Transcript Slide 1

The Future of Glioblastoma Therapy:
Multi-modality with Multiple Targets
Gautam Prasad
Resident Physician
Grand Rounds: May 15, 2009
Outline
• Case Presentation
• Moving Beyond Local Therapy
• Potential Molecular Targets for GBM and a Case Example
• XL765 – a dual PI3K/mTOR inhibitor
• Preclinical Data with XL765
• Model Systems
• In vitro – cytotoxicity and downstream molecular changes
• In vivo – survival and disease burden in mice
• Clinical Data with XL765
• Future Directions
Case Presentation
• Pt J.E. is a 33M RH physician
• Initial presentation and work-up
• 1/09 – began experiencing worsening L frontal HA; pt reports
awakening at night w/ pain accompanied by N/V
• 2/9/09 – CT Head: 8.8 x 5.9 cm R frontal lobe mass w/ mass effect
and R  L shift
• 3/3/08 – Pre-operative MRI
4/6/08 – Post-op MRI - GTR
Case Presentation
• Standard treatment (Stupp, NEJM 2005):
60 Gy + 75 mg/m2 of TMZ  4 week break  6 additional cycles of TMZ
Median Survival: 14.6 months (12.1 months control)
Median Surival w/ Methylated MGMT promoter: 21.7 months (15.3 months
control)
No Clinical Trials Available!
Given age and KPS pt has < 4 years survival in all likelihood.
Outline
• Case Presentation
• Moving Beyond Local Therapy
• Potential Molecular Targets for GBM and a Case Example
• XL765 – a dual PI3K/mTOR inhibitor
• Preclinical Data with XL765
• Model Systems
• In vitro – cytotoxicity and downstream molecular changes
• In vivo – survival and disease burden in mice
• Clinical Data with XL765
• Future Directions
Local Therapy Alone is not the Solution
“Isolation and characterization of human malignant glioma cells
from histologically normal brain”
Department of Neurosurgery, Washington University (J Neurosurgery 1997)
3 adults with supratentorial GBMs had resections in addition to biopsies
of “normal” brain ≥ 4 cm from tumor
* Normal brain biopsy
Local Therapy Alone is not the Solution
Tumor
cells?
Gross
Tumor
Yes
“Normal” Cultured
Brain
Glia
No
No
GFAP
+
+
+
Growth
Rate
19%/day
36%/day
~5%/day
Motility
3.92
4.18
Karyotype Neoplastic
Neoplastic
Normal
Local Therapy Alone is not the Solution
Conclusions
Obviously a limited study (n = 3), but several interesting findings:
1. Histopathologic examination of frozen sections to determine
what constitutes “normal” brain may be misleading
2. “Normal” brain in GBM patients may be infiltrated by tumor
cells (?stem cells) as evidence by examination in culture
3. Focusing on the gross tumor (+ margin) alone will probably
prevent death by herniation in the short-term but is not very
what about increasing long-term survival?
Outline
• Case Presentation
• Moving Beyond Local Therapy
• Potential Molecular Targets for GBM and a Case Example
• XL765: a dual PI3K/mTOR inhibitor
• Preclinical Data with XL765
• Model Systems
• In vitro – cytotoxicity and downstream molecular changes
• In vivo – survival and disease burden in mice
• Clinical Data with XL765
• Future Directions
GBM – No Shortage of Potential Targets
Source: Argyriou AA and Kalofonos HP 2009, Mol Med
PI3K Signaling Pathway
EGF
IRS1
PI3K
PIP2
EGFR
PIP3
PTEN
AKT
mTOR
Rictor GßL
PRAS40
mTOR
Raptor GßL
4EBP1
p70S6K
Cell growth
Survival
PI3K Signaling Problems in GBMs
1. EGFR amplified (~40%)
2. EGFR overexpressed (~60%)
3. LOH 10q (~70%)
4. PI3K mutated/amplified (~20%)
Source: Redmond KJ and Kleinberg LR 2009, Principles & Practice of Oncology
GBM – Targeting mTOR (Clinical Trial)
Antitumor Activity of Rapamycin in a Phase I Trial for
Patients with Recurrent PTEN-Deficient Glioblastoma
Tim F. Cloughesy, Koji Yoshimoto, Phioanh Nghiemphu, et. al.
PLoS Medicine, Jan 2008
165 pts in
original cohort
14 PTEN deficient
pts selected
GBM – Targeting mTOR (Clinical Trial)
After one week of treatment with Rapamycin:
1. 7 of 14 (50%) of pts had a substantial reduction in mTOR levels which
coorelated well with tumor proliferation (p = 0.005)
2. Tumor cells harvested from non-responders did respond to rapamycin ex
vivo. Therefore there was nothing intrinsic in the cells themselves that
caused resistance.
3. 7 of 14 (50%) of pts had up-regulation of Akt (loss of negative feedback)
which led to shortened time-to-progression (p = 0.05).
Conclusion: There is value in inhibition of the Akt/PI3K
pathway through mTOR but as the TTP curves show,
combination with a second inhibitor (e.g. PI3K or EGFR)
would be valuable.
PI3K Signaling Pathway
EGF
IRS1
PI3K
PIP2
EGFR
PIP3
PTEN
AKT
mTOR
Rictor GßL
PRAS40
mTOR
Rapa
Analogs
Raptor GßL
4EBP1
p70S6K
Resistance to Rapamycin
via p70S6K:IRS pathway
Cell growth
Survival
Outline
• Case Presentation
• Moving Beyond Local Therapy
• Potential Molecular Targets for GBM and a Case Example
• XL765: a dual PI3K/mTOR inhibitor
• Preclinical Data with XL765
• Model Systems
• In vitro – cytotoxicity and downstream molecular changes
• In vivo – survival and disease burden in mice
• Clinical Data with XL765
• Future Directions
XL765: A Potent PI3K/mTOR Inhibitor
Inhibition of Class I PI3K isoforms and mTOR
ATP competitive and reversible binding
Family
Kinase
IC50 (nM)
PI3K
39
PI3K
113
PI3K
43
Class IB
PI3K
9
Class III
VPS34
9000
DNA-PK
150
mTOR
157
XL765
PI3K
XL765
Class IA
AKT
mTOR
PRAS40
Rictor
XL765
mTOR
Raptor
4EBP1
PI3K
p70S6K
S6
PIKK (PI3K-related)
Highly selective in panel of > 120 kinases
GBM Xenografts: Clinical and Biological Data
Xenograft
Clinical Information
EGFR
PTEN
GBM6
65M, Frontal, OS 13 mo
VIII
wt
GBM8
74F, Frontal, OS 16 mo
wt
null
GBM12
68M, Occiptal, OS 3 mo*
wt
wt
GBM GS-2
57M, Occipital, 2nd resection
wt
null
GBM 39
51M, Frontal, OS 20 mo
VIII
wt
* Pt died of pulmonary embolus (NED at time)
60%
40%
20%
20
.0
16
.0
12
.0
8.
0
4.
0
2.
0
0%
N
16
.0
20
.0
8.
0
12
.0
4.
0
2.
0
1.
0
0.
5
0%
80%
1.
0
40%
20%
100%
GBM 8 (EGFR wt, PTEN null)
IC50 = 4.0 µM
0.
5
80%
60%
120%
eg
at
iv
e
100%
Relative Cell Viability
GBM 6 (EGFR VIII, PTEN wt)
120%
IC50 = 7.5 µM
Ne
ga
tiv
e
Relative Cell Viability
In vitro - XL 765 Effects on Cell Viability
120%
100%
GBM GS-2 (EGFR wt, PTEN null)
IC50 = 4.0 µM
80%
60%
40%
20%
XL 765 (in µM)
XL 765 (in µM)
20
.0
16
.0
12
.0
8.
0
4.
0
2.
0
1.
0
0.
5
eg
at
iv
e
0%
N
20
.0
12
.0
8.
0
4.
0
2.
0
1.
0
0.
5
80%
60%
40%
20%
0%
Relative Cell Viability
XL 765 (in µM)
GBM 12 (EGFR wt, PTEN wt)
120%
IC50 = 2.0 µM
100%
Ne
ga
ti v
e
Relative Cell Viability
XL 765 (in µM)
In vitro - Downstream Changes in the PI3K Pathway
pAktser473
pPRAS40thr246
pS6ser235/236
p4EBP1thr37/46
Actin
XL 765 (µM)
1
2
4
8
16
EGFR wt, PTEN null
Control
Control
EGFR VIII, PTEN wt
GBM12
XL 765 (µM)
1
2
4
8
16
GBM GS-2
EGFR wt, PTEN wt
XL 765 (µM)
1
2
4
8
16
EGFR wt, PTEN null
Control
GBM
Control
GBM 6
XL 765 (µM)
1
2
4
8
16
76
5
+
ar
+
XR
T
XR
T
T
T
* *
20
Al
l
3
ar
XR
XR
Te
m
od
ar
+
+
20
Al
l3
+
od
76
5
Te
m
G
y
uM
8
ar
80
0
76
5
od
uM
100
Te
m
od
ar
Te
m
od
*
*
+
Te
m
8
*
76
5
40
M
60
00
u
80
ar
8
100
8G
y
120
XR
T
76
5
40
Te
m
od
*
60
XR
T
*
tr
ol
0
M
XL
GBM6 (EGFR VIII, PTEN wt)
.0
u
GBM12 (EGFR wt, PTEN wt)
Relative Cell Viability
*
tro
l
Co
n
3
ar
Al
l
Te
m
od
XR
T
20
Relative Cell Viability
+
ar
+
XR
T
* *
Co
n
20
3
ar
T
* *
Al
l
Te
m
od
76
5
od
+
uM
Gy
40
76
54
+
XR
T
Te
m
ar
80
0
8
*
XL
76
5
od
ar
+
XR
40
+
*
76
5
od
XR
T
60
Te
m
80
uM
Te
m
uM
* *
76
5
ar
80
0
60
od
100
G
y
120
uM
2.
5
ro
l
80
8
2.
5
76
5
Co
nt
100
XR
T
76
5
tr
ol
XL
Relative Cell Viability
120
Te
m
XL
Co
n
Relative Cell Viability
In vitro - XL 765 + TMZ + XRT on Cell Viability
120
GBM8 (EGFR wt, PTEN null)
* p < 0.05
80
*
*
0
GBM GS2 (EGFR wt, PTEN null)
*
0
0
PI3K Signaling Pathway
EGF
XL765
IRS1
PI3K
PIP2
EGFR
PIP3
PTEN
XL765
AKT
mTOR
Rictor GßL
PRAS40
mTOR
XL765
Raptor GßL
4EBP1
p70S6K
Cell growth
Survival
In vivo - Methodology
Nude mouse with serially
passaged subcutaneous
xenograft
Xenograft removed and diced
Intracranial injection of xenograft
In vivo - Methodology
Agent Route
Control
Oral gavage w/ Ora-Care Plus
XL 765
(XL)
TMZ
Oral gavage w/ XL dissolved in
sterile saline
Oral gavage w/ TMZ dissolved in
Ora-Care Plus
Erlotinib Oral gavage w/ ERL dissolved in
sterile saline
(ERL)
Single lateral Cs-137 beam through
XRT
head; body shielded w/ Pb
In vivo Methodology
Day
1
Intracranial injection of xenograft
5-20
Mice optically imaged and sorted into
groups of 10
20-30
Mice treated with XL765 bid and/or
TMZ qd by oral gavage
50-60
Repeat treatments
** Mice optically imaged 3/week during first 2 months and
weighed daily during treatment
In vivo – Control vs XRT
Total: Area Flux = 6.91471e+07
Total: Area Flux = 1.97694e+08
GBM 12 (EGFR wt, PTEN wt)
Day 18
3.50E+06
Control
Average Radiance
3.00E+06
ROI 3=3.1335e+07
ROI 2=4.5634e+07
ROI 3=3.4216e+06
ROI 1=1.3218e+07
ROI 1=1.1075e+08
RO
ROI 2=1.9004e+07
ROI 4=5.5907e+06
XRT 2 Gy MWF
Control
2.50E+06
XRT
2.00E+06
1.50E+06
1.00E+06
5.00E+05
p = 0.15
0.00E+00
7
12
Tx #1
17
22
27
Click # SM20081006122331
Series:
Mon, Oct 06, 2008 12:23:45
Experiment: GBM12
Bin:M (8), FOV25, f1, 30s
Label: 388, 86, 393, 100 Series
Click # SM20081006115606
Filter: Open
Comment:
Mon, Oct 06, 2008 11:56:18
Exper
Camera: IVIS 13040, SI620EEV
Analysis Comment:
Bin:M (8), FOV25, f1, 30s
Label
Filter: Open
Comm
Camera: IVIS 13040, SI620EEV
Analy
Days s/p implantation
Total: Area Flux = 2.48469e+09
Total: Area Flux = 6.01055e+08
In vivo – XL765 ± TMZ
Imag
Min = -1.72
Max = 1.66
p/sec/cm
WARNING: Saturated Luminescent Image
GBM 39 (EGFR VIII, PTEN wt)
MGMT hyper-methylated
8.00E+06
ROI 2=2.454e+08
ROI 1=9.0168e+08
Average Radiance
ROI 3=2.9723e+07
ROI 1=4.3235e+06
100
ROI 4=1.47
80
Control
ROI 3=1.0863e+08
XL
60
Control
Total: Area Flux = 1.64832e+07
XL
6.00E+06
Day
46
ROI 4=1.3079e+09
ROI 2=2.7163e+08
40
Total: Area Flux = 6.5042e+07
TMZ
20
XL + TMZ
4.00E+06
ROI 2=3.3204e+07
Color
Min = 5.96
ROI 2=7.3402e
Max = 1.19
R
p = 0.001
ROI 1=3.3586e+06
TMZ
2.00E+06
Click # SM20090320121757
Fri, Mar 20, 2009 12:18:10
Bin:M (8), FOV25, f1, 5s
Filter: Open
Camera: IVIS 13040, SI620EEV
Series:
Click # SM20090320122520
Experiment: GBM39
Fri, Mar 20, 2009 12:25:33
Bin:M (8), FOV25,Label:
f1, 5s
Comment:
Filter: Open
Analysis
Comment:
Camera: IVIS 13040,
SI620EEV
ROI 3=6.2986e+05
ROI 1=2.7215e+05
bkg sub
flat-fielded
cosmic
XL+TMZ
Series:
Experiment: G
Label:
Comment:
Analysis Com
0.00E+00
17 22 27 32 37 42 47 52 57 62
Tx #1
Tx #2
p = 0.0002
Days s/p implantation
p = 0.063
Total: Area Flux = 2.48469e+09
In vivo – XL ± ERL
GBM 39 (EGFR VIII, PTEN wt)
MGMT hyper-methylated
Total: Area Flux = 6.01055e+08
Imag
Min = -1.72
Max = 1.66
p/sec/cm
WARNING: Saturated Luminescent Image
Day
46
ROI 4=1.3079e+09
ROI 2=2.7163e+08
ROI 2=2.454e+08
ROI 1=9.0168e+08
ROI 3=2.9723e+07
ROI 1=4.3235e+06
80
Control
8.00E+06
100
ROI 4=1.47
ROI 3=1.0863e+08
XL
60
Control
6.00E+06
40
Total: Area Flux = 4.52977e+08
Total: Area Flux = 6.06837e+07
Image
Min = -1.6611e+09
Max = 1.3777e+08
p/sec/cm^2/sr
ERL
XL + ERL
10
4.00E+06
ROI 4=2.7683e+06
Im
Min = -3.
Max
20= 5.
p/sec/
50
Color
Min = 5.96
Max = 1.19
ROI 2=6.8719e+06
ROI 4=2.6526e+07
p = 0.001
ROI 2=2.9054e+06
40
8
ROI 1=5.8971e+05
3=4.6397e+07
ROIROI
1=3.3164e+08
ROI 3=7.7691e+07
ROI 5=4.057e+06
ERL
2.00E+06
Series:
Click # SM20090320122520
Experiment: GBM39
Fri, Mar 20, 2009 12:25:33
Bin:M (8), FOV25,Label:
f1, 5s
Comment:
Filter: Open
Analysis
Comment:
Camera: IVIS 13040,
SI620EEV
0.00E+00
6
Click # SM20090320121757
Fri, Mar 20, 2009 12:18:10
Bin:M (8), FOV25, f1, 5s
Filter: Open
Camera: IVIS 13040, SI620EEV
ROI 5=1.4211e+07
bkg sub 30
XL+ERL
flat-fielded
6
x10
Average Radiance
XL
4
cosmic
Series:
20
Experiment: G
Label:
Comment:
10
Analysis Com
2
17 22 27 32 37 42 47 52 57 62
Tx #1
Colo
Min = 2.
Max = 5.
Tx #2
p < 0.0001
Days s/p implantation
Click # SM20090320124719
Fri, Mar 20, 2009 12:47:32
Color Bar
Min = 1.1835e+05
Max = 1.0558e+07
p = 0.97
Series:
bkg sub
Experiment: GBM39
flat-fielded
bkg sub
flat-fielde
cosmic
In vivo Survival – Control vs. XRT
100
80
Group
Median
Survival
60
Control
25
XRT
32
40
20
Control
XRT
0
20
25
30
Time (Days)
35
40
Group
p
HR
XRT
vs
Control
0.001
13.3 (2.7-65.3)
In vivo Survival – XL ± TMZ
100
80
60
40
Control
TMZ
XL
XL+TMZ
20
0
40
50
60
70
Time (Days)
80
90
Group
Median
Survival
Control
54.5
XL
67.5
TMZ
82.5
XL+TMZ
N/R
Groups
p
HR
XL
vs
Control
0.06
2.8 (1.0-7.8)
TMZ
vs
Control
0.0001
12.4 (3.5-43.9)
XL+TMZ
vs
TMZ
0.08
4.6 (1.1-19.4)
In vivo Survival – XL ± ERL
Group
Median
Survival
Control
54.5
XL
67.5
ERL
77
XL+ERL
78.5
Group
p
HR
Control
Erl
XL
XL+Erl
XL
vs
Control
0.06
2.8 (1.0-7.8)
0.0002
11.0 (3.1-38.9)
50
ERL
vs
Control
XL+ERL
vs
ERL
0.44
0.6 (0.2-1.6)
100
80
60
40
20
0
40
60
70
Time (Days)
80
90
Preclinical Data Summary
In Vitro
• XL765 results in concentration-dependent cytotoxicity alone and is
supra-additive when combined with conventional agents.
•In addition, the PI3K/mTOR pathway is specifically inhibited as
demonstrated by Western Blot.
In Vivo
•XL765 given as monotherapy in mice with intracranial GBM
xenografts resulted in improved survival.
•Combination of XL765 with TMZ resulted in a trend for decreased
tumor growth and survival.
•Combination of XL765 with Erlotinib did not demonstrate any additive
effects in the model we tested.
Outline
• Case Presentation
• Moving Beyond Local Therapy
• Potential Molecular Targets for GBM and a Case Example
• XL765 – a dual PI3K/mTOR inhibitor
• Preclinical Data with XL765
• Model Systems
• In vitro – cytotoxicity and downstream molecular changes
• In vivo – survival and disease burden in mice
• Clinical Data with XL765
• Future Directions
Clinical Data in Humans
Phase I dose-escalation study
Presented at the joint EORTC-NCI-AACR conference in Geneva 10/08
Vall d’Hebron Hospital (Barcelona, Spain), Karmanos Cancer Center
(Detroit, MI), START Medical Oncology (San Antonio, TX)
1. 29 patients w/ metastatic or unresectable solid tumor for which no
further effective measures exist
2. No chemotherapy, radiotherapy, or biological agents within 30 days
3. Primary objective: saftey and tolerability
4. Secondary objectives: PK/PD/preliminary efficacy
Clinical Data – Doses & Status
Clinical Data - Toxicity
Clinical Data - Response
Note decrease in phospoAkt and phospho-4EBP1 in
patient hair follicles after
treatment
Clinical Data - Conclusions
1. XL765 was generally well-tolerated w/ GI complaints being most
common; no MTD reached
2. XL765 showed pharmacodynamic response in hair follicles, skin cells,
and in cases of tumor biopsy.
Phase I dose-escalation study of XL765 + TMZ in adults w/
malignant gliomas
University of California Los Angeles and Memorial Sloan-Kettering
Cancer Center
•
Patients need to be on Temozlomide already at a dose of 200
mg/m2/day on days 1-5 of 28 day cycle
•
Patients must have completed four cycles w/o unacceptable toxicity
•
NO progression on temozlomide
•
Currently accruing
Future Directions
1. IHC examination of treated in vivo
xenografts
2. In vivo model using XL 765 + Erlotinib
3. Clinical Trial @ UCSF
• Phase I
• Fixed dose XL 765 + escalating Erlotinib
doses
Haas-Kogan Lab
Haas-Kogan Lab (the reality)
Acknowledgements
Daphne Haas-Kogan
Michael Prados
Theo Sottero
Xiaodong Yang
Sabine Mueller
C. David James
Mei-Yin Polley
Tomoko Ozawa
Raquel Santos
Dana Aftab