Ahmad Al Moujahed, Fotini Nicolaou, Thanos D. Papakostas,

Joan W. Miller, Evangelos Gragoudas and Demetrios G. Vavvas

Angiogenesis Laboratory,Massachusetts Eye and Ear Infirmary,

Harvard Medical School

Uveal Melanoma Cells Are Inhibited by AICAR, an Exercise Mimetic, Partially Through Activation of

AMP-Dependent Kinase

May 8, 2013ARVO Meeting

Commercial disclosure

✴Ahmad Al Moujahed: None✴Fotini Nicolaou: None✴Thanos Papakostas: None✴Joan Miller: Massachusetts Eye and Ear Infirmary:Patent;

Novartis: Personal Financial Interest; Alcon: Consultant; KalVista Pharmaceuticals: Consultant

✴Evangelos Gragoudas: QLT Phototherapeutics, Inc. Patent✴Demetrios Vavvas: MEEI: Patent; Kala

pharmaceuticals:Consultant; Roche:Consultant; Genentech: Consultant

✴ Most common primary intraocular malignancy in adults.

✴ Survival rates for uveal melanoma remain poor.

✴ Up to 50% of patients will develop metastatic disease, even 10–15 years after diagnosis, which invariably leads to death.

✴ The median survival of patients with metastasis is less than 6 months.

✴ Although current therapies achieve good local control, treatment for metastatic disease has poor results, and is associated with significant morbidity.

Uveal Melanoma

WE WANTED TO INVESTIGATE NON-CHEMOTHERAPEUTIC DRUGS AS ANTI-CANCER

AGENTS

5-aminoimidazole- 4-carboxamide riboside (AICAR)

AICAR (ACADESINE or

ZMP)

AMP

✴ AICAR (5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside) is an analog of AMP.

✴Widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change.

✴ Is an exercise mimetic drug with anti-proliferative properties.

AMP-activated protein Kinase (AMPK)

✴AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase.

✴ IT IS THE MAJOR ENERGY SENSOR OF THE CELL

✴ It is regulated by hypoxia, exercise, ischemia, heat shock, and long-term starvation.

✴One of its upstream protein kinase LKB1 is known to be a tumor suppressor involved in Peutz-Jegher syndrome.

✴Downstream effectors of AMPK also involve the tumor suppressor tuberous sclerosis complex (TSC2) and the mammalian target of rapamycin (mTOR).

Exercise, Energy Depletion, HormonesATPAMP

αAMPKγ β

CaMKK LKB1

ACC malonyl-CoA FA

CPTI

F.A. oxidation Mitochondria

TSC

mTORGLUT4

PFK

The World Anti-Doping Code

THE 2009 PROHIBITED LIST

INTERNATIONAL STANDARD

The official text of the Prohibited List shall be maintained by WADA and shall be published in English and French. In the event of any conflict between the English and French versions, the English version shall prevail.

This List shall come into effect on 1 January 2009

The Prohibited List 2009 20 September 2008

PROHIBITED METHODS M1. ENHANCEMENT OF OXYGEN TRANSFER The following are prohibited: 1. Blood doping, including the use of autologous, homologous or heterologous

blood or red blood cell products of any origin. 2. Artificially enhancing the uptake, transport or delivery of oxygen, including

but not limited to perfluorochemicals, efaproxiral (RSR13) and modified haemoglobin products (e.g. haemoglobin-based blood substitutes, microencapsulated haemoglobin products).

M2. CHEMICAL AND PHYSICAL MANIPULATION 1. Tampering, or attempting to tamper, in order to alter the integrity and

validity of Samples collected during Doping Controls is prohibited. These include but are not limited to catheterisation, urine substitution and/or alteration.

2. Intravenous infusions are prohibited except in the management of surgical

procedures, medical emergencies or clinical investigations. M3. GENE DOPING The transfer of cells or genetic elements or the use of cells, genetic elements or pharmacological agents to modulating expression of endogenous genes having the capacity to enhance athletic performance, is prohibited. Peroxisome Proliferator Activated Receptor � (PPAR�) agonists (e.g. GW 1516) and PPAR�-AMP-activated protein kinase (AMPK) axis agonists (e.g. AICAR) are prohibited.

The Prohibited List 2009 20 September 2008

6

AICAR as anti-proliferative agent

Adenosine Kinase

Adenosine

Receptors

AICA

AICA

ZMP

A M P K

X

X 5-Iodotubercidin

DPY

Aicar: Simplified Mechanism Of Action

OCM3

0

50

100

150 Day 3Day 5

AICAR mM

Cel

l gro

wth

(% o

f con

trol)

0 1 2 4

MEL92.1

0

50

100

150 Day3Day5

0 1 2 4AICAR mM

Cel

l gro

wth

(% o

f con

trol)

AICAR Inhibits the Growth of Human Uveal Melanoma Cells

Uveal melanoma cell lines were treated for 3 and 5 days with various concentrations of AICAR (1–4 mM), and cell viability was measured by MTT assay.

Inhibition of AICAR entry into the cell abolishes its effect on cell growth. Inhibition of conversion of AICAR into ZMP

partially abolishes the effect on cell growth

OCM3

0

50

100

150 Day 3Day 5

Control AICAR DPY DPY+AICAR

*

Cel

l gro

wth

(% o

f con

trol)

MEL92.1

0

50

100

150 Day3 Day 5

Control AICAR DPY DPY+AICAR

**

Cel

l gro

wth

(% o

f con

trol)

OCM3

0

50

100

150 Day 3Day 5

Control AICAR Iodo Iodo+AICAR

NS

**

Cel

l gro

wth

(% o

f con

trol)

MEL92.1

0

50

100

150 Day 3Day 5

Control AICAR Iodo Iodo+AICAR

*

Cel

l gro

wth

(% o

f con

trol)

Antiproliferative effects of AICAR are associated with activation of AMPK pathway

P-ACC

Control AICAR DPY + AICAR

0

1

2

3

4

5

CONTROL AICAR 2mM DPY+AICAR

fold

incr

ease

**

P-ACC

Control

0

2

4

6

8

**

Control AICAR 2mM DPY+AICAR

fold

incr

ea

se

P-ACC

Control AICAR Iodo + AICAR

0

5

10

15

20

fold

incr

ease

*

CONTROL AICAR 2mM Iodo+AICAR

P-ACC

Control AICAR Iodo + AICAR

0

2

4

6

8

10

CONTROL AICAR 2mM Iodo+AICAR

**

fold

incr

ease

OCM3 MEL 92.1AICAR DPY + AICAR

Day 3

Apopto

sis G1 S G20

20

40

60

Day 5

Apopto

sis G1 S G20

20

40

60

80 ControlAICAR 1mMAICAR 2mM

Day 1

Apopto

sis G1 S G20

20

40

60

80

MEL 92.1 Day 3

Apopto

sis G1 S G20

20

40

60

80Day 5

Apopto

sis G1 S G20

20

40

60

80ControlAICAR 1mMAICAR 2mM

AICAR causes cell cycle arrest in S phase

Flow cytometry assessment of cell cycle using propidium iodide staining.

AICAR decreases the levels of cyclins A1 and D1 in Uveal Melanoma cells

OCM3

Cyclin

A1

Cyclin

A2

Cyclin

E1

Cyclin

E2

Cyclin

D1

Cyclin

D3

0.0

0.5

1.0

1.5

2.0

Control

AICAR 1mM

AICAR 2mM

***R

elat

ive

expr

essi

on

MEL 92.1

Cyclin

A1

Cyclin

A2

Cyclin

E1

Cyclin

E2

Cyclin

D1

Cyclin

D3

0.0

0.5

1.0

1.5

2.0Control

AICAR 1mM

AICAR 2mM

Rel

ativ

e ex

pres

sion

* *

Quantitative RT-PCR analysis

AICAR down-regulates 4E-BP1 phosphorylation but not S6 Kinase and partially induces the autophagy marker LC3B in uveal melanoma cells

0 1 2OCM3

0 1 2MEL 92.1

P-4EBP1

0.0

0.5

1.0

1.5

fold

incr

ease

Control AICAR 1mM AICAR 2mM

**

4EBP1

0.0

0.5

1.0

1.5

Control AICAR 1mM AICAR 2mM

**

fold

incr

ease

0 1 2 0 1 2

LC3B ILC3B1I

P-4E-BP1

LC3B

0.0

0.5

1.0

1.5

2.0

fold

incr

ease

Control AICAR 1mM AICAR 2mM

NSNS

LC3B

0.0

0.5

1.0

1.5

2.0

Control AICAR 1mM AICAR 2mM

***

fold

incr

ease

0 1 2

p-S6

0 1 2

AICAR (mM)

AICAR (mM)

AICAR (mM)

AICAR does NOT affect the levels of CDK inhibitor p27, p21, tumor suppressor protein p53, and PCNA

AICAR (mM) 0 1 2

OCM3

0 1 2

MEL 92.1

p21

p27

PCNA

p53

CDK2

Phospho-p44/42 MAPK

0 1 2

OCM3

0 1 2

MEL 92.1

AICAR does NOT affect the levels of the cyclin-dependent kinases CDK2 and CDK4, and MAPK pathway.

CDK4

AICAR (mM)

Conclusion

✴ Our results indicate that the exercise mimetic AICAR inhibits uveal melanoma cell growth partially via an AMPK-dependent mechanism.

✴ AICAR may have potential, as a novel targeted therapy, in treating patients with uveal melanoma.

Financial Support

✴ Research to Prevent Blindness

Thank you for your attention!

Syria