Functional effect of DOT1L pharmacological inhibition in ERα-positive Ovarian Cancer cells
ABSTRACT: Estrogen Receptor alpha (ERα), a nuclear receptor with transcriptional activity, is a master regulator of estrogen signaling, widely known as therapeutic target in hormone-responsive breast cancer (BC). Moreover, ERα is highly expressed in approximately 80% of High Grade Serous Ovarian Cancer (HGSOC), the most common epithelial ovarian carcinoma. Despite some promising clinical trials evaluating endocrine therapy in this type of tumor, the role of ERα is still unknown. Epigenetic changes, such as DNA methylation, are emerging as key contributing factors to carcinogenesis. Disruptor of telomeric silencing-1-like (DOT1L), the only known histone methyl transferase capable to produce H3K79 mono, di and tri-methylation, modulates ERα actions in hormone-responsive BC. Considering this evidence, ERα-DOT1L association was confirmed in ERα-positive OC cells, PEO1 and PEO4, by Co-IP. DOT1L pharmacological inhibition by EPZ004777 (EPZ) revealed the involvement of this epigenetic enzyme in cell proliferation, cell cycle progression and apoptosis. Transcriptome profiling after ICI (a Selective Estrogen Receptor Degrader) and EPZ treatment, in both cell lines, has underlined a deep impact of both compounds on ERα-modulated genes, including the down-regulation of ERα itself. On the other hand, functional analysis showed that commonly affected transcripts are involved in different cellular processes, such as cancer cell survival, chemoresistance and cell cycle progression. Moreover, ChIP-qPCR performed on ERα promoter highlighted ERα and DOT1L co-localization, both in PEO1 and in PEO4 cells, which was reduced after EPZ treatment, suggesting a role of this complex on receptor transcriptional activity. In addition, drug combination studies performed with EPZ and ICI showed an additive effect in cell growth inhibition. Taken together, these results suggest DOT1L as a potential therapeutic target in the treatment of OC.
Project description:Paired platinum-sensitive & platinum-resistant cell lines PEO1 and PEO4 (respectively) were derived from the same patient. Cell line cultures were profiled with the HT-HGU133a GeneChip to investigate chemotherapy resistance related expression of genes marked with different histone modifications in a primary ovarian tumour (clinical data for the primary ovarian tumour is available as Series supplementary file). 4 replicates of each of the PEO1 and PEO4 cell line were profiled using the HT-HGU133a GeneChip
Project description:DOT1L-dependent H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, DNA repair, modulation of constitutive heterochromatin, and transcriptional activation. Overall design: We used microarrays to detail the global program of gene expression in C021 melanoma cells (DOT1L wild type) and C025 melanoma cells (DOT1L mutant) treated with DMSO vehicle control, and in C021 cells treated with the DOT1L inhibitor EPZ-5676.
Project description:Purpose: To characterize transcriptional changes associated with inhibition of Dot1l in 2 inv(16) patient AML samples Methods: We sequenced mRNA from patient samples that were exposed to 5 uM EPZ004777 or DMSO control for 7 days. Results: Inhibition of Dot1l leads to gene expression changes in genes related to cell growth and cell cycle. Examination of mRNA levels between cells treated with 5 uM EPZ004777 or DMSO control
Project description:To identify DOT1L targets, associated signaling pathways and networks in chondrocytes, we used genome-wide gene expression microarray analysis in human articular chondrocytes of 5 different donors (without known or documented joint disease) treated with EPZ-5676 or vehicle for 4 days. It is known that DOT1L inhibitors require longer time of treatment in order to show effect and influence the expression of MLL target genes in leukemia cells, but we opted for this relatively short inhibition time to be able to identify early changes induced by DOT1L inhibition. Human articular chondrocytes were obtained from 5 non-OA hip fracture patients. The cells were treated with 3 μM EPZ-5676 or vehicle (DMSO) for 4 days.
Project description:Purpose: To characterize the genome-wide distribution of H3K79me2 in human leukemia cell lines treated with the Dot1l inhibitor EPZ004777 or control Methods: We performed Chip-seq for the H3K79me2 on the leukemia cell lines Mutz3, Loucy and Molm14 after 6 days in culture in the presence of 3uM EPZ004777 or DMSO control Results: H3K79me2 is completely erased from key target genes such as the HOXA cluster. Conclusions: Exposure of Mutz3, Loucy and Molm14 to 3uM EPZ004777 erases H3K79 methylation globally as well as on key loci ChIP-Seq for H3K79me2 on human leukemia cell lines exposed for 6 days to the Dot1l inhibitor EPZ004777