A screen for epigenetic reprogramming reveals LSD1 inhibitor as a potential intervention to promote differentiation in pancreatic cancer


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Frances Willenbrock1,James Wantling1,Eric O'Neill1
1University of Oxford, Oxford, UK

Abstract

Background

Pancreatic ductal adenocarcinoma (PDAC) responds poorly and rapidly gains resistance to conventional chemotherapeutics. Since this resistance may derive from proliferation of stem-cell like cells, inducing differentiation in these cells should increase their susceptibility to therapy. Epigenetic silencing of the set of genes responsible for differentiation commonly occurs and there is therefore potential to “reprogram” these cells by altering their epigenetic status. Our work looks at the effect of demethylase inhibitors in stabilising a more differentiated status in pancreatic cancer.

Method

We established a screen in which the PDAC cell line PSN1 was stably transfected with a reporter consisting of the RASSF1A promoter region linked to luciferase. Hypermethylation of the RASSF1A promoter is associated with adverse prognosis. This screening is therefore suitable as a proxy for detecting genome-wide demethylation events upon therapeutic intervention. Clones responding to treatment with the DNA methyltranferase-1 inhibitor, , by increasing luciferase activity were further screened for synergy with known inhibitors of epigenetic modifiers. The effect of inhibitors on the differentiation status of parental PSN1 cells was validated by qPCR and western blotting.

Results

We identified lysine-specific demethylase 1 inhibitor, GSK-LSD1, as a positive hit in this screen. GSK-LSD1 increased expression levels of E-cadherin, claudin 1 and occludin mRNA and decreased Zeb2 mRNA expression. By optimising scheduling and media conditions we achieved durable responses in cellular differentiation status upon drug withdrawal, indicating we are rewiring the epigenome (e.g. gene specific DNA methylation, increased H3K4me2, and decreased H3K9me2) to stably alter cell phenotype. 

Conclusion

Our screen is a rapid, sensitive method for detecting drug combinations that alter the epigenome in cancer cells. Use of GSK-LSD1 increased E-cadherin and other markers of differentiation, indicating that cells switch phenotype. Altering tumour cell phenotype to be well differentiated is likely to be an effective strategy to improve prognosis and responses to therapy.