Silencing of Mutant AR Co-activators as a Therapeutic

Approach

Manuela Basso, Assistant ProfessorUniversity of Trento, Centre for

Integrative Biology

Trento, Italy

Basso’s Lab

Pennuto’s Lab

Italian Meeting with researchers and Scientists

November 12th 2016, MilanApril 18th 2015, TrentoNovember 2013, Padova

The fourth meeting is coming soon….

March 27th-29th 2015

Naarden, The Netherlands

A new one coming up soon

Neuron and muscle

degeneration

QQQQQQQCAGCAGCAGCAGCAGCAG…

>36 repeats

HD Mutant ARAR gene

SBMA is caused by expansion of a polyglutamine tract in AR

Chr X

p q

AR gene

CAGCAGCAG…

8-29 repeats

WT QQQQ ARAR gene

Healthy neuron and muscle

AR post-translational modifications

Polyglutamine tract

Phosphorylation

Acetylation

SUMOylation

Palazzolo et al., 2007Scaramuzzino et al., 2015

Montie et al., 2012Chua et al., 2015

S96

R2

10

S21

5

S79

2

1 920555 670

AR

R methylation

R7

89

R7

87

R2

12

Qn DBD LBDNTD

K6

30

K6

32

K6

33

S38

5

S51

8

Arginine methylation is catalyzed by Protein Arginine Methyltransferases (PRMTs)

PRMT1 PRMT2 PRMT3 PRMT4 PRMT6PRMT8

Type IPRMTs

PRMT5 PRMT9

Type IIPRMTs

PRMT7

Type IIIPRMT

Basso and Pennuto, 2015

Basso & Pennuto, Exp Neurol 2015

Arginine methylation and serine phosphorylation are mutually exclusive and both modulate AR function

Does PRMT6 modify toxicity in vitro?

Scaramuzzino et al., 2015

Does PRMT6 modify toxicity in vivo?

In collaboration with the lab of Udai Pandey, University of Pittsburgh

AR X KD PRMT6

UAS-AR0Qor

UAS-AR52Q

GMR-Gal4; DART8 silencing

Scaramuzzino et al., 2015

PRMT6 modifies AR toxicity in vivo

AR is transcription factor

https://en.wikipedia.org/wiki/Androgen_receptor#/media/File:Human_androgen_receptor_and_androgen_binding.svg

PRM

T66

????

PRMT6 specifically transactivates AR

Scaramuzzino et al., Neuron 2015

AR transactivation by PRMT6 is enhanced by polyglutamine expansion

3.3-fold

5.3-foldARE Luc

AR

Testosterone

Is it possible to reduce the activity of PRMT6 and preserve the physiological functions of AR?

SBMA pathogenesis is modified through arginine methylation of polyQ-expanded AR

Silence PRMT6 by novel selective inhibitors to assess the rescue of polyQ-expanded AR

2

Silence PRMT6 by an artificial microRNA to assess the rescue of polyQ-expanded AR in vivo

1

Hypothesis

1a

Debasmita Tripathy, postdoctoral fellow

Identify the best artificial miRNA for PRMT6

The artificial miRNA 13.1 silences PRMT6 of nearly 50%

Fold

ch

ange

(o

ver

ne

gati

ve c

on

tro

l)

The artificial miRNA 13.1 reduces AR functionFo

ld c

han

ge (

ove

r n

ega

tive

co

ntr

ol)

Fold

ch

ange

(o

ver

ne

gati

ve c

on

tro

l)

1b

Mathilde ChivetPennuto’s lab

New transgenic mouse model

Chivet et al., in preparation

Life span is reduced in mice overexpressing expanded AR

Chivet et al., in preparation

Weight body and strength are reduced in mice overexpressing expanded AR

Chivet et al., in preparation

Motor coordination is also reduced in mice overexpressing expanded AR

In progress:

Artificial miRNA for PRMT6

Behavior assessment

In collaboration with Giuseppe Ronzitti, Genethon

2

PRMTs

SAM /AdoMet

Me

Adox / MTA

Novel pharmacological inhibitorsMS023 and MS049

In collaboration with Masoud Vedadi, University of Toronto

The novel inhibitors reduce AR-dependent transactivation

1-Which are other AR coactivators?

2-Is it feasible to reduce the over-activation of AR to correct its transcriptional activity but maintain its physiological functions?

Where are we going?

Maria Pennuto’s LabUniversity of Padova

Mathilde Chivet (Postdoctoral fellow)Carlo Rinaldi’s LabOxford University

Acknowledgements

Alice Migazzi, PhD student Mathi Chivet, postdoc

Debasmita Tripathy, postdoc

Giuseppe RonzittiGenethon, France

Chivet et al., in preparation