Understanding epigenetics: The potential rationale for BET - PowerPoint PPT Presentation

Understanding epigenetics: The potential rationale for BET inhibition in management of CVD August 25, 2018 Munich, Germany Jorge Plutzky, MD Director, Preventive Cardiology Cardiovascular Division Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts

Coordinated Programs In Cardiometabolic States? Physiology Pathology • Central obesity • Fit • Diabetes • Normotensive • Hypertension • Insulin sensitivity • Inflammation • Less inflammation • Alzheimer’s Disease • Longevity • Cancer - - + + Gene A Gene C Gene J Gene X Gene Z

Coordinated Programs In Cardiometabolic States? Physiology CV Disease • Central obesity • Fit • Diabetes • Normotensive • Hypertension • Lower Triglycerides • Hypertriglyceridemia • Higher HDL • Low HDL • Insulin sensitivity • Insulin resistance • Less inflammation • Inflammation • Endothelial function • Endothelial dysfunction • Longevity • Coagulation Modulating key proximal transcription factors • Myocardial injury - - + + Gene A Gene C Gene J Gene X Gene Z

Energy Balance PPAR RXR

Energy Balance Lipids: Glucose Obesity Triglycerides (Fatty Acids) PPAR RXR Diabetes Dyslipidemia Inflammation Atherosclerosis

PPARs in Transcriptional Regulation PPARs and Gene Regulation

LPS Cytokines AngII Matrix Lipids AGEs Hemodynamics A key integrator of inflammation NF- k B and atherosclerosis Inflammatory targets Collins, Cybulsky JCI 2001

PPARs in Transcriptional Regulation Challenges in PPARs and Gene Regulation - Store vast genetic material in nucleus - Access specific genetic stretches to enable transcription

10,000 fold 2 nm decrease in size 30 nm 300 nm 700 nm 1400 nm

Transcription Factors Heterochromatin Transcription impeded Euchromatin Transcription Factor Access Transcription Possible

CHROMOSOME FIBRE NUCLEOSOME EPIGENETIC CODE DNA histone mark chromatin fibre Plutzky, El Assam, Circ Res, 2016

CHROMOSOME FIBRE NUCLEOSOME EPIGENETIC CODE DNA acetylases, methylases phosphylases … histone mark deacetylases, demethylases dephosphylases … chromatin fibre Plutzky, El Assam, Circ Res, 2016

CHROMOSOME FIBRE NUCLEOSOME EPIGENETIC CODE DNA histone mark Acetylated lysines: BETs chromatin fibre Plutzky, El Assam, Circ Res, 2016

Coordinated Programs In Cardiometabolic States? Physiology CV Disease • Central obesity • Fit • Diabetes • Normotensive • Hypertension • Low TG/High HDL • High TG/ Low HDL • Insulin sensitivity • Inflammation • Less inflammation • Endothelial dysfunction • Endothelial function • Coagulation • Longevity • Myocardial injury Epigenetics: BETs Master Protein Transcription Factors mRNA Enhancers Promoters Gene J Gene X Gene Z Response elements

Bromodomains • Structural motif (110 aa) • Interacts with acetylated lysines on histones • ~50 bromodomain-containing protein family Bromodomain and Extra-Terminal Domain (BETs): Sub-family of proteins containing tandem bromodomains 1 74-185 345-457 632-710 801 BRD2 BD BD E 1 2 T 1 34-145 307-419 562-640 726 BRD3 BD BD E 1 2 T 58-159 349-461 600-678 1362 1 BRD4 BD BD E 1 2 T 1 27-138 268-380 500-578 947 BRDT* BD BD E 1 2 T

BET Proteins: Integral to Transcription Complex Assembly Along Gene Body BET BET Inhibitor Protein Selectivity BET Protein 4 (BRD4) BET Inhibitor Nature Reviews Mol Cell Biology 13, 543-547 16 Bradner, Nature 2010

Current Issues in Epigenetics/BETs: Super Enhancers

Cell States: Transformation Health Disease Differentiation Activation Adaptation Maladaptive Pathologic BETs Epigenetics Therapeutic? Programs that define cell state

Organ: functional, dynamic Cell States Interface: Circulation + Organs Transducer Endothelium Rest Inflammation Cell State Transitions

Multiple stimuli: Physical, chemical Cell States Rapid Reset Pathologic Essential: Host defenses Endothelium Rest Inflammation Cell State Transitions Transcriptional Programs Super-Enhancers BET Epigenetic Readers Chromatin Remodeling

Genetics DIABETES Cell States Exposures Hyperglycemia : Cig AGEs HTN Dyslipidemia Maternal- Fetal Inflammation Diet NF k B Epigenetic Readers: BETs Modified, Reddy, Natarajan, Epigenetics: Development & Disease , Subcellular Biochemistry 61, p435, 2013

Pressure Overload: BET Bromodomains Mediate Transcriptional Pause Release In Heart Failure Cell States Transverse Aortic Constriction Anand, Brown, Plutzky, Bradner, Haldar et al Cell 154: 569-582, 2013

BET bromodomain inhibition significantly decreases LDLr -/- atherosclerosis VEH JQ1 6 * % Lesion Area 4 - 12 Weeks of HC/HF Diet - JQ1 50mg/kg, IP daily 2 0 VEH JQ1 Oil Red O Mac3 CD4 VCAM1 VEH Brown, Plutzky et al JQ1 Mol Cell, 2013

Chromatin Immunoprecipitation coupled with deep sequencing: ChIP-Seq Crosslink Enrich for Massively protein to Harvest cells protein-bound parallel DNA DNA binding and fragment DNA fragments sequencing sites in living DNA with antibodies cells or tissue: Myocardium Endothelium

TNF- a induces BRD4 recruitment to specific EC enhancers, enabling execution of the inflammatory NF k B transcriptional program - TNF a p65 + TNF a Start site - TNF a BRD4 + TNF a VCAM1

TNF- a induces BRD4 recruitment to specific EC enhancers, enabling execution of the inflammatory NF k B transcriptional program TNF a gained SE 10.0 10kb p65 ChIP-Seq rpm/bp TNF a (-) 10.0 TNF a (+) 10.0 BRD4 ChIP-Seq rpm/bp TNF a (-) 10.0 TNF a (+) 3.0 H3K27ac ChIP-Seq rpm/bp TNF a (-) 3.0 TNF a (+) 3.5 H3K4me3 ChIP-Seq rpm/bp TNF a (-) Chemokine 3.5 TNF a (+) CCL2 15.0 RNA Pol II ChIP-Seq rpm/bp TNF a (-) 15.0 TNF a (+) chr17:29,571,987 chr17:29,622,217 CCL2

Dynamic, rapid remodeling of super-enhancers in Ecs directs a broad, canonical pro-atherosclerotic program Change in BRD4 Log2 TNF a (+) vs. TNF a (-) gn al ฀5 0 5 0 s i TNF a gained SEs B R D 4 SELE n VCAM1 i chan ge CCL2 100 by n=152 ed 200 ran k E C s n 300 i o n s regi n=124 S up er฀ en han cer TNF a lost SEs 400 SOX18 500

Apabetalone Reduces Atheroma in Aorta of ApoE-/- Mice Chow High Fat (42% kcal) Diet Chow: TD 2016 +/- WK apabetalone WK 33 WK WK 19 8 9 Animal HF Diet Chow Diet Necropsy Arrival Aortic sinus Whole aorta Placebo Apabetalone 20 Whole aorta  of -31%  of -40% (p<0.016) 15 (p<0.045) 11.081-607-040 Plaque/whole area (%) +/- SE 10 15.081-608-519 Aortic sinus 5 8.092-895-111 0 16.040-776-379 PlaceboApabetalone PlaceboApabetalone (150 mg/kg) (150 mg/kg) 28 Jahagirdar Atherosclerosis 2015

Tandem BET bromodomains allows for inhibitor selectivity: Distinct expression effects: Apabetalone (selective) vs. JQ-1 (pan) BET inhibition (+)- JQ1 (+)- JQ1 (+)- JQ1 PNAS, 2013 110 (49):19754-9. 29

 BRD4: Log D2 vs D0 BRD4 Regulates the Adipogenic Program Pre-Adipo DAY 0 PPAR g CEBP a PTN PPAR g Adipo DAY 2 PPAR g PTN PTN PNAS, 2018 Enhancers Ranked By BRD4 Signal

BET Inhibition BET Action Pathologic Transcriptional Programs

Inflammation: Metabolism: Cardiovascular: M F T cells Adipocytes b Cells Renal: Pltlets ECs VSMCs Cardiomyocytes Hepatocytes Tubular BET Inhibition BET Action cells Apabetalone Injury Inflammation Indirect Indirect Direct Atherosclerosis Pathologic Transcriptional Programs