Cardiotoxicity of Dasatinib and its Regulation

Through miR19b

Bhagyashree M. Shelar
Integrated Master in Biotechnology
MIT1323

Project Guide: Dr. Dhandapany Perundurai

Institute for Stem Cell Biology and Regenerative Medicine, Bangalore

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INTRODUCTION

Anti-Cancer
drugs Cardiotoxicity

Raf1

2
 HYPOTHESIS

Raf1

miR19b
Anit-miR19b
Normal Heart

Whether miR19b play role in

Cardiotoxicity ?

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 DASATINIB
SIDE EFFECT
Dose dependent side effect widely causes
• Cardiac Failure
•Cardiovascular disorder
•Atrial flutter
• Hypertension

Mostly causes Hypertrophic Cardiomyopathy

N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-
yl]amino]-1,3-thiazole-5-carboxamide

Widely used drug in cancer predominantly in CML.

Targets Raf-1 and B-Raf hetrodimerization

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 Objectives

•To study Dasatinib dose and Raf-1 mutation dependent effect at

cell morphology, RNA and Protein level.

•To study effect of anti-miR19b on Raf-1 mutation and Dasatinib

dose at cell morphology, RNA and Protein level

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 WORKFLOW

Transfection
Transfection
(Raf1 mutants S259A )
(Raf1 mutants S259A )

anti-miR19b
Dasatinib
Treatment
Dasatinib Treatment

3)WesternBlot
3)Western Blot

1)Phalloidin staining 2)qRT-PCR

1)Phalloidin staining 2)qRT-PCR 6
Results

7
 Positive SDM clone for S259A position

Reference
sequence

TCC(Serine)-GCC(Alanine)
Sequencing confirmation for positive S259A SDM

8
Cell size
Cell size increases
decreasesobserved
observedin
inmutant
mutanttreated
treatedwith
with
Dasatinib and anti-miR19b
Dasatinib

Raf -1WT
Raf WT+anti-miR19b Raf-1Raf WT+10nM
WT+10nM Dasatinib Raf-1Raf WT+30nM
WT+30nM Dasatinib
Dasatinib+anit-miR19b Dasatinib+anti-miR19b

S259A
S259A+anti-miR19b S259A+10nM Dasatinib
S259A+10nM S259A+30nM Dasatinib
S259A+30nM
Dasatinib+anti-miR19b
Phalloidin staining Dasatinib+anti-miR19b
for cell size measurement
Phalloidin staining for cell size measurement 9
Scale:10um
Expression
Expression level
level of
of miR19bafter
miR19b in presence
treatment
of Raf1
with over
anti-
expression and
miR19b
mutants S259A

Mutation
Mutation and
and Dasatinib
Dasatinib dose
dose dependent
dependent expression
expression level
level of
of miR19b
miR19b

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 Reducedand
Mutation Eexpression
Dasatiniblevel
doseofdependent
Hypertrophic Markerlevel
expression after
of
anti-miR19b
Hypertrophictreatment
Marker

Atrial Natriuretic peptide

Secreted and activated

Protective Role

ANP expression
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ANP expression
 Reducedand
Mutation Eexpression
Dasatiniblevel
doseofdependent
Hypertrophic Markerlevel
expression after
of
anti-miR19b
Hypertrophictreatment
Marker

BNP activates ANP

Atrial Natriuretic peptide

Secreted and activated

Protective Role

BNP
BNPexpression
expression
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Mutation
Reducedand Dasatiniblevel
Eexpression doseofdependent expression
Hypertrophic Markerlevel of
after
Hypertrophictreatment
anti-miR19b Marker

β-MHC – fetal life

Low ATPase activity

Up-regulated under stress

condition

β-MHC
β-MHC expression
expression
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 Dasatinibis iswell
Dasatinib welltolerated
toleratedininHEK293T
C2C12 Cell
Cell

24r 24hr 24hr 24hr 48hr 48hr 48hr 48hr 72hr 72hr 72hr 72hr
CT 3nM24r 5nM24hr
10nM 24hr
CT 3nM
48hr 5nM
48hr10nM48hr
CT 3nM
72hr5nM72hr
10nM 72h
CT 10nM 30nM CT 10nM 30nM CT 10nM 30nM
p-Erk1/2
p-Erk1/2

Total-Erk1/2
Total-Erk1/2

c-Raf c-Raf

GAPDHβ-actin

DasatinibDose
Dasatinib DoseStandardization
Standardizationatatdifferent
differenttime
timepoints
pointsininHEK293T
C2C12 cell
cell

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 DASATINIB is toxic for H9C2 Cells

pRaf1(S338)

Raf1

GAPDH

Western Blot analysis for Raf-1(p-S338) expression

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Increase
Decrease
phosphorylation
phosphorylationof of
ErkErk1/2
1/2 ininthe
thepresence
presenceof
over expressed Raf-1 and 30nm Dasatinib in H9C2 cells
anti-miR19b

pErk 1/2
pErk 1/2
Total Erk 1/2
Total Erk 1/2

pS338-Raf1
pS338-Raf1

Total
Total Raf1
Raf1

β-Actin
β-Actin

WesternBlot
Western Blot analysis
analysis for
forpErk1/2
pErk1/2expression
expression
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 Decrease
Increase phosphorylation
phosphorylation of of
ErkErk
1/21/2ininthe
thepresence
presenceof
over expressed Raf1anti-miR19b
S259A mutation and 10nm of
Dasatinib in H9C2 cells

pErk 1/2
pErk 1/2
Total Erk 1/2
Total Erk 1/2

pS338-Raf1
pS338-Raf1

Total Raf1
Total Raf 1

β-Actin
β-Actin

Western Blot analysis for pErk1/2 expression

Western Blot analysis for pErk1/2 expression 17
 DISCUSSION

miR19a/b

mRNA

Anti-HCM Protein
Atrogin and Muscle RING -finger protein 1

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Song 1
DW et.al,
 CONCLUSION

S259A + Dasatinib

Anti-miR19b
High expression of HCM Marker + High expression of miR 19b
&
High Expression of pErk1/2
Low expression of HCM Marker + Low expression of miR 19b
&
Low Expression of pErk1/2
Cell size increase

Cell size is retained to normal

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 FUTURE PROSPECTIVE

•Study Cardiotoxicity of Dasatinib in Human Induce Pluripotent Stem

Cells

•Drug modelling to reduce cardiotoxicity or increase cardiac safety index

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 BIBLIOGRAPHY
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 ACKNOWLEDGEMENT

•I thank my Institute for improving and developing my skills throughout

the degree programme.

•Dr. Dhandapany Perundurai for giving me the opportunity to work in his

lab.

•Dr. Anupam Mittal for guiding me throughout the project.

•All Dhan lab members .

•My friends Akansha , Mahima, Kamini, Indra, Anushree,

Annu, Deepanshu for sharing anxieties, excitement and
innumerable cup of coffee.

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Thank You

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