Abstract 4440: Diffuse intrinsic pontine glioma epigenetic marks change depending on cell culture conditions: Implications for investigating epigenetically directed therapy

Abstract

Abstract Methylation of cytosine is viewed as repressive of transcription. 5-methylcytosine (5mC) can be removed from DNA by activity of ten-eleven translocation (TET) methylcytosine dioxygenase. The first step in erasing the repressive 5mC mark is conversion to 5-hydroxymethylcytosine (5hmC) by TET. Subsequent steps lead to replacement of 5hmC by cytosine, providing a mechanism by which 5mC-mediated repression can be erased. We have previously identified 5hmC and 5mC as being imbalanced in primary tumor tissue samples from the universally fatal pediatric malignancy diffuse intrinsic pontine glioma (DIPG) compared normal brain and glioblastoma (GBM). The aberration in DNA methylation along with known aberrant histone methylation in DIPGs, likely creates an active transcriptional/translational state and potentiates tumor growth. We hypothesized that the elevation of 5hmC in DIPG is secondary to increased TET activity. We found two different epigenetic signatures for DIPG cells depending on their culture conditions. When cultured in serum-free media as neurospheres, to our surprise, we found cells had low 5hmC, in contrast to what we observed in primary tumor and in orthotopic xenograft tumors. We hypothesized that 5hmC status may vary depending on differentiation state, and found that when DIPG cells were differentiated on matrigel for 14 days in the presence of serum, 5hmC levels increased and were similar to that observed in primary tissue and xenograft tumors. Differentiation was confirmed by upregulation of GFAP (glial marker) and MAP2 (neuronal marker) expression and the elaboration of long processes. We also found that DIPG cells in the serum/matrigel/differentiated state had increased TET1 and TET2 mRNA expression relative to undifferentiated neurospheres (2.6 and 2.1 fold increase respectively for TET1 and TET2 mRNA expression). As a control for adherent versus nonadherent growth, we also plated DIPG cells on laminin in EGF/FGF media, and we found that the serum-differentiated cells upregulated TET1 and TET2 mRNA 6.7 and 3.3 fold respectively, compared to laminin-plated cells, providing further support that differentiation affects epigenetic marks in DIPG. In contrast to DIPG, GBM cells did not exhibit similar changes in TET mRNA expression when differentiated, emphasizing the uniqueness of the DIPG epigenetic signature. Our data shows that neurosphere cell culture models of DIPG may not be true epigenetic representations of DIPG tumor in vivo and the predicted response to potential therapeutic options may differ based on the epigenetic state used for preclinical testing. Citation Format: Sama Ahsan, Michael Haffner, Charles Eberhart, Eric Raabe. Diffuse intrinsic pontine glioma epigenetic marks change depending on cell culture conditions: Implications for investigating epigenetically directed therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4440.