The Future of Glioblastoma Therapy: Multi-modality with Multiple Targets Gautam Prasad Resident...

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.






• Model Systems





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


Gross Tumor

“Normal” Brain

Cultured Glia

Tumor cells?

Yes No No

GFAP + + +Growth

Rate19%/day 36%/day ~5%/day

Motility 3.92 4.18

Karyotype Neoplastic Neoplastic Normal


ConclusionsObviously 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?




• XL765: a dual PI3K/mTOR inhibitor


• Model Systems





GBM – No Shortage of Potential Targets

Source: Argyriou AA and Kalofonos HP 2009, Mol Med

PIP2

PI3K

PIP3

EGFR

PTEN

IRS1

SurvivalRictor

mTOR

GßL

4EBP1

PRAS40

Raptor

p70S6K

Cell growth

mTOR

AKT

GßL

PI3K Signaling PathwayEGFEGF

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.

PIP2

PI3K

PIP3

EGFR

PTEN

IRS1

SurvivalRictor

mTOR

GßL

4EBP1

PRAS40

Raptor

p70S6K

Cell growth

mTOR

AKT

GßL

PI3K Signaling Pathway

EGFEGF

Resistance to Rapamycinvia p70S6K:IRS pathway

Rapa Analogs




• XL765: a dual PI3K/mTOR inhibitor


• Model Systems





Family Kinase IC50 (nM)

PI3K

Class IA

PI3K 39

PI3K 113

PI3K 43

Class IB PI3K 9

Class III VPS34 9000

PIKK (PI3K-related)DNA-PK 150

mTOR 157

XL765: A Potent PI3K/mTOR Inhibitor

Inhibition of Class I PI3K isoforms and mTOR

ATP competitive and reversible binding

Highly selective in panel of > 120 kinases

p70S6K

4EBP1

PRAS40

mTOR

AKTmTOR

PI3K

Raptor

Rictor

S6

XL765

XL765

XL765

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)

0%

20%

40%

60%

80%

100%

120%

XL 765 (in µM)

Rel

ativ

e C

ell V

iabi

lity

0%

20%

40%

60%

80%

100%

120%

Negat

ive

0.5

1.0

2.0

4.0

8.0

12.0

16.0

20.0

XL 765 (in µM)

Rel

ativ

e C

ell V

iab

ility

0%20%40%60%80%

100%120%

XL 765 (in µM)

Rel

ativ

e C

ell V

iabi

lity

0%

20%

40%

60%

80%

100%

120%

Negat

ive

0.5

1.0

2.0

4.0

8.0

12.0

16.0

20.0

XL 765 (in µM)

Rel

ativ

e C

ell V

iab

ility

GBM 6 (EGFR VIII, PTEN wt) IC50 = 7.5 µM

In vitro - XL 765 Effects on Cell Viability

GBM 8 (EGFR wt, PTEN null) IC50 = 4.0 µM

GBM 12 (EGFR wt, PTEN wt) IC50 = 2.0 µM

GBM GS-2 (EGFR wt, PTEN null) IC50 = 4.0 µM

Co

ntr

ol

XL 765 (µM)

1 2 4 8 16

pAktser473

pPRAS40thr246

pS6ser235/236

p4EBP1thr37/46

Actin

GBM 6 GBM GBM12 GBM GS-2

EGFR VIII, PTEN wt EGFR wt, PTEN null EGFR wt, PTEN wt EGFR wt, PTEN null

Co

ntr

ol

XL 765 (µM)

1 2 4 8 16 Co

ntr

ol

XL 765 (µM)

1 2 4 8 16 Co

ntr

ol

XL 765 (µM)

1 2 4 8 16

In vitro - Downstream Changes in the PI3K Pathway

In vitro - XL 765 + TMZ + XRT on Cell Viability

GBM12 (EGFR wt, PTEN wt)

GBM6 (EGFR VIII, PTEN wt) GBM8 (EGFR wt, PTEN null)

GBM GS2 (EGFR wt, PTEN null)

0

20

40

60

80

100

120

Rel

ativ

e C

ell V

iabi

lity

* **

* **

0

20

40

60

80

100

120

Rel

ativ

e C

ell V

iabi

lity

* *

*

0

20

40

60

80

100

120

Rel

ativ

e C

ell V

iabi

lity

**

* * *

* p < 0.05

0

20

40

60

80

100

120

Rela

tive

Cell

Viab

ility

**

* * *

PIP2

PI3K

PIP3

EGFR

PTEN

IRS1

SurvivalRictor

mTOR

GßL

4EBP1

PRAS40

Raptor

p70S6K

Cell growth

mTOR

AKT

GßL

PI3K Signaling Pathway

EGFEGF

XL765

XL765

XL765

Nude mouse with serially passaged subcutaneous xenograft

Xenograft removed and diced

Intracranial injection of xenograft

In vivo - Methodology

In vivo - Methodology

Agent Route

Control Oral gavage w/ Ora-Care Plus

XL 765

(XL)

Oral gavage w/ XL dissolved in sterile saline

TMZ Oral gavage w/ TMZ dissolved in Ora-Care Plus

Erlotinib

(ERL)

Oral gavage w/ ERL dissolved in sterile saline

XRT Single lateral Cs-137 beam through head; body shielded w/ Pb

Intracranial injection of xenograft

In vivo Methodology

Day

1

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

Days s/p implantation

Ave

rag

e R

adia

nce

Click # SM20081006115606Mon, Oct 06, 2008 11:56:18Bin:M (8), FOV25, f1, 30sFilter: OpenCamera: IVIS 13040, SI620EEV

Series: Experiment: GBM12Label: 396, 98, 80, 79Comment: Analysis Comment:

20

15

10

5

x10

6

ImageMin = -4.6141e+08Max = 4.3052e+07

p/sec/cm^2/sr

Color BarMin = 1.0372e+05Max = 2.0743e+07

bkg subflat-fieldedcosmic

ROI 1=1.1075e+08ROI 2=4.5634e+07ROI 3=3.4216e+06ROI 4=3.7889e+07

Total: Area Flux = 1.97694e+08

Day 18

Control XRT

Click # SM20081006122331Mon, Oct 06, 2008 12:23:45Bin:M (8), FOV25, f1, 30sFilter: OpenCamera: IVIS 13040, SI620EEV

Series: Experiment: GBM12Label: 388, 86, 393, 100Comment: Analysis Comment:

5

4

3

2

1

x10

6

ImageMin = -3.076e+08Max = 1.4487e+07

p/sec/cm^2/sr

Color BarMin = 29091

Max = 5.8182e+06


ROI 1=1.3218e+07ROI 2=1.9004e+07ROI 3=3.1335e+07

ROI 4=5.5907e+06


Tx #1

GBM 12 (EGFR wt, PTEN wt)

p = 0.15

Tx #1 Tx #2


Ave

rag

e R

adia

nce

In vivo – XL765 ± TMZGBM 39 (EGFR VIII, PTEN wt)

MGMT hyper-methylated

Day 46

Click # SM20090320121757Fri, Mar 20, 2009 12:18:10Bin:M (8), FOV25, f1, 5sFilter: OpenCamera: IVIS 13040, SI620EEV

Series: Experiment: GBM39Label: Comment: Analysis Comment:

100

80

60

40

20

x10

6

ImageMin = -1.7221e+08Max = 1.6611e+09

p/sec/cm^2/sr



WARNING: Saturated Luminescent Image

ROI 1=9.0168e+08

ROI 2=2.454e+08

ROI 3=2.9723e+07

ROI 4=1.3079e+09




40

30

20

10

x10

6

ImageMin = -1.2919e+09Max = 4.4818e+07

p/sec/cm^2/sr



ROI 1=4.3235e+06

ROI 2=2.7163e+08

ROI 3=1.0863e+08

ROI 4=1.4715e+08

ROI 5=6.9324e+07


Control XL

p = 0.001



20

15

10

5

x10

6

ImageMin = -3.4741e+07Max = 9.2281e+08

p/sec/cm^2/sr



ROI 1=3.3586e+06

ROI 2=3.3204e+07

ROI 3=6.2986e+05

ROI 4=2.747e+07

ROI 5=3.7987e+05




12

10

8

6

4

2

x10

6

ImageMin = -3.3431e+07Max = 1.1074e+09

p/sec/cm^2/sr



ROI 1=2.7215e+05

ROI 2=7.3402e+06

ROI 3=2.0087e+06

ROI 4=3.889e+06

ROI 5=2.9732e+06


TMZ XL+TMZ

p = 0.0002 p = 0.063

In vivo – XL ± ERL

Tx #1 Tx #2


Ave

rag

e R

adia

nce



10

8

6

4

2

x10

6

ImageMin = -1.6611e+09Max = 1.3777e+08

p/sec/cm^2/sr



ROI 1=5.8971e+05

ROI 2=6.8719e+06

ROI 3=4.6397e+07

ROI 4=2.7683e+06

ROI 5=4.057e+06




50

40

30

20

10

x10

6

ImageMin = -3.6912e+08Max = 5.7511e+07

p/sec/cm^2/sr



ROI 1=3.3164e+08

ROI 2=2.9054e+06

ROI 3=7.7691e+07

ROI 4=2.6526e+07

ROI 5=1.4211e+07


GBM 39 (EGFR VIII, PTEN wt)

MGMT hyper-methylated

Day 46



100

80

60

40

20

x10

6

ImageMin = -1.7221e+08Max = 1.6611e+09

p/sec/cm^2/sr



WARNING: Saturated Luminescent Image

ROI 1=9.0168e+08

ROI 2=2.454e+08

ROI 3=2.9723e+07

ROI 4=1.3079e+09




40

30

20

10

x10

6

ImageMin = -1.2919e+09Max = 4.4818e+07

p/sec/cm^2/sr



ROI 1=4.3235e+06

ROI 2=2.7163e+08

ROI 3=1.0863e+08

ROI 4=1.4715e+08

ROI 5=6.9324e+07


Control XL

p = 0.001

ERL XL+ERL

p < 0.0001 p = 0.97

Group Median Survival

Control 25

XRT 32

In vivo Survival – Control vs. XRT

Group p HR

XRT

vs

Control

0.001 13.3 (2.7-65.3)

20 25 30 35 40

100

80

60

40

20

0

Time (Days)

Su

rviv

al

pro

ba

bil

ity

(%

)

ControlXRT

40 50 60 70 80 90

100

80

60

40

20

0

Time (Days)

Sur

viva

l pro

babi

lity

(%)

ControlTMZXLXL+TMZ


Control 54.5

XL 67.5

TMZ 82.5

XL+TMZ N/R

In vivo Survival – XL ± TMZ

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)

40 50 60 70 80 90

100

80

60

40

20

0

Time (Days)

Sur

viva

l pro

babi

lity

(%)

ControlErlXLXL+Erl

In vivo Survival – XL ± ERL


Control 54.5

XL 67.5

ERL 77

XL+ERL 78.5

Group p HR

XL

vs

Control

0.06 2.8 (1.0-7.8)

ERL

vs

Control

0.0002 11.0 (3.1-38.9)

XL+ERL

vs

ERL

0.44 0.6 (0.2-1.6)

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.






• Model Systems





Clinical Data in HumansPhase I dose-escalation studyPresented 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 phospo-Akt and phospho-4EBP1 in patient hair follicles after treatment

Clinical Data - Conclusions1. 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 Directions1. 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)

AcknowledgementsDaphne Haas-Kogan Michael Prados

Theo Sottero

Xiaodong Yang

Sabine Mueller

C. David James Mei-Yin Polley

Tomoko Ozawa

Raquel Santos

Dana Aftab

The Future of Glioblastoma Therapy: Multi-modality with Multiple Targets Gautam Prasad Resident...

Documents

Transcript of The Future of Glioblastoma Therapy: Multi-modality with Multiple Targets Gautam Prasad Resident...