Nessa Carey

Epigenetics – The Toxicological Sleeper Agent.com

nessa.carey@pfizer

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 0 The Mission

• What is ?

• Epigenetic drugs

• Transgenerational inheritance

• The implications of epigenetics for toxicology

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. Epigenetics – The Phenomenon

• Epi – at, on, besides, in addition to ......

• Any situation where two genetically identical entities are phenotypically discordant

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 2 Epigenetics – The Molecules

• Chemical modifications to chromatin  DNA  Histone • These don’t affect the gene sequence • They do affect how genes are expressed

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 3 Molecular Epigenetics

Dunn & Kingston, 2007

One way or another, there is an effect on , which means one way or another, there is an effect on the binding or activity of RNA polymerase II, or on post- transcriptional processes such as splicing. Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 4 DNA Methylation

• DNA methylation occurs at position C5 within CpG dinucleotides. • 5meC constitutes <1% of nucleotides. • CpGs are not randomly distributed in the genome. • CpGs are methylated in repeats and 3’ regions of genes, but often not in CpG islands. • There are estimated to be about 26,000-45,000 CpG islands, many of which tend to be located in the regions of housekeeping genes and some tissue specific genes. 60% of human genes have CpG islands.

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 5 Histone Modifications

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 6 Epigenetic Modifications – Compare And Contrast....

DNA Histones • Methylation (and • Multiple forms of derivatives) modification • Only on cytosines • Multiple amino acids • Represses gene • Variable effects on activity gene activity • Extremely stable • Transient to stable • Cellular transmission • Cellular transmission well understood poorly understood

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 7 Epigenetic Drugs – Why?

• Most human disease is not associated with changes in gene sequence • Most human disease is associated with abnormal gene regulation • Epigenetic modifications are one of the key mechanisms for maintaining patterns of gene (mis)expression • Realignment of abnormal epigenetic modifications may re-establish and stabilise appropriate gene expression patterns

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 8 Epigenetic Drugs- What?

• DNA • Histone deactylase methyltransferase inhibitors inhibitors O NH OH HN

NH2 O

N N

NH O

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 9 Toxicity Of Epigenetic Drugs

• Nausea • Anaemia • Thrombocytopaenia • Neutropaenia • Leukopaenia

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 10 So, What’s The Problem?

• DNA is not the only carrier of information from one generation to the next • How do we know this?

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 11 Not All DNA Is Created Equal

• Impossible to create mammals by fusing two sperm nuclei or two egg nuclei in an enucleate egg • Seems odd, given that two sperm or two eggs would have exactly the same DNA as an egg and a sperm • Mammalian DNA is marked with its parent-of-origin • Only occurs at a relatively small number of specific genes • Process known as imprinting • Probably evolved as a mechanism to balance out the competing drivers from maternal and paternal developmental agendas • The information on parent-of-origin is not transmitted in the DNA sequence • It is transmitted by chromatin modifications, usually DNA methylation, that regulate expression of genes from specific chromosomal loci

• Shows that epigenetic information can be transmitted from one generation to the next

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 12 Fat Yellow Mice

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 13 Passing It On

• of parent influences phenotype of offspring

• Transmission of phenotype isn’t perfect

• Can the phenotype of offspring be affected by environmental exposure?

Morgan et al., Nature 1999

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 14 Alcohol And Epigenetics

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Exposure of a dark agouti mother to alcohol led to a greater proportion of dark offspring, with increased DNA methylation at the agouti-associated retrotransposon.

Environmental agents can alter the transmission of epigenetically-mediated traits.

Kaminen-Ahola et al., PLoS Genet. 2010 Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 15 And Not Just Alcohol

In utero exposure to genistein, a soy-derived phyto-oestrogen, alters transmission of coat colour in agouti mice

But – confounding effects of maternal transmission......

Dolinoy et al., Environ Health Perspect 2006

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 16 Fathers And Food And Epigenetics

These effects, just like those found in the alcohol-treated agouti mice, occur at too high a rate to be due to mutation.

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 17 But Is This Really Epigenetic?

• A true transgenerational effect would manifest in offspring from sperm never exposed to dietary modification • It remains to be determined whether paternal F2 of low- -diet/high fat diet males also exhibit and transmit metabolic

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 18 Vinclozolin

• Effects seen at much too high Abdominal injection a rate to be caused by of vinclozolin into pregnant rat mutation

• Effects transmitted beyond the Every day Days 8-15 F1 males sterile F1 males fertile but with original animals exposed to the Decreased sperm count stimulus (directly or indirectly) Decreased sperm motility Increased testicular apoptosis • Later work reported alterations in DNA methylation in the testes. Effect continues until at least F4

Anway et al., Science 2005

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 19 Hmmm

• Most of the compounds known to have epigenetic effects are not direct inhibitors of epigenetic proteins

• In some cases they are probably working by altering the availability of e.g. methyl donors

• In other cases we have no real idea how they cause epigenetic alterations

• There are hints from human systems that we may be susceptible to similar effects . Foetal alcohol syndrome . Dutch hunger winter . Overkalix feast or famine

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 20 The Next Frontiers In Epigenetic Drug Discovery

• Improved epigenetic drugs for oncology . BRD4 . DOT1L . EZH2 . Isoform-specific HDACi • Epigenetic drugs for non-oncology indications . Chronic . Mendelian

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 21 What We Need To Think About

Effects on Transgenerational toxicity drug metabolism pathways Predicting e.g. altered longevity of expression adverse effect of Cyp450 genes Assay types

Mechanism- based toxicity Genome regions

Compound- Population based versus toxicity individual risk Reproductive toxicity

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 22 With Thanks To

Pfizer Academia Mark Bunnage Harvard Anne Phelan Caroline Benn MGH Rob Rollins University of Cambridge University of Edinburgh University of CellCentric Pennsylvania USC Babraham Institute

nessa.carey@pfizer

Confidential and Internal to Pfizer - subject to works council and/or union consultations and other legal requirements. 23