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 • Fit • Central obesity • Normotensive • Diabetes • Insulin sensitivity • Hypertension • Less inflammation • Inflammation • Longevity • Alzheimer’s Disease • Cancer
+ - - + Gene A Gene C Gene J Gene X Gene Z Coordinated Programs In Cardiometabolic States? Physiology CV Disease • Fit • Central obesity • Normotensive • Diabetes • Lower Triglycerides • Hypertension • Higher HDL • Hypertriglyceridemia • Insulin sensitivity • Low HDL • Less inflammation • Insulin resistance • Endothelial function • Inflammation • Longevity • Endothelial dysfunction Modulating key proximal transcription factors • Coagulation • Myocardial injury
+ - - + Gene A Gene C Gene J Gene X Gene Z Energy Balance
PPAR RXR Energy Balance
Lipids: Glucose Triglycerides (Fatty Acids) Obesity
PPAR RXR Dyslipidemia Diabetes
Inflammation
Atherosclerosis PPARs in Transcriptional Regulation PPARs and Gene Regulation LPS Cytokines Matrix Lipids AngII AGEs Hemodynamics
A key integrator of inflammation NF-kB and atherosclerosis
Inflammatory targets
Collins, Cybulsky JCI 2001 ChallengesPPARs inin Transcriptional Regulation
- Store PPARsvast genetic and material Gene in nucleus Regulation - 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 • Fit • Central obesity • Normotensive • Diabetes • Low TG/High HDL • Hypertension • Insulin sensitivity • High TG/ Low HDL • Less inflammation • Inflammation • Endothelial function • Endothelial dysfunction • Longevity • Coagulation • 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 1 58-159 349-461 600-678 1362
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 Protein Inhibitor 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 Epigenetics Pathologic BETs Therapeutic? Programs that define cell state Organ: functional, dynamic Cell States Interface: Circulation + Organs Transducer
Endothelium Rest Cell State Transitions Inflammation 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 DIABETES Genetics Cell States Exposures Hyperglycemia : Cig AGEs HTN Dyslipidemia Maternal- Fetal Inflammation Diet NFkB
Epigenetic Readers: BETs
Modified, Reddy, Natarajan, Epigenetics: Development & Disease, Subcellular Biochemistry 61, p435, 2013 Pressure Overload: BET Bromodomains Mediate Cell States Transcriptional Pause Release In Heart Failure
Transverse Aortic Constriction
Anand, Brown, Plutzky, Bradner, Haldar et al Cell 154: 569-582, 2013 BET bromodomain inhibition significantly decreases LDLr -/- atherosclerosis VEH JQ1 VEH JQ1 6 6
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Q J Mol Cell, 2013 Chromatin Immunoprecipitation coupled with deep sequencing: ChIP-Seq
Crosslink Enrich for protein to Massively Harvest cells protein-bound DNA binding parallel DNA and fragment DNA fragments sites in living sequencing DNA with antibodies cells or tissue:
Myocardium Endothelium TNF-a induces BRD4 recruitment to specific EC enhancers, enabling execution of the inflammatory NFkB transcriptional program
- TNFa p65 + TNFa
Start site - TNFa BRD4 + TNFa VCAM1 TNF-a induces BRD4 recruitment to specific EC enhancers, enabling execution of the inflammatory NFkB transcriptional program TNFa gained SE
10.0 p65 ChIP-Seq 10kb
p b
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10.0 BRD4 ChIP-Seq
p b
/ TNFa (-)
m
p r 10.0 TNFa (+)
3.0 H3K27ac ChIP-Seq
p b
/ TNFa (-)
m
p r 3.0 TNFa (+)
3.5 H3K4me3 ChIP-Seq
p b
/ TNFa (-)
m
p r Chemokine 3.5 TNFa (+)
15.0 RNA Pol II ChIP-Seq
CCL2 p b
/ TNFa (-)
m
p r 15.0 TNFa (+)
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
Log TNFa (+) vs. TNFa (-)
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u 500 S Apabetalone Reduces Atheroma in Aorta of ApoE-/- Mice
Chow High Fat (42% kcal) Diet Chow: TD 2016 +/- WK WK WK apabetalone WK 33 8 9 19
Animal HF Diet Chow Diet Necropsy Arrival
Whole aorta Aortic sinus Placebo Apabetalone 20
of -31% of -40% (p<0.016) 11.081-607-040 15 (p<0.045)
Wholeaorta 15.081-608-519
SE 10 -
(%) +/ (%) 5 Plaque/whole area area Plaque/whole
8.092-895-111 16.040-776-379 Aortic Aortic sinus 0 Placebo PlaceboApabetalone Apabetalone (150 mg/kg) (150 mg/kg) Jahagirdar Atherosclerosis 2015 28 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 Regulates the Adipogenic Program BRD4: Log D2 vs D0
Pre-Adipo DAY 0 PPARg
CEBPa PTN PPARg
Adipo DAY 2
PPARg PTN
PTN PNAS, 2018 Enhancers Ranked By BRD4 Signal BETBET Inhibition Action
Pathologic Transcriptional Programs Cardiovascular: Inflammation: Metabolism: MF T cells Cardiomyocytes ECs VSMCs Pltlets Adipocytes b Cells Renal: Hepatocytes Tubular BETBET Inhibition Action cells
Apabetalone
Inflammation Injury
Indirect Direct Indirect Atherosclerosis Pathologic Transcriptional Programs