Epigenetic Therapeutics: Mechanisms, Modalities and Outcomes for Human Health

1. Epigenetic Therapeutics: Mechanisms, Modalities and Outcomes for Human Health Marc Muskavitch Sr. Director, Epigenetics September 10, 2014

2. Epigenetic Therapeutics Epigenetics(“over” or “upon” - genetics) is the study of (heritable) changes in gene function that depend on mechanisms other than changes in DNA sequence Consensus 2013 “an epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” Berger et al., 2008 “the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms” Holliday, 1990 ‘‘the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being”Waddington, 1942

3. Epigenetic Therapeutics Chromatin Biology Histone Writers/Erasers Histone Readers/Remodelers DNA Methylation RNA Biology Long Noncoding RNAs Micro RNAs RNA Editing Arrowsmith et al Nat Rev Drug Disc 2012 Wahlestedt Nat Rev Drug Disc 2013

4. Epigenetic Therapeutics • Epigenetics and disease • Mutations in genes encoding epigenetic modifiers • (e.g., HDAC, HAT, DNMT, MECP) and ncRNAs • are associated with human disease • Antagonists of histone deacetylases are moving into the clinic • Anti-sense RNA-based therapies are moving into the clinic • Ensemble therapeutics • Epigenetic therapeutics can be considered “ensemble therapeutics” because drugs directed against an epigenetic function will affect the expression/function of multiple targets • Chromatin writers/erasers/readers interact with multiple sites • Antisense RNAs target multiple mRNAs and/or ncRNAs

5. Epigenetic Therapeutics • Histones and DNA • are modified by a variety of enzymes • HAT: histone • acetyl transferase • HDAC: histone deacetylase • HMT (KMT): histone methyltransferase • HDM (KDM): histone • demethylase • DNMT: DNA methyltransferase Peterson and LanielCurrBiol 2004 Yandell The Scientist 2014

6. Epigenetic Therapeutics Open (active/transcribed) and closed (inactive/ nontranscribed) configurations of chromatin Klug et al 2011

7. Epigenetic Therapeutics miRNA regulation and RNA interference lead to targeted RNA degradation microRNA (miRNA) precursors are transcribed in the nucleus, then processed into mature miRNAs by Dicer miRNAs bind to the RNA-induced silencing complex (RISC) and catalyze degradation of messenger RNAs and noncoding RNAs Klug et al 2011

8. Epigenetic Therapeutics Dicer cleaves double-strand RNA (including double-strand RNA viruses) into 21 nucleotide fragments These 21 ntsilencing RNAs (siRNAs) bind to the multicomponent RISC RISC targets for degradation messenger RNAs and noncoding RNAs, complementary to the bound siRNA Anti-viral defense system Klug et al 2011

9. Diseases associated with mutations in epigenetic modifiers GosActaNeurobiolExp 2013

10. Epigenetic Therapeutics Chromatin Biology Histone Writers/Erasers Histone Readers/Remodelers DNA Methylation Arrowsmith et al Nat Rev Drug Disc 2012

11. HDAC and sirtuin inhibitors in clinical development Arrowsmith et al Nat Rev Drug Disc 2012

12. Long noncoding RNAs implicated in human disease Wahlestedt Nat Rev Drug Disc 2013

13. Epigenetic Therapeutics RNA Biology Long Noncoding RNAs Micro RNAs RNA Editing Wahlestedt Nat Rev Drug Disc 2013

14. Anti-sense RNA therapeutic pipeline at Isis Pharmaceuticals http://www.isispharm.com/Pipeline/index.htm

15. Epigenetic Therapeutics

16. Epigenetic Therapeutics Modalities for epigenetic therapies Small molecules: antagonists and agonists of epigenetic modifiers (oral, subcutaneous, intravenous) Biologics (antibodies, factors): inhibitors or substitutes for epigenetic modifiers (oral, subcutaneous, intravenous) Antisense oligonucleotides (ASOs): reduction of RNA levels by RNA interference (subcutaneous, intravenous, intrathecal) Cell and gene therapy: genome editing (ZFN, TALEN, CRISPR), antisense, noncoding or coding RNA delivery (viral vectors)

17. Epigenetic Therapeutics Drug development pipeline (in part)

18. Epigenetic Therapeutics • HDACi approved by FDA for clinical use • Vorinostat • Cutaneous T cell lymphoma (Merck) • Romedepsin • Cutaneous T cell lymphoma (Gloucester Pharmaceuticals) • Belinostat • Peripheral T celllymphoma (Spectrum Pharmaceuticals) • HDACi side effects/liabilities • Fatigue • Nausea/diarrhea • Thrombocytopenia • Cardiotoxicity (prolonged QT interval, hERG effects)

19. Epigenetic Therapeutics • HDAC6-selective inhibitor in clinical trials • Ricolinostat • (AcetylonPharmaceuticals) • Indications • Multiple myeloma • With bortezomib or lenalidomide, and dexamethasone • Relapsed-and-refractory multiple myeloma • With pomalidomide and dexamethasone • Relapsed/refractory lymphoid malignancies

20. Epigenetic Therapeutics • DNMTi approved by FDA for clinical use • Azacitidine(Pharmion Corporation) • Decitabine(InnoPharma) • Myelodysplastic syndrome • DNMTi side effects/liabilities • Neutropenia • Thrombocytopenia • Anemia • Pneumonia • Fatigue

21. Epigenetic Therapeutics • ASOs approved by FDA for clinical use • Mipomersen (ApoB) (Isis Pharmaceuticals/Genzyme) • Familial hypercholesterolemia • Fomivirsen (Isis Pharmaceuticals/Ciba Vision Corporation) • Cytomegalovirus retinitis • ASO side effects/liabilities • Prolonged activated partial thromboplastin time (aPTT) • Activation of alternative complement pathway • Pro-inflammatory reactions • Elevation of hepatic enzymes (ALT, AST)

22. Epigenetic Therapeutics Common drugs with known/possible epigenetic impacts Csoka and Szyf Med Hypoth 2009

23. Epigenetic Therapeutics • Epigenetic drugs and pregnancy/breastfeeding • Should epigenetic therapeutics be avoided during conception, pregnancy and breastfeeding? • For vorinostat, romedepsin, belinostat: • US FDA Pregnancy Category D: There is positive evidence of human fetal risk based on adverse reaction data from investigational or marketing experience or studies in humans, but potential benefits may warrant use of the drug in pregnant women despite potential risks. • A decision should be made to discontinue breastfeeding or discontinue the drug, taking into account the importance of the drug to the mother. Csoka and Szyf Med Hypoth 2009

24. Epigenetic Therapeutics Chromatin Biology Histone Writers/Erasers Histone Readers/Remodelers DNA Methylation RNA Biology Long Noncoding RNAs Micro RNAs RNA Editing Arrowsmith et al Nat Rev Drug Disc 2012 Wahlestedt Nat Rev Drug Disc 2013

25. Epigenetic Therapeutics • Epigenetics and disease • Mutations in genes encoding epigenetic modifiers • (e.g., HDAC, HAT, DNMT, MECP) and ncRNAs • are associated with human disease • Antagonists of histone deacetylases are moving into the clinic • Anti-sense RNA-based therapies are moving into the clinic • Ensemble therapeutics • Epigenetic therapeutics can be considered “ensemble therapeutics” because drugs directed against an epigenetic function will affect the expression/function of multiple targets • Chromatin writers/erasers/readers interact with multiple sites • Antisense RNAs target multiple mRNAs and/or ncRNAs

26. Epigenetic Therapeutics: Mechanisms, Modalities and Outcomes for Human Health Marc Muskavitch Sr. Director, Epigenetics September 10, 2014