Transcript Signal transduction and development radiation sensitizers/protectors Dennis Hallahan, MD Elizabeth & James McDonnell Distinguished Professor Head, Dept of Radiation Oncology Washington University St Louis Mallinckrodt Institute.

Signal transduction and development
radiation sensitizers/protectors
Dennis Hallahan, MD
Elizabeth & James McDonnell Distinguished Professor
Head, Dept of Radiation Oncology
Washington University St Louis
Mallinckrodt Institute of Radiology
Siteman Cancer Center
Radiation Chemistry
micro seconds
Non-homologous end joining: SCID
SCID tumor
vasculature is
radiosensitive
Shinohara, Cancer Res June 2005
Vanderbilt-Ingram Cancer Center
SCID vs. C57BL6 tumor volume
C57BL6
SCID
C57BL6 RT
SCID RT
Growth delay
RadiotherayI
Kinetics of repair (human
cells)
Pharmacokinetics of radiation
sensitizing drugs
Percent of peak
100
90
80
70
60
50
40
30
20
10
0
0
1
Irradiation
2
3
Hours
4
5
6
DNA-PK Inhibition
Colony formation
Tumor growth delay
IC87361 radiosensitizes wt but not
SCID cells
Improved tumor growth delay by
IC87361
LLC
Vanderbilt-Ingram Cancer Center
B16F0
Pro-survival signaling in irradiated vascular
endothelium
Ionizing
radiation
GPCR
(GPR4)
RTK
(Flk-1)
 


Ggb
PI3K
ERK1/2
Ga
GTP
PI3Kg
Akt
ERK1/2
AA
Arachidonyltrifluoromethyl Ketone (AACOCF 3)
Methyl Arachidonyl Fluorophosphonate (MAFP)
Cox1/2
Isoprostanes,
prostaglandins
Cell
death
Viability
Inflammation

cPLA2
LPC
Receptor Tyrosine Kinase Inhibition
Increased Spectrum of RTK
inhibition
SU11248
Known Spectrum of RTK
inhibition
VEGFR2 Flt
SU5416
SU6668
SU11248
Gleevec
AG1478
AEE787
PTK 788
+
+
+
+
+
+
+
+
+
+
PDGFR
c-Kit
+
+
+
+
+
+
+
FGFR ErbB1/2
+
-
+
+
Why do RTK inhibitors prevent
radiation-induced Akt-phosphorylation?
• Scaffold for adaptor protein binding
• RTKs initiate signal transduction
• SU6668 & SU11248 are nonspecific
X
Akt phosphorylation by radiation
in absence of serum/GFs
Endothelial cells
P-AktAkt-
Gy:
0
1
3
6
9
15
30
60
12
P-Akt-
Total AktMinutes: 0
5
Vanderbilt-Ingram Cancer Center
120
Can tumor microvasculature be
made more radiation responsive?
3 Gy
alone
0 hours
SU11248 +
3 Gy
Vanderbilt-Ingram Cancer Center
48 hours
Endothelial cell death
RTK inhibitor with radiation
Analysis of Lipid Second Messengers
60
50
444.4
446.3
10
18:1 LPC
538.6
526.3
528.4
532.4
518.4
504.3
475.3
454.4
520.5
502.3
482.2
468.2
20
050209 samples
524.3
494.3
40
30
NL: 7.77E5
20:2 LPC
70
522.3
18:0 LPC
14:0 LPC
80
18:3 LPC
16:1 LPC
90
18:2 LPC
496.3
100
16:0 LPC
Variability in positive mass spectra of
control sample (444.4 -606.3)
490.3
0
100
577.6
544.4
603.3
548.4
566.3
584.4
552.4
592.5
606.3
NL: 6.68E5
522.2
496.3
Sample 2
pos#1-51 RT:
0.01-0.99 AV: 51
T: + p Q1MS [
350.00-1200.00]
50
494.3
16:1 LPC
90
40
80
30
444.5
70
449.4
453.4
20
60
10
468.4
482.2
476.3
502.2
496.2
502.1
504.2
524.2
524.4
538.6
518.4
548.4
526.2
504.1
478.3
494.3
522.1
528.3
578.3
592.3
606.4
518.2
522.3
496.3
476.3
544.1
469.1
449.3
455.1
457.4
482.0
490.3
516.2
505.2
508.2 520.2
581.1
10
60
570.1
530.0
NL: 7.16E5
560.3
528.1
480.3
30
40
603.4
577.5
560.4
551.2
90
100
566.4
548.1
40
50
0
050713 samples
544.4
Sam ple 26
pos #1-51 RT:
0.01-0.99 AV: 51
T: + p Q1MS [
350.00-1200.00]
550.0
100
20
70
NL: 3.96E5
526.2
50 0
80
20:2 LPC
60
100
18:3 LPC
16:0 LPC
70
18:1 LPC
520.4
18:0 LPC
90
80
Sample 3
pos#1-51 RT:
0.01-0.99 AV: 51
T: + p Q1MS [
350.00-1200.00]
562.2
577.3
576.3
589.3
592.1
601.2
Sample 1
pos#1-51 RT:
0.00-0.98 AV: 51
T: + p Q1MS [
350.00-1200.00]
524.3
494.2
502.2
524.1
526.2
NL: 7.29E5
538.4
Sam ple 25
pos #1-50 RT:
Excerpt from a Lipid Array in negative
ionization mode (time course)
2 min
16:1 LPS
22:6 LPG
34:2 PE
34:2 PG
36:3 PG
40:4 PG
34:2 PI
36:1 PI
5 min
15 min
30 min
60 min
418.16
0
0
1
0
3
419.16
1
1
-1
1
0
494.23
-1
0
2
1
3
495.20
1
0
0
1
0
555.23
1
0
0
2
4
556.22
0
-1
0
0
2
581.26
0
0
-1
3
2
582.27
0
0
0
1
2
594.37
3
0
-1
-1
0
595.33
-1
1
1
0
0
691.30
0
0
1
0
3
692.36
0
2
0
0
1
698.36
1
2
0
-3
0
699.31
2
1
0
0
0
704.34
2
0
3
0
0
705.30
1
0
-2
0
1
714.28
0
0
3
-3
2
715.29
1
1
0
-2
-1
745.32
-1
1
2
-3
1
746.31
1
0
0
-1
1
757.34
3
2
2
-3
0
758.33
2
0
1
-2
-1
771.32
-1
1
0
-3
-1
772.32
1
1
1
-1
0
825.35
-3
0
1
-3
-1
826.36
-1
1
2
-3
-1
833.31
0
1
0
-3
-2
834.31
0
0
0
0
0
858.34
2
1
-1
3
-1
859.34
-1
0
0
-1
-1
863.34
-3
1
1
0
-1
X-ray-induced cPLA2 activation
PLA2 inhibitors enhance efficacy of
radiotherapy
Pro-survival signaling in irradiated vascular
endothelium
Ionizing
radiation
GPCR
(GPR4)
RTK
(Flk-1)
 


Ggb
PI3K
ERK1/2
Ga
GTP
PI3Kg
Akt
ERK1/2
AA
Arachidonyltrifluoromethyl Ketone (AACOCF 3)
Methyl Arachidonyl Fluorophosphonate (MAFP)
Cox1/2
Isoprostanes,
prostaglandins
Cell
death
Viability
Inflammation

cPLA2
LPC
Inhibition of cPLA2 leads to Mitotic
Catastrophe 24-48 hours after treatment
Control
AACOCF3
MAFP
IR
AACOCF3+IR
MAFP+IR
DAPI
Tubulin-FITC
Merged
Inhibition of cPLA2 leads to Mitotic
Catastrophe 24-48 hours after treatment
Multinucleated/giant cells
(fold increase of control)
8

7

6
5

4

3
Control
IR
AACOCF3
AACOCF3+IR
MAFP
MAFP+IR

2
1
0
24 h
48 h
72 h
Control cPLA2+/+
IR cPLA2+/+
Control cPLA2-/-
IR cPLA2-/-
Inhibition of cPLA2 leads to cyclin B1
accumulation 24-48 hours after treatment
Pretreatment
3 Gy
EtOH
-
AACOCF3
+
-
+
MAFP
-
+
cyclin B1
24 h
actin
cyclin B1
48 h
actin
Inhibition of cPLA2 results in a delayed
program cell death 72-96 hours after treatment
IR
AACOCF3
AACOCF3+IR
MAFP
MAFP+IR
Apoptosis (fold increase of control)
Control

Control
IR
10
AACOCF3
AACOCF3/IR
8

MAFP
MAFP/IR
6
4


2
0
24 h
48 h
72 h
96 h
Inhibition of cPLA2 results in a delayed
program cell death 72-96 hours after
treatment
Apoptotic cells (% of total)
80

Control cPLA2+/+
IR cPLA2+/+
60
Control cPLA2-/IR cPLA2-/-

40
20
0
24 h
48 h
72 h
96 h
cPLA2 inhibition decreases migration in
irradiated endothelial cells (gash closure )
0 Gy
EtOH
AACOCF3
MAFP
0 Gy
3 Gy
120
EtOH
Cell density (%)
100
AACOCF3
MAFP
80

3 Gy60


40
20
0
co
l
ro
t
n
y
3
Gy
Gy
FP
CF
A
3
3
3G
O
+
+
M
C
F3
FP
C
A
AA
M
CO
A
A
Inhibition of cPLA2 attenuates endothelial
tubule formation
0 Gy
EtOH
0 Gy
AACOCF3
MAFP
3 Gy
3 Gy
Tubule/HPF (total number)
50
EtOH
40
AACOCF3
MAFP
30

20

10
0
c
t
on
l
ro
y
F3
Gy
Gy
FP
C
A
3
3
3G
+
M
CO
P+
3
F
F
A
A
OC
MA
C
AA
Treatment Protocol
for Tumor Mouse Models
● C57BL6 mice with Lewis Lung carcinoma in hind limb
● IP of 70% EtOH or AACOCF3 (10 mg/kg) prior to tumor irradiation
with 3 Gy
● Treatment once per day for 5 consecutive days
● 24 hours post-treatment: analysis via Power Doppler sonography
and vWF
cPLA2 inhibition decreases blood flow in
irradiated tumors
Control
IR
20
AACOCF3
IR
AACOCF3/IR
Vascular Index, %
Control
15
10

AACOCF3
AACOCF3/IR

5
0
control
IR
AA
AA/IR
cPLA2 inhibition (AACOCF3) decreases
growth of irradiated tumors
3
Tumor volume (mm )
250
control
3 Gy
2.0 mg/kg AACOCF3
200
2.0 mg/kg AACOCF3+ 3 Gy
150
100
50
0
5
6
7
8
9
10
11
12
13
14
15
16
Time after tumor innoculation (days)
17
18
Small molecule inhibitors of GSK3b
attenuate neurocognitive deficits that
result from brain irradiation
Medulloblastoma
Whole Brain irradiation
Neurocognitive deficit
•
•
•
•
•
•
Hippocampal SGZ of Dentate
Short term memory
Learning
Verbal and spatial
Medulloblastoma Survivors
Leukemia survivor unemployment NEJM’03
Radiation-induced apoptosis in
neuronal progenitor cells: SGZ
0 Gy
Mallinckrodt Institute
Washington University St Louis
7 Gy
Siteman Cancer Center
Apoptosis in hippocampal neurons
Piknotic cells/HPF
250
200
150
100
50
0
0
1
2
3
4
5
6
8
10
Radiation dose, Gy
Mallinckrodt Institute
Washington University St Louis
Siteman Cancer Center
p53 phosphorylation and Bax expression
Mallinckrodt Institute
Washington University St Louis
Siteman Cancer Center
Cancer dies post-mitotic not PCD
Thotala, Cancer Research 2008
GSK3 inhibition alters Bax & Bcl2
in intestinal crypt cells
Radiation-induced
Apoptosis in
intestinal crypts
is attenuated by
GSK3 inhibition
Intestinal Irradiation