , Nguyen Hoa , Matusow Bernice , Bhagat Tushar , Yu Yiting , Shellooe Rafe , Burton Elizabeth , Habets Gaston , Greally John , Kenny Paraic , Sosman Jeffrey , Bollag Gideon , West Brian L. , Verma Amit 
Published: August 2014

Abstract
Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metastasis, but this previously has not been studied at a genome wide level in melanoma. We analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it with healthy melanocyte controls. We observed widespread demethylation in malignant melanoma, preferentially outside of CpG islands. The epigenomic loss of methylation was independent of mutational status of BRAF, RAS and Kit. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with very few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways that were aberrantly expressed and regulated by loss of DNA methylation. Strikingly, the colony stimulating factor-1 receptor (CSF1R, c-fms) was aberrantly expressed and hypomethylated in nearly all cases. CSF1R is a transmembrane tyrosine kinase receptor that predominantly regulates macrophages, osteoclasts, and microglia, but is known to sometimes be aberrantly expressed by malignant cells in Hodgkins lymphoma. The expression of CSF1R on malignant melanocytes was validated by immunohistochemical analysis of primary tumors. In a melanoma cell line (A2058) we found through PCR sequencing of the cDNA 5' untranslated region that the CSF1R can be expressed through an aberrant promoter, as has been described for Hodgkin lymphoma. A custom Taqman assay was developed for this unique transcript, and then used to detect the transcript in 4 of 40 samples in a panel of melanoma biopsies, suggesting that aberrant CSF1R expression in melanoma is not uncommon. Expression of CSF1R protein in A2058 cells was confirmed by FACS using anti-CD115 antibodies, and by Western blot using antibodies directed to the C-terminus. Expression of the ligand CSF-1 was also found in A2058 cells by both ELISA and Taqman assays. Inhibition of A2058 cell growth by PLX3397, a clinically relevant small molecule inhibitor of CSF1R kinase, could be observed in 3D cell culture, indicating that under some conditions an autocrine stimulation of growth occurs. shRNA mediated knockdown of CSF1R also demonstrated decreased colony size and increased apoptosis in 3D culture conditions. The invasiveness of A2058 cells was decreased after treatment with PLX3397 or anti-CSF1 antibodies, suggesting a role for melanoma cancer cell expression of CSF1R in metastasis. Since A2058 cells possess an oncogenic BRAF mutation, co-inhibition of CSF1R and BRAF was tested and resulted in synergistic blockade of xenograft growth. The CSF1R inhibitor, PLX3397, is under investigation in clinical trials for breast, glioma, and other cancers, and these data present a preclinical rationale for its study in malignant melanoma.
Citation Format: Yongkai Mo, Orsolya Giricz, Caroline Hu, Kimberly Dahlman, Sanchari Bhattacharyya, Hoa Nguyen, Bernice Matusow, Tushar Bhagat, Yiting Yu, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John Greally, Paraic Kenny, Jeffrey Sosman, Gideon Bollag, Brian L. West, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma, and reveals aberrant CSF-1 receptor expression as a regulator of malignant growth and invasion inhibited by PLX3397. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A26.