Tumor organoids from individual patients with metastatic colorectal cancer can help predict response to irinotecan with or without 5-fluorouracil.

At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development. Methylation of DNA and histones exert profound and inherited effects on gene expression. These occur without changes to the underlying DNA sequence and are considered epigenetic effects. Disrupting epigenetic states can cause developmental abnormalities and cancer. Very little is known about how locations in the mammalian genome are chosen to receive these chemical modifications, or how their placement is regulated. We have identified a DNA sequence that acts as a methylation programmer at the Rasgrf1 locus in mice. It is required for methylation of nearby DNA sequences and can also influence the levels of local histone methylation. The methylation programmer has different effects on paternally and maternally derived chromosomes, directing DNA methylation on the paternal allele and histone H3 lysine 27 trimethylation on the maternal allele. These two methylation marks are not only mutually exclusive; they are also mutually antagonizing, whereby one blocks the placement of the other. Manipulations that cause aberrant changes in the levels of one of these marks had the opposite effect on the other mark. These observations identify novel mechanisms that specify epigenetic states in vivo and provide a framework for understanding how pathological epigenetic changes can arise, including those emerging at tumor suppressors during carcinogenesis.

Bladder cancers are a leading cause of death from malignancy. Molecular markers might predict disease progression and behaviour more accurately than the available prognostic factors. Here we use whole-genome sequencing to identify somatic mutations and chromosomal changes in 14 bladder cancers of different grades and stages. As well as detecting the known bladder cancer driver mutations, we report the identification of recurrent protein-inactivating mutations in CDKN1A and FAT1. The former are not mutually exclusive with TP53 mutations or MDM2 amplification, showing that CDKN1A dysfunction is not simply an alternative mechanism for p53 pathway inactivation. We find strong positive associations between higher tumour stage/grade and greater clonal diversity, the number of somatic mutations and the burden of copy number changes. In principle, the identification of sub-clones with greater diversity and/or mutation burden within early-stage or low-grade tumours could identify lesions with a high risk of invasive progression.

Resistance to targeted cancer drugs is thought to result from selective pressure exerted by a high drug dose. Partial inhibition of multiple components in the same oncogenic signalling pathway may add up to complete pathway inhibition, while decreasing the selective pressure on each component to acquire a resistance mutation. We report here testing of this Multiple Low Dose (MLD) therapy model in EGFR mutant NSCLC. We show that as little as 20% of the individual effective drug doses is sufficient to completely block MAPK signalling and proliferation when used in 3D (RAF + MEK + ERK) or 4D (EGFR + RAF + MEK + ERK) inhibitor combinations. Importantly, EGFR mutant NSCLC cells treated with MLD therapy do not develop resistance. Using several animal models, we find durable responses to MLD therapy without associated toxicity. Our data support the notion that MLD therapy could deliver clinical benefit, even for those having acquired resistance to third generation EGFR inhibitor therapy.

Significance: Cytotoxic CD8 ⁺ T cells control infectious diseases as well as cancer. Memory T cells are a subset of CD8 ⁺ T cells that have previously encountered and rapidly responded to a specific antigen. Here we identified the epigenetic writer DOT1L as a central regulator of T cell physiology. In the absence of DOT1L, which methylates histone H3K79 at active genes, cytotoxic T cells prematurely acquired memory features without having encountered an antigen. However, loss of DOT1L in the T cell lineage led to a compromised immune response. Our findings highlight the importance of epigenetic signals in regulating T cell differentiation. Understanding these epigenetic signals is important for developing strategies to modulate T cell immunity.