epigenetic mechanisms targeting alp: a pathway for prevention?€¦ · epigenetic mechanisms...

Epigenetic mechanisms

targeting ALP:

A pathway for prevention?

Marta Ruiz-Ortega, PhD

Madrid, Spain

June 15, 2019 - Budapest, Hungary

“Vascular calcification in kidney disease:

Epigenetics as a novel approach?”

Epigenetic mechanisms targeting alkaline

phosphatase (ALP):

A pathway for prevention?

Marta Ruiz-Ortega, PhD

Professor. Medicine Department.

IIS-Fundación Jiménez Díaz

Universidad Autónoma de Madrid. Spain.

ERA-EDTA Congress, Budapest 2019

EPIGENETICS IN RENAL DISEASES: A mechanism of renal damage

CKD and VC

EPIGENETIC CHANGESDNA Methylation

Histone modificationmiRnas changes

GENETIC

ENVIRONMENT

- Age

- Obesity and nutrients

- Toxic exposure

(contamination)

- Exercise

Metabolic disorders

Elevated cytokines

inflammation, oxidation

or uremic toxins

DNA methylation : CpGChromosome

Chromatin fiber

Nucleosome

Chemical reactions in the histone tails

Epigenetics: changes in cromatin structure without changes in DNA sequence

Histone Modifications

Acetylation (Lys)Methylation (Lys, Arg)

Phosphorylation (Thr, Ser)

Ubiquitination (lys)

Deamination (Arg---citrulline)Sumoylation

ADP-ribosylation

Chrotonylation

Ac-CoA

CoA

KATs

K

NAD+/H2O

KDACs

Histones

Transcription factors

Acetylation: A mechanism involved in gene transcription regulation

Acetylated Histones: Changes in Chromatin Structure

“OPEN”

Acetylated Transcription factors

“ACTIVE”

Reversible Reaction: Potential target for treatment

K

BETs

BETs “Bromodomain andExtra-Terminal domain family”

K

Proteins(histones or transcription factors)

BETs

BET proteins bind to acetylated lysine

on histones and other nuclear proteins to regulate the transcriptional program

Histones

acetylated

Transcriptions

Factors

TF

PP

P

P

P

P

P

P

P

AcBRD4

CycT1

CDK9

RN

AP

IIAc

TF

Ac

BRD4

AcAcAcAc

Ac

Ac

p300/CBP

HDACs

HDACs

Promoter

GENE TRANSCRIPTION

AcAcAcAc

Ac

BRD4

BET proteins as epigenetic therapeutic targets

Ac

These small molecules compete for the bromodomain binding pocket and displace BET

proteins from binding to acetylated lysine residues on histone tails and transcription factors

BET inhibitors; potential therapeutic targets

Filippakopoulos P et al. Nature 2010; 468:1062

iBET

BC

ZA

BET protein

(BRD2,3 and 4)

Bromodomains

Hydrophobic pocket

Apabetalone (RVX-

208)

BD2 Specific

BET inhibitors: beneficial effects in many preclinical studies

P

Cell cycle control

Proliferation

Differentiation

Inflammation

BDs

iBET

GENE TRANSCRIPTION

BET inhibitors; potential therapeutic targets

BET inhibitors: beneficial effects in many preclinical studies

TF

Inhibition of

Ac

Ac Ac

immune pathologies

BET inhibitors: beneficial effects in

proliferative disorders

fibrotic diseases

chronic inflammatory diseases

Vascular calcification?

CHRONIC KIDNEY DISEASE / MINERAL BONE DISORDER

Vascular calcifications in CKD

Other factors involved in CKD

(oxidation, metabolic disorders, cytokines,

inflammation or uremic toxins)

EPIGENETIC CHANGESVSMC

InhibitorsFetiun A

MGP, Osteopontin

Pyrophosphatase

InducersHyperphosphatemia

Hypercalcemia

Vascular

Calcifications

and Stiffness

Osteoblasts-like VSMC

Phenotype conversion

Could BET inhibition be a potential

therapeutic target for vascular

calcification?

Several ongoing clinical trials have shown that

Apabetalone reduced cardiac events on CVD patients and

had favorable effects on kidney function in CKD patients.

RVX-208, the most specific BD2 inhibitor, has been shown to reduce serum

alkaline phosphatase (ALP) in CKD patients with a history of cardiovascular

events (Kulikowski et al. Kidney and Blood Pressure Research, 2018:43(2);449–457),

suggesting that ALP modulation can be a therapeutic target to inhibit vascular

calcification progression.

Induction of Runx2, osteopontin, ALP

Downregulation of SM22

EPIGENETICS DRUGS?

Osteogenic medium

(b-glycerophosphate, +)



Epigenetic mechanisms in vascular calcifications in CKD

Calcium-rich

Hydroxyapatite

formation

Reprogramming pro-calcific pathways

HyperphosphatemiaHypercalcemia

Gilham et al. Atherosclerosis. 2019;280:75-84

VSMC

In vitro studies in human VSMCs

cultured for 12 days with iBETs


BET inhibitors oposses osteogenic calcification of VSMCs

iBET diminished Calcium Deposition

in VSMCs

Alizarin red staining

BET inhibitors downregulate osteogenic genes in VSMCs

Gilham et al.

Atherosclerosis.

2019;280:75-84

Apabetalone inhibits Alkaline phosphatase

Apabetalone inhibits TNAP protein

in VSMCs

cultured in osteogenic conditions


Apabetalone

downregulates ALPL gene

in VSMCs

cultured in osteogenic conditions

Proposed mechanism of Alkaline phosphatase in tissue mineralization

2) post-translational

modifications

3) Released

in membrane-bound

BALP-rich

matrix vesicles

1) Transcription

activation

4) BALP inactivates

the mineralization

inhibitors inorganic

pyrophosphate (PPi)

and osteopontin

5) Generation of a

pro-calcific

extracellular milie

6) ALP binds directly

to collagen type I,

which forms a

scaffold for the

propagation of

matrix mineralization

Scheme of Haarhaus et al Nature Reviews Nephrol JULY 2017 | VOLUME 13

Extracellular space

VSMC Osteoblasts-like VSMC


Beneficial effects of BET inhibitors on vascular calcifications in CKD

Apabetalone


Inhibition of Alkaline Phosphatase

InhibitorsOsteopontin

Pyrophosphatase

Inhibits pro-calcification genes (ALP,RUNX,…)

38 genes were uniquely associated with BRD4-rich enhancers in

osteogenic conditions; 11 were previously associated with calcification.



CHIP-SEQ EXPERIMENTS GeneinProximitytoBRD4-richEnhancer

Proteinname ProteinFunction

C6 ComplementC6 Celllysis

IL1R1 Interleukin-1ReceptorTypeI Cytokinereceptor

PKDCC ProteinKinaseDomainContaining,

Cytoplasmic.

Secretedtyrosine-protein

kinase

TIMP4 TIMPMetallopeptidaseInhibitor4 Inhibitorofthematrix

metalloproteinases

ZBTB16 ZincFingerAndBTBDomainContaining16

Zincfingertranscriptionfactor

Apabetalone reduced gene expression

VSMC

Vascular

calcifications


(oxidation, metabolic disorders,

cytokines, inflammation, LDL ox

or uremic toxins)

EPIGENETIC CHANGES


Apabetalone increased high density lipoproteins (HLD) in patients with

cardiovascular diseases (Nicholls et al. Am J Cardiovasc, 2018:18:109-115). HLD

inhibits IL-6 mediated calcification in vitro (Parhami et al.,Cir Res 2002:570-576)

DNA genomic array

TNFα

3 hours

+ JQ1

0

10

20

30

RN

A le

vels

(n-f

old

) *

+ TNFα

#

*

P-TEF-independent: CSF2

NF-KB regulated

P-TEF-dependent: CCL20

0

20

40

60

80

*

#

*

+ TNFα

RN

A le

vels

(n-f

old

)

BET inhibitors exert anti-inflammatory actions: gene downregulation

Tubular epithelial cells

Ruiz-Ortega J Am Soc Nephrol. 2017;28(2):504-519

IL-6

CCL-2

CCL-5

Control

TNFα

SiRNA

Control

SiRNA

BDR4

0

5

10

15

20

25

30

35

40

*

*

*

#

#

#

*

*

*

mR

NA

levels

(n-f

old

)

BDR4 gene silencing in cytokine-stimulated cells

ChIP assay in TNF-stimulated renal cells

BDR4 binds to proinflammatory gene promoters


PBDs

iBET

Ac Ac

SOMAscan® Analysis of Plasma ProteomeBaseline analysis: IPA Canonical Pathway: Top 5 Pathways Upregulated

CKD Baseline p-value

CKD

Apabetalone p-value

Dendritic Cell Maturation 3.46 1.1 E-06 -4.12 9.6 E-13

IL-6 Signalling 3.21 9.1 E-10 -3.46 2.9 R-08

Th1 Pathway 3.16 2.2 E-09 -3.32 5.4 E-07

NF-kB Signalling 3.05 5.6 E-07 -3.46 1.3 E-06

Acute Phase Response Signalling 2.89 7.2 E-11 -3.46 7.5 E-07

A Phase I, Open-Label, Parallel Group Study to Evaluate the Safety and Pharmacokinetics of a

Single Oral Dose of RVX000222 in Subjects with Severe Renal Impairment



Bioinformatic analysis of CHIP-SEQ EXPERIMENTS: BrD4 cooperate with 7 transcription factors

TF

iBET

Ac

GENE TRANSCRIPTION

ActiveNF-B

p50

p65

IB

P

Proteasomedegradation

ACK310

p50

p65

IB

P

Nucleus

ACK310

p50

p65

PSer536

AC

p65

BRD4 BDs

BET inhibitor

Proteasomedegradation

ACK310

p65

RN

AP

II

p6

5

TNF-αIL-6IFN-γIL-17ROR-γT

BET inhibitors and Nuclear factor kappa B pathway

NF-KB inhibition


Inhibition of

Zou Z, el at. Oncogene 33(18): 2395-2404, 2014

Wu et al., J Biol Chem 288(50): 36094-36105, 2013

NucleiP

Smad2/3Smad4

Smad7Cellular Membrane

TRII

TRI

TGF

TRII

TRI

TGF

Smad4

Smad2/3P

Smad2/3

Sm

ad

4

Sm

ad

2/3

P300

Collagen

CTGF

SMAD 7

TGF-β

Differentation

iBETs inhibit Smad pathway activation in fibrotic diseases

iBET

GENE TRANSCRIPTION

Inhibition of

Ac AcAc

Ac

Ding et al. PNAS 2015: 112(51), 15713–18.

iBET inhibits STAT- 3 activation in immune-mediated pathologies

RORγT

STAT-3

TH17

Cells

CD4+

Cells

Inhibition of IL17A gene

transcriptionDifferentiation

iBET

iBET

Studies in immune-mediated glomerulonephritis in mice

Control NTS NTS+JQ1

Renal IL-17A

Ruiz-Ortega and cols J Am Soc Nephrol. 2017;28(2):504-519

Inhibition

of STAT-3

Cheung et al. J Am Soc Nephrol., 2011; 22(5), 802–809

Mele et al. J Exp Med, 2107:(11): 2181–90.

Histones acetylated

TF

AcAcAcAc

Ac

BET proteins as epigenetic therapeutic targetsBeneficial effects of BET inhibitors on VC: CHIP-SEQ EXPERIMENTS

Pro calcific genes

ALP

RUNX

Inhibition of

Enlarged enhacers: Open chromatin

Activated Transcriptions Factors

OSTEOGENIC

CONDITIONS iBET

AcAcAcAc

Ac

TF

iBET

iBET

Ac

OSTEOGENIC

CONDITIONS

VSMC Osteoblasts-like VSMC



serum levels of inorganic Phosphate/calcium

CKD

Apabetalone

Inhibits pro-calcification genes (ALP,RUNX,…)

and transcription factors (NF-KB, Smad, STAT others)



(oxidation, metabolic disorders, cytokines,

inflammation or uremic toxins)

Calcium-rich

Hydroxyapatite

formation

Inhibition of Alkaline Phosphatase

InhibitorsOsteopontin

Pyrophosphatase

epigenetic mechanisms targeting alp: a pathway for prevention?€¦ · epigenetic mechanisms...

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