2540 Background: We reported that immunohistochemistry (IHC) expression of the platinum/copper transporter CTR1 in human tumors correlated inversely with DNA methylation and was increased by the DNA demethylating agent decitabine (D. Stewart et al, Proc ASCO 2008). Decitabine also increased expression of folate transporters in platinum-resistant cell lines that have decreased expression of folate transporters and of small GTPases such as RHOA that regulate endocytosis (D Shen et al. Br J Cancer 2004). Methods: Tumors were biopsied pre decitabine and on cycle 1 day 12 in 31 patients with refractory malignancies in a phase I study of decitabine given days 1–5 ± 8–12 each cycle. We used IHC to assess the folate carriers FOLR1 and RFC1, the glucose transporter GLUT4 and the endocytosis regulating small GTPase RHOA. Scores of 0–300 were calculated by multiplying staining intensity (0–3) by % cells staining. LINE assays were used to assess % global DNA methylation. Results: DNA methylation did not correlate with FOLR1, RFC1 and GLUT4 scores but did correlate inversely with RHOA (r= -0.58, p=0.006). Median tumor IHC scores post vs pre decitabine were 80 vs 80 (range, 0–270 vs 0–210, p=0.89) for FOLR1, 90 vs 90 (range, 15–300 vs 0–300, p=0.17) for RFC1, 0 vs 0 (range 0- 140 vs 0–70, p=0.61) for GLUT4, and 77.5 vs 50 (range, 0–210 vs 0–210, p=0.03) for RHOA. If only tumors with pre decitabine scores <150 were included, post vs pre RFC1 differences were significant (95 vs 80, p=0.004). Outcomes were similar whether or not 4 melanoma patients (in whom melanin pigment decreased the reliability of IHC scoring) were included. Conclusions: As previously noted with the copper/platinum transporter CTR1, treatment with the DNA demethylating agent decitabine was associated with a significant increase in tumor expression of the endocytosis regulator RHOA in resistant human tumors. Expression of the folate transporter RFC1 was also increased if only patients with levels that were initially low were included. This suggests that decitabine should be tested for its ability to increase uptake of chemotherapy in resistant tumors. Promoter methylation for these transporters has not yet been assessed. Supported in part by NIH grant UO1 CA062461–10 & R21 CA112895–01A1. No significant financial relationships to disclose.

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Breast cancer is clinically and molecularly complex disease driven by aberrant genetic and epigenetic alterations. Epigenetic alterations in particular DNA methylation changes are one of the most important events involved in breast cancer initiation and progression. Previous reports identified many aberrant DNA methylation signatures associated with molecular subtypes of breast cancer and over 100 candidate genes with promoter hypermethylation in breast cancer. However, it remains elusive which of these genes with promoter hypermethylation play “driver” role in tumorigenesis. In previous studies, the average gain of DNA methylation across all cancer samples compared to the average DNA methylation in normal samples has been the criterion to select for potential targets. However, known tumor suppressor driver genes regulated by methylation are relatively infrequently altered in target cancers. Therefore, we propose the paradoxical hypothesis that identifying hypermethylated cancer drivers require focusing on infrequent rather than frequent events. Hence, to identify these potential driver genes, we developed an algorithm with two unique properties. First, unlike previous studies we focused on targets that gained DNA methylation relatively infrequently (10-40%) and that lost expression in breast cancer. Second, using this algorithm, we distinguished cancer dependent gain of DNA methylation from age-dependent gain of methylation. To discern age dependent and independent DNA methylation changes, we generated DNA methylation sequencing data on 29 normal purified breast epithelium (age range 33-82 years old). Furthermore, to study the biological effects of the overexpression or downregulation of these genes, we generated DNA methylation sequencing data on 6 breast cancer cell lines. We also used DNA methylation and RNA expression datasets (675 cancer, 100 normal) available through the TCGA. Using our algorithm, we identified 53 genes with age independent promoter hypermethylation and loss of expression in TCGA tumor samples. To begin testing the biological effects of these driver genes, we performed canonical pathway enrichment analyses using Ingenuity Pathway Analysis software. We also investigated the mutational status of these genes and their molecular subtype enrichment. Based on these analyses, we picked 12 genes (C10orf125, RUNX3, YOD1, FXYD5, SMOC1, SLC16A5, RNLS, DKK1, PNPLA3, FZD10, RND2, and PLCB1) for further study. We stably overexpressed these potential driver genes in different breast cancer cell lines. Twelve genes out of the 12 tested, slowed cell proliferation and 9 decreased anchorage independent growth. We further validated these driver genes by knocking them out in normal human mammary epithelial cells using CRISPR/Cas9 tool. The loss of these genes, increased cell proliferation rate in normal human mammary epithelial cells compared to the control cells. In conclusion, based on our preliminary data, using bioinformatics tools as well as functional assays, we identified epigenetically altered breast cancer driver genes. Identifying and deciphering true epigenetic cancer drivers could potentially lead to the development of therapeutic drugs targeting these genes and/or targeting pathway dependence. Citation Format: Panjarian S, Madzo J, Slater C, Jelinek J, Chen X, Issa J-P. Identification of epigenetically silenced breast cancer driver genes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-05-03.