Targeting histone modifications as a novel approach

Targeting histone modifications as a novel approach for a selective treatment for Diffuse Intrinsic Pontine Glioma

Background:
Diffuse intrinsic pontine glioma (DIPG) is a tumour located in the pons of the brain stem which affects mostly young children. DIPG has a zero percent survival rate with a median overall survival of children diagnosed with DIPG of approximately 9 months, which makes it one of the most devastating paediatric malignancies. Clinical studies conducted over the past 20 years, most of them using drugs approved for adults, have failed to demonstrate any significant improvement in survival for these patients. In a variety of human cancers, epigenetic alterations have emerged as common hallmarks and key factors in tumour initiation and progression. Interestingly, in about 90% of DIPGs, a somatic mutation in the H3F3A gene has been identified resulting in replacement of lysine 27 by methionine in its encoded histone H3.3 protein (H3.3K27M). This mutation has been recently shown to be the driver mutation in DIPG. Expression of this mutation has a dominant effect leading to a global reduction of methylation of histone H3 lysine 27 (H3K27), an important mark for the modulation of gene expression. In addition, as a results of the global H3K27 reduction in methylation, DIPG epigenome show increased H3K27 acetylation. The high frequency and the specificity of the mutation (K27M) make it an attractive therapeutic target for the treatment of DIPGs.

Aims/Objectives:
Our preliminary data indicates that expression of the histone mutants H3R26M and H3K56M have a detrimental effect specifically on cells expressing the K27M mutation. We will address then the following questions: How the two mutants are affecting specifically cells expressing K27M mutation. Do these two mutants interfere with the hallmarks of DIPG cells? Is there specific regulatory pathways that are affected by these mutants? To answer these questions we have set up the following aims:

Aim 1: To characterise the effect of H3R26M and H3K56M mutants in DIPG cells.
Aim 2: To characterise the effect of H3R26M and H3K56M on the main hallmark of DIPG.
Aim 3: To characterise the effect of H3R26M and H3K56M on the transcriptional profile of DIPG.

Methods:
Aim 1: The results of our screening clearly indicate that expression of the histone mutants H3R26M and H3K56M results in a decrease presence of cells expressing these mutants specifically in combination with the expression of H3.3K27M. However the mechanism that explain this effect is unknown. We will therefore first characterise the effect of expression of the two mutants on the mouse cell model that we have used for the screening. We will determine the effect of expression of the two mutants on cell growth, apoptosis, and differentiation, as they are likely to be the main mechanisms explaining our screening results. Expression of H3R26M and H3K56M will be performed using the lentiviral constructs used for the screening. We will use the following methods to identify the mechanisms: (i) cell proliferation will be assessed by MTS assays and EdU incorporation. (ii) Caspase activation assay will be evaluated using Caspase-Glo 3/7 assay. (iii) Differentiation will be assessed by immunostaining cells with stem cell markers. Similar experiments will be performed using DIPG cells derived from patients.
Aim 2: The main hallmarks of DIPG cells are an overall reduction of H3K27 methylation, an overall increase of H3K27 acetylation and an overall reduction of DNA methylation which altogether explain the overall increased transcriptional level in this cancer. As histone marks cross-talk to each other, we hypothesise that mechanisms used by H3R26M and H3K56M to selectively kill DIPG cells involve changes of H3K27 methylation and/or acetylation. This might happen on specific subset of genes or on the overall genome. We will therefore assess the level of H3K27 methylation and acetylation level in our model upon expression of H3R26M and H3K56M. We will first characterise the level of histone H3K27 methylation and acetylation by western-blot at selected time frame to determine the range of interference of these mutants and the dynamic. In order to identify genomic region that are specifically affected by the expression of H3R26M and H3K56M, we will perform ChIP-seq experiments using H3K27me3 and H3K27ac antibodies. A clear crosstalk in between H3K27 methylation and DNA methylation has been previously showed. Indeed studies confirmed that DIPG cells have an overall reduction in global DNA methylation. We hypothesise that H3R26M and H3K56M expression might influence the level of DNA methylation globally or at specific loci. We will therefore assess whether expression of the mutants affect the level of DNA methylation by performing DNA methylation array using the Illumina DNA methylation profiling array. These data will be analysed in light of the ChIP-seq data to identify possible cross-talk in between perturbation of H3K27 methylation and acetylation induced by H3R26M and H3K56M.
Aim 3: Transcriptional regulation is a complex and dynamic process of which histone modifications are a key component. In order to identify transcriptional changes that explain the effect of expression of H3R26M and H3K56M specifically in cells expressing H3.3K27M mutation, we will perform a whole-transcriptome analysis. The conditions will be the same as the one used for the ChIPseq analysis enabling us to identify the key genes affected by the expression of H3R26M and H3K56M specifically in cell expressing the mutation K27M.

By gaining key insights into mechanisms used H3R26M and H3K56M to selectively kill cells expressing the K27M mutant, these experiments have the potential to highlight new targets for further drug development to treat DIPG.

References:
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