Abstract Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer-related deaths in the U.S., is associated with a pronounced collagenrich stromal reaction that has been shown to contribute to chemoresistance. We recently published that PDAC cells grown in the collagen microenvironment are resistant to gemcitabine-induced proliferation and checkpoint arrest (Dangi-Garimella et al, Cancer Research 2011). The effects were mediated through upregulation of membrane type 1-matrix metalloproteinase (MT1-MMP)- and ERK1/2-dependent expression of high mobility group A2 (HMGA2), a well-known chromatin remodeling protein. We have now found that relative to cells grown on tissue culture plastic, PDAC cells grown in 3-D collagen display increased acetylation of lysines K9 and K27 on histone H3 along with a reduction in the levels of the heterochromatin binding protein HP1-α, suggesting that cells in collagen have a more open chromatin state. Decreasing MT1-MMP or HMGA2 expression or inhibiting ERK1/2 activity attenuates the effect of collagen on K9 and K27 acetylation. Collagen also increases Aurora kinase B-dependent phosphorylation of S10 on histone H3, which has been shown to reduce HP1-α binding to the chromatin and thereby promote more open chromatin state. 3-D collagen also increases expression of the histone acetyl transferase enzymes pCAF and GCN5. Finally, cells in 3D matrix demonstrate increased H2AX and a reduced tailing with the comet assay following gemcitabine treatment, suggesting that there is increased DNA repair associated with the more open chromatin state when cells are in the 3-D collagen microenvironment. Overall, our results increase our understanding of how growth in 3-D collagen contributes to chemoresistance and identify potential therapeutic targets against this deadly cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C52.