The PI3-Kinase Pathway Thomas W. Grunt Signaling Networks Program Division of Oncology Department of Medicine I Comprehensive Cancer Center Medical University Vienna & Ludwig Boltzmann Cluster Oncology

The PI3K/AKT/mTOR Pathway (incl. neg. Feedback Loops)

Manning, Cantley. Cell. 2007;129:1261-74

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The PI3K/AKT/mTOR Pathway (incl. neg. Feedback Loops)

Carnero. Curr Pharm Design. 2010;16:34-44

Growth Factors IGF-1, EGF, TGFα, VEGF, etc

Growth Factors and the mTOR Pathway

PI3-K

•

– –

PTEN Oxygen, energy, and nutrients

Ras/Raf

Akt/PKB

mTOR

Abl ER TSC2 TSC1

Ras/Raf pathway kinases

mTOR

S6K1 4E-BP1 S6

Protein Production Cell Growth and Proliferation

4

elF-4E

Intracellular protein Central controller of cell growth and proliferation

•

mTOR signaling is often deregulated in cancer

•

Downstream inhibition of mTOR has potential for

–

Antiproliferative effects on tumor cells

– –

Angiogenesis inhibition Enhancement of the effects of chemotherapy

Angiogenesis

This slide shows only a few of the many kinases involved in these signaling pathways

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Signaling Through mTOR

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The PI3K family and generation of phosphatidylinositol-3,4,5-trisphosphate

Liu et al. Nature Rev Drug Discovery. 2009;8:627-44

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Feedback Regulation of Akt by Rictor-mTOR (mTORC2)

Cheng et al. Oncogene. 2005;24:7482-92

The PI3K/AKT/mTOR Pathway and Cancer ¾ PI3K–Akt pathway inhibitors are in clinical development for cancer (e.g. dual PI3K–mTOR inhibitors, PI3K inhibitors, Akt inhibitors and mTOR inhibitors). ¾ PI3K–Akt pathway activated in many cancers (e.g. by receptor tyrosine kinases, mutation, amplification). ¾ Most effective drug used to inhibit this pathway depends on mechanism of PI3K–Akt activation. ¾ PI3K–Akt pathway inhibitors might have single-agent activity in breast cancers with ERBB2 amplifications or PIK3CA mutations. These drugs might also be effective in overcoming acquired resistance to therapies that target receptor tyrosine kinases. ¾ PI3K–Akt pathway inhibitors probably most effectivein combination with other targeted drugs, such as MEK inhibitors.

Engelman. Nature Rev. Cancer. 2009;9:550-62

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The mTOR Pathway and Cancer • mTOR is a central controller of cell growth and proliferation in normal cells • mTOR pathway deregulation causes loss of growth control in cancer • mTOR controls responses essential to cancer cells – mTOR regulates cell growth and division – mTOR influences responses to DNA damage – mTOR regulates angiogenesis – mTOR responds to antiestrogens – mTOR controls translation

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Somatic genetic mutations activating the PI3K-Akt pathway

Engelman. Nature Rev. Cancer. 2009;9:550-62

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Incidence of genetic alterations in the PI3K pathway in cancer

Liu et al. Nature Rev Drug Discovery. 2009;8:627-44

The mTOR Pathway Is Deregulated in Many Cancers Brain Thyroid

Oral SCC Breast

Lung Blood Kidney Ovary Prostate

Pancreas Colon Uterus

Skin Sarcoma

12

6

mTOR-Linked Pathway Deregulations in Selected Cancers EGFR, 32%–60%1 p-Akt, 23%–50%18 Ras, 30%12 PTEN, 24%22 HER2, 5%30 PI3-K, 4%13 TGFα/TGFβ1, 60%–100%35 VHL, 30%–50%36,37 IGF-1/IGF-IR, 39%-69%9 p-Akt, 38%38 PTEN, 31%39 TSC1/TSC240

Lung

Breast

NET

Kidney Colon

p-Akt, 42%16 PTEN, 15%–41%25 HER2, 30%–36%26,27 PI3-K, 18%–26%27,28 EGFR, 6%29 TSC1/TSC231,32 IGF-1/IGF-1R33 VHL34 Ras, 50%12 p-Akt, 46%15 PTEN, 35%41 PI3-K, 20%–32%13,41 EGFR, 8%42 HER2, 3%42

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The PI3K/AKT/mTOR Pathway and Cancer: Main Targets for Therapeutic Intervention

van der Heijden, Bernards. Clin Cancer Res. 2010;16:3094-9

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Targeting the PI3K/AKT/mTOR Pathway in Cancer a | Inhibitors that target key nodes in the phosphoinositide 3-kinase (PI3K) signalling pathway, including receptor tyrosine kinases (RTKs), PI3K, AKT and mammalian target of rapamycin (mTOR), have reached clinical trials. Dual inhibitors that target both PI3K and RTK or PI3K and mTOR may provide more potent therapeutic effects in suppressing the PI3K signalling. Combinations of PI3K and RAF– mitogen-activated protein kinase (MAPK) inhibitors may achieve more effective clinical results. b | Inhibitors in clinical development that target the PI3K or related pathways are shown. EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HER2, human epidermal growth factor receptor 2 (also known as ERBB2); MEK, mitogen-activated protein kinase kinase; VEGFR, vascular endothelial growth factor receptor. *Bevacizumab targets VEGFA instead of VEGFR directly. ‡Both AZD8055 and OSI-027 are ATP-competitive mTOR inhibitors that target the mTOR complexes mTORC1 and mTORC2.

Liu et al. Nature Rev Drug Discovery. 2009;8:627-44

Summary of Drugs Targeting the PI3K Pathway in Clinical Trials for Cancer Treatment

Liu et al. Nature Rev Drug Discovery. 2009;8:627-44

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mTOR Inhibition May Enhance the Antitumor Effects of Other Therapies Radiation

Chemotherapy

mTOR Inhibition ErbB Inhibitors

Antiestrogens

Antiangiogenics

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mTOR Inhibition May Enhance the Antitumor Effects of Other Therapies (cont) Agent

Rationale

ErbB inhibitors

Defects in the mTOR signaling pathway may counter the effects of ErbB inhibitors on cell growth and proliferation. Combined treatment has been beneficial in preclinical studies1

Cytotoxic chemotherapy

Cytotoxic drugs such as the platinum derivatives, taxanes, anthracyclines, and gemcitabine have shown improved antitumor effects in preclinical models when used in combination with mTOR inhibitors2-4

Antiangiogenic agents

mTOR inhibition affects angiogenesis through mechanisms that enhance and complement those of anti-VEGF/anti-VEGFR signaling inhibitors5

Antiestrogens

Defects in the mTOR signaling pathway may render estrogendependent tumor cells resistant to antiestrogens and aromatase inhibitors. Combinations effective preclinically6-8

Radiation

In preclinical studies, mTOR inhibition enhances cell killing induced by radiation, possibly by interfering with repair of damage to DNA9

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mTOR Inhibition Blocks Cell Cycle Progression at the G1–S Restriction Point

M G2 mTOR

G1 S

Restriction point

19 From Israels and Israels. Oncologist. 2000;5:510-513, with permission.

mTOR Inhibition Enhances the Activity of Many Chemotherapeutic Agents • Preclinical evidence suggests that mTOR inhibition could also enhance the activity of

– Paclitaxel, carboplatin, vinorelbine, doxorubicin, or gemcitabine in breast cancer1

– Immunomodulatory agents in multiple myeloma2 – Gemcitabine in pancreatic cancer3 – Doxorubicin in PTEN-negative prostate cancer4

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1. Mondesire et al. Clin Cancer Res. 2004;10:7031-7042. 2. Raje et al. Blood. 2004;104:4188-4193. 3. Bruns et al. Clin Cancer Res. 2004;10:2109-2119. 4. Grünwald et al. Cancer Res. 2002;62:6141-6145.

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mTOR Inhibition May Enhance the Cytotoxicity of DNA-Damaging Agents • DNA damage caused by agents Cisplatin

such as cisplatin activates p53

DNA

• p53 triggers DNA repair, which

Cross-linked DNA

Cycle Arrest DNA Repair Cell Death Cell Death Cell Death

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allows the cell to survive, or, failing that, p53 initiates cell death • mTOR regulates production of p21, a cell cycle inhibitor that allows DNA repair • mTOR inhibition blocks p21 translation, forcing cell death even when the DNA damage is otherwise nonlethal • mTOR inhibition can enhance the activity of certain drugs such as cisplatin and other platinum derivatives

Survival

Beuvink et al. Cell. 2005;120:747-759.

mTOR Inhibition Decreases Angiogenesis • mTOR regulates HIF-1α and HIF-2α expression • HIF-1 and HIF-2 are transcription factors for hypoxic stress-related genes • HIF-1α/2α are normally degraded by VHL protein • HIF-1 and HIF-2 condition the tumor to adapt to growth under hypoxic conditions and promote angiogenesis and metastasis

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HIF = hypoxia-inducible factor; VHL = von Hippel-Lindau protein.

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mTOR Tumor Cell Growth and Angiogenesis Growth factors

Endothelial cell

Smooth muscle cell (pericyte)

Cancer cell PI3-K PI3-K

PTEN

PI3-K

Akt/ PKB TSC2

TSC1

Akt/ PKB

Akt/ PKB

mTOR

mTOR mTOR

Protein production

HIF-1α HIF-2α

VHL

Cell growth and proliferation

Cell growth and proliferation

Angiogenic growth factors Cell growth and proliferation

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Angiogenesis Inhibitors and mTOR Inhibitors May Act Synergistically Primary orthotopic (ear) B16 melanoma

Fractional Tumor Volume (V/Vo, mean ± SEM)

8

Vehicle, n = 6 1 mg/kg mTOR inhibitor, po q24h, n = 6 100 mg/kg VEGFR inhibitor, po q24h, n = 6 1 mg/kg mTOR inhibitor + VEGFR inhibitor, n = 6 5 mg/kg mTOR inhibitor + VEGFR inhibitor, n = 6

6

4

* *

2

*P < .05 vs vehicle controls and single agents.

0 7

14

21

Days Post Tumor Cell Injection

*O’Reilly et al. Proc Am Assoc Cancer Res. 2005;46:715. Abstract 3038.

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mTOR Inhibition Enhances Effects of Antiestrogenic Agents • Akt activation predicts a worse outcome for breast cancer patients treated with endocrine therapy1 • Activated Akt mediates resistance to antiestrogen therapy related to HER2 overexpression2,3 • mTOR inhibition restores responses to tamoxifen in breast cancer cells with high levels of Akt activity4 • Synergistic in vitro and in vivo effects have been seen with combined antiestrogen therapy and mTOR inhibition5

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1. Perez-Tenorio et al. Br J Cancer. 2002;86:540-545. 2. Campbell et al. J Biol Chem. 2001;276:9817-9824.. 3. Kurokawa and Arteaga. Clin Cancer Res. 2003;9(suppl):511s-515s. 4. de Graffenried et al. Clin Cancer Res. 2004;10:8059-8067. 5. Zhang et al. Proc Am Assoc Cancer Res. 2003;44(2nd ed):739. Abstract 3715.

Dual mTOR and Aromatase Inhibition Induces Apoptosis in Breast Cancer Models

Apoptotic Cells, %

25 20 15 10 5 0 0

2

0 100 nM AI

2

0

2

500 nM AI

mTOR Inhibitor (nM)

The combination of an mTOR inhibitor and an aromatase inhibitor (AI) increases induction of apoptosis, compared with 2 nM of the mTOR inhibitor alone or 100 nM or 500 nM of the AI alone

P < .05 (Friedman test).

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Modified from Boulay et al. Clin Cancer Res. 2005;11:5319-5328, with permission.

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Summary Rationale for Targeting mTOR • mTOR, an intracellular protein, acts as a central regulator of multiple signaling pathways that may mediate abnormal growth and proliferation • mTOR provides a stable genetic target • mTOR activity is influenced by nutrient and energy levels and by signaling through pathways often deregulated or overexpressed in cancer, such as – Cell surface receptors, such as EGFR/HER, IGFR, VEGFR – The PI3-K/Akt survival pathway, in which the PTEN tumor suppressor gene is often lost – Ras-Raf – Intracellular receptors, such as ER and PR 27

Summary Rationale for Targeting mTOR (cont) • Aberrant signaling through upstream pathways can activate mTOR inappropriately, promoting – Abnormal cell growth, proliferation, and angiogenesis – Survival of cancer cells in the nutrient- and oxygendepleted tumor environment • Targeting deregulated pathways has been a successful clinical strategy in cancer • Combination therapy targeting mTOR and deregulated pathways may provide enhanced anticancer activity

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Phospho-AKT correlates with ErbB drug resistance Breast cancer

Pelitinib sensitive SKBR3

µM Pelitinib

resistant T47D

0 0.1 1 2 4 8

0 0.1 1 2 4 8

pAKT(Ser473) pAKT(Thr308) AKT pERK1,2 ERK1,2 Actin

• Resistant cells reveal drugrefractory PI3K signaling • MAPK signaling is uncoupled from resistance

Canertinib sensitive SKBR3 µM Canertinib

resistant T47D

0 0.1 1 2 4 6 10 0 0.1 1 2 4 6 10

pAKT(Ser473) pAKT(Thr308) AKT pERK1,2 ERK1,2 Actin

thway MAPK pa

Ovarian cancer

PI3K /AKT path wa y

Pelitinib

sensitive HOC7 µM Pelitinib 0 0.1 1 2 4 8 pAKT(Ser473) pAKT(Thr308) AKT pERK1,2 ERK1,2 Actin

intermediate SKOV3

resistant CAOV3

0 0.1 1 2 4 8

0 0.1 1 2 4 8

sensitive

resistant Brünner-Kubath et al., BCRT, Nov 2010

Constitutively active AKT induces ErbB drug resistance ErbB drug sensitive SKBR3 breast cancer cells Signaling pathways MAPK pathway

Cell growth

MEK1,2 Pelitinib IC50 (µM)

pERK1,2 ERK1,2

PI3K/AKT pathway pAKT(Ser473) AKT pGSK3β

mTOR

Controls

pS6

ca ca MEK AKT

Transfections

ErbB drug sensitive ErbB drug resistant

α,β-Tubulin Pelitinib

***

SK BR SK 3 BR SK 3L BR TX 3p cD N A3 SK BR 3G SK FP BR 3M E SK K1 BR 3A KT 1

pmTOR

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

-

+

-

Controls

+

-

+

-

+

caMEK caAKT

thway MAPK pa

Transfections PI3K /AKT path w ay

Brünner-Kubath et al., BCRT, Nov 2010

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Dominant negative AKT induces ErbB drug sensitivity ErbB drug resistant T47D breast cancer cells PI3K/AKT signaling pathway

Cell growth

pAKT(Ser473) Pelitinib IC50 (µM)

AKT pS6 α,β-Tubulin Pelitinib

-

-

+

+

Controls

-

+

dnAKT

Transfections ErbB drug sensitive ErbB drug resistant

16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0

*** Controls

dnAKT

Transfections thway MAPK pa

PI3K /AKT path w ay

Brünner-Kubath et al., BCRT, Nov 2010

PI3K inhibitors overcome ErbB drug resistance - I 120 110 100 90 80 70 60 50 40 30 20 10 0

Cell Number (% of Control)

PI3K inhibitors sensitize the cells against ErbB drugs

Cell Number (% of Control)

ErbB drug resistant T47D breast cancer cells

Pelitinib Pelitinib+BEZ235 Pelitinib+Rapamycin Pelitinib+Akti-1/2

0

1

2

3

4

5

6

7

120 110 100 90 80 70 60 50 40 30 20 10 0

8

Canertinib Canertinib+BEZ235 Canertinib+Rapamycin Canertinib+Akti-1/2

0

1

2

thway MAPK pa

120 110 100 90 80 70 60 50 40 30 20 10 0

Pelitinib Pelitinib+AZD6244 Pelitinib+UO126

0

1

2

3

4

5

Pelitinib (µM)

3

4

5

6

7

8

6

7

8

Canertinib (µM) Cell Number (% of Control)

MAPK inhibitors do NOT sensitize the cells against ErbB drugs

Cell Number (% of Control)

Pelitinib (µM)

6

7

8

120 110 100 90 80 70 60 50 40 30 20 10 0

Canertinib Canertinib+AZD6244 Canertinib+UO126

0

1

2

3

4

5

Canertinib (µM)

PI3K /AKT path w ay

Brünner-Kubath et al., BCRT, Nov 2010

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PI3K inhibitors overcome ErbB drug resistance - II ErbB drug resistant T47D breast cancer cells 0,07

IC50

0,06

NVP-BEZ235 (µM)

The PI3K/mTOR inhibitor NVP-BEZ235 synergizes with ErbB drugs

NVP-BEZ235 (µM)

0,07

0,05 0,04 0,03 0,02 0,01 0,00

IC50

0,06 0,05 0,04 0,03 0,02 0,01 0,00

0

2

4

6

8

10

12

14

16

0

2

Pelitinib (µM)

IC50

8

10

12

14

16

IC50

60,0

U0126 (µM)

50 40 30 20 10

thway MAPK pa

6

Canertinib (µM)

60

U0126 (µM)

The MEK inhibitor U0126 does not synergize with ErbB drugs

4

50,0 40,0 30,0 20,0 10,0

0

0,0 0

2

4

6

8

10

12

14

16

Pelitinib (µM)

0

2

4

6

8

10

12

Canertinib (µM)

PI3K /AKT path w ay

Brünner-Kubath et al., BCRT, Nov 2010

Signaling and metabolism: Interaction between the ErbB/PI3K/AKT/mTOR pathway and fatty acid synthase • ErbB membrane receptor tyrosine kinases are upstream activators of PI3K/AKT/mTOR • Fatty acid synthase (FASN) is key enzyme in fatty acid production – Overexpressed in many tumors – FASN inhibitors block tumor growth

• ErbB- and FASN-pathways cross-talk - ErbB drugs inhibit FASN and vice versa - FASN drugs silence PI3K/AKT/mTOR

• Combination of both drug types yield strong antitumor effects

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FASN inhibitor downregulates PI3K signaling and cooperates with ErbB blockers C75

OVCAR-3 +

SKOV-3 +

HEY -

+

p-AKT

FASN inhibitor C75 • Dephosphorylates/downregulates AKT and S6 • Stimulates protein ubiquitination/proteasomal degradation • Has less effect on ERK signaling

AKT p-S6 S6 p-ERK1/2 ERK1/2

Ubiquitin

Actin

Cell Number (% of Control)

40 µM C75 alone 10µM ErbB inhibitor alone C75 and ErbB inhibitor together

3 days

40

SKOV3 5 days

Combination of FASN inhibitor C75 with • ErbB drugs (e.g. pelitinib, canertinib) reveal strongly improved antitumor effects

35 30 25 20 15 10 5

1 2

1 2

1 2

1 2

C 75 an C7 +C er 5 an tini er b tin ib C

P C 75 eli 75 +P tin el ib iti ni b C

75 Can C7 +C er 5 an tin er ib tin ib C

C

C 75 Pel 75 +P itin el ib iti ni b

0

Grunt et al. BBRC. 2009;385:454-9 Tomek et al. Submitted, 2010

Conclusions • Drug-refractory phosphorylation of AKT is a biomarker for ErbB drug resistance of breast and ovarian cancer cells • PI3K/AKT is more important than MAPK for breast and ovarian cancer growth and survival • Silencing of PI3K/AKT can overcome ErbB drug resistance in breast and ovarian cancer

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