The growth inhibitory effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines.

1. 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, 2013
ARVO Meeting

2. 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

3. ✴ 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

4. WE WANTED TO INVESTIGATE NON-
CHEMOTHERAPEUTIC DRUGS AS ANTI-CANCER
AGENTS

5. 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.

6. 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).

7. Exercise, Energy Depletion, Hormones
ATP
AMP
αAMPK
γ β
CaMKK LKB1
ACC malonyl-CoA FA
CPTI
F.A. oxidation
Mitochondria
TSC
mTOR
GLUT4
PFK

10. 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

11. AICAR as anti-proliferative agent

13. Adenosine
Kinase
Adenosine
Receptors
AICA
AICA
ZMP
A M P K
X
X 5-Iodotubercidin
DPY
Aicar: Simplified Mechanism Of Action

14. OCM3
0
50
100
150 Day 3
Day 5
AICAR mM
Cellgrowth(%ofcontrol)
0 1 2 4
MEL92.1
0
50
100
150 Day3
Day5
0 1 2 4
AICAR mM
Cellgrowth(%ofcontrol)
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.

15. 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 3
Day 5
Control AICAR DPY DPY+AICAR
*
Cellgrowth(%ofcontrol)
MEL92.1
0
50
100
150 Day3
Day 5
Control AICAR DPY DPY+AICAR
**
Cellgrowth(%ofcontrol)
OCM3
0
50
100
150
Day 3
Day 5
Control AICAR Iodo Iodo+AICAR
NS
**
Cellgrowth(%ofcontrol)
MEL92.1
0
50
100
150 Day 3
Day 5
Control AICAR Iodo Iodo+AICAR
*
Cellgrowth(%ofcontrol)

16. 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
foldincrease
**
P-ACC
Control
0
2
4
6
8
**
Control AICAR 2mM DPY+AICAR
foldincrease
P-ACC
Control AICAR Iodo + AICAR
0
5
10
15
20
foldincrease
*
CONTROL AICAR 2mM Iodo+AICAR
P-ACC
Control AICAR Iodo + AICAR
0
2
4
6
8
10
CONTROL AICAR 2mM Iodo+AICAR
**
foldincrease
OCM3 MEL 92.1
AICAR DPY + AICAR

17. Day 3
Apoptosis
G
1
S
G
2
0
20
40
60
Day 5
Apoptosis
G
1
S
G
2
0
20
40
60
80 Control
AICAR 1mM
AICAR 2mM
Day 1
Apoptosis
G
1
S
G
2
0
20
40
60
80
MEL 92.1 Day 3
Apoptosis
G
1
S
G
2
0
20
40
60
80
Day 5
Apoptosis
G
1
S
G
2
0
20
40
60
80
Control
AICAR 1mM
AICAR 2mM
AICAR causes cell cycle arrest in S phase
Flow cytometry assessment of cell cycle using propidium iodide staining.

18. AICAR decreases the levels of cyclins A1 and
D1 in Uveal Melanoma cells
OCM3
C
yclin
A1
C
yclin
A2
C
yclin
E1
C
yclin
E2
C
yclin
D
1
C
yclin
D
3
0.0
0.5
1.0
1.5
2.0
Control
AICAR 1mM
AICAR 2mM
*
*
*
Relativeexpression
MEL 92.1
C
yclin
A1
C
yclin
A2
C
yclin
E1
C
yclin
E2
C
yclin
D
1
C
yclin
D
3
0.0
0.5
1.0
1.5
2.0
Control
AICAR 1mM
AICAR 2mM
Relativeexpression
* *
Quantitative RT-PCR analysis

19. AICAR down-regulates 4E-BP1 phosphorylation but not S6 Kinase
and partially induces the autophagy marker LC3B in uveal melanoma cells
0 1 2
OCM3
0 1 2
MEL 92.1
P-4EBP1
0.0
0.5
1.0
1.5
foldincrease
Control AICAR 1mM AICAR 2mM
*
*
4EBP1
0.0
0.5
1.0
1.5
Control AICAR 1mM AICAR 2mM
**
foldincrease
0 1 2 0 1 2
LC3B I
LC3B1I
P-4E-BP1
LC3B
0.0
0.5
1.0
1.5
2.0
foldincrease
Control AICAR 1mM AICAR 2mM
NS
NS
LC3B
0.0
0.5
1.0
1.5
2.0
Control AICAR 1mM AICAR 2mM
*
**
foldincrease
0 1 2
p-S6
0 1 2
AICAR (mM)
AICAR (mM)
AICAR (mM)

20. 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

21. 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)

22. 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.

23. Financial Support
✴ Research to Prevent Blindness

24. Thank you for your attention!
Syria