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, +)
Osteoblasts-like VSMC
Phenotype conversion
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
Gilham et al. Atherosclerosis. 2019;280:75-84
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
Gilham et al. Atherosclerosis. 2019;280:75-84
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
Phenotype conversion
Beneficial effects of BET inhibitors on vascular calcifications in CKD
Apabetalone
Reprogramming pro-calcific pathways
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.
Beneficial effects of BET inhibitors on vascular calcifications in CKD
Gilham et al. Atherosclerosis. 2019;280:75-84
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
Other factors involved in CKD
(oxidation, metabolic disorders,
cytokines, inflammation, LDL ox
or uremic toxins)
EPIGENETIC CHANGES
Osteoblasts-like VSMC
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
Ruiz-Ortega J Am Soc Nephrol. 2017;28(2):504-519
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
Beneficial effects of BET inhibitors on vascular calcifications in CKD
Gilham et al. Atherosclerosis. 2019;280:75-84
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
Ruiz-Ortega J Am Soc Nephrol. 2017;28(2):504-519
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
Phenotype conversion
Beneficial effects of BET inhibitors on vascular calcifications in CKD
serum levels of inorganic Phosphate/calcium
CKD
Apabetalone
Inhibits pro-calcification genes (ALP,RUNX,…)
and transcription factors (NF-KB, Smad, STAT others)
Reprogramming pro-calcific pathways
Other factors involved in CKD
(oxidation, metabolic disorders, cytokines,
inflammation or uremic toxins)
Calcium-rich
Hydroxyapatite
formation
Inhibition of Alkaline Phosphatase
InhibitorsOsteopontin
Pyrophosphatase
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