mTOR: a new pathway to target in oncology

1. mTOR: a new pathwayto target in oncology MadlainaBreuleux,PhD NovartisPhrmaAG Basel,Switzerland

2. The mTOR pathway and cancer mTOR (mammalian “Target Of Rapamycin”): • A high molecular weight serine-threonine kinase • Senses and responds to cellular nutrient and energy levels • Influences protein translation regulating G1 progression, S-phase entry, and ultimately cell growth and proliferation • Functions downstream of the PI3 kinase / AKT pathway, which is often deregulated in human cancer • mTOR pathway deregulation causes loss of growth control in cancer

3. mTOR: A central controller of tumor cell growth

4. mTOR: A controller of angiogenic processes

5. RAD001 (Everolimus): An oral mTOR pathway inhibitor • Broad antiproliferative and antitumor properties • Inhibits the mTOR pathway • Sensor of physiological signals • “Downstream“ of PI3K / AKT pathway • RAD001 activity associated with overactivation of the PTEN / PI3K / AKT pathway • Inhibits tumor cell growth • Delays G1/S phase progression • Sensitises tumor cells to targeted and chemotherapeutic agents • Anti-angiogenic properties • Direct and indirect activity • Phase IB/II clinical trials in oncology Model of RAD001 binding to intracellular receptor (FKBP-12) to form complex inhibiting mTOR pathway

6. p-AKT levels correlate with RAD001 sensitivity

7. RAD001: Broad in vivo antitumor activity

8. mTOR inhibition decreases angiogenesis • mTOR regulates HIF-1α and HIF-2α expression (transcription factors mediating hypoxia-induced gene expression) • 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 HIF = hypoxia-inducible factor; VHL = von Hippel-Lindau protein.

9. Evidence of antiangiogenic activity • Tumor xenograft–bearing mice: single 5 mg/kg oral dose • RAD001 plasma levels never exceed the in vitro IC50 for HCT116 (colon) or KB-31 (epidermoid) tumor cells • However, both HCT116 and KB-31 xenografts are sensitive to RAD001 in vivo at this dose • RAD001 levels exceed the in vitro IC50 for VEGF- or FGF-stimulated human umbilical vein endothelial cultures (HUVECs) • RAD001 inhibits tumor cell VEGF production in vitro and decreases tumor and plasma VEGF levels in animal models • RAD001 selectively inhibits VEGF-dependent angiogenic response in vivo, and reduces microvessel density in tumors derived from sensitive or resistant cell lines • These data suggest an antiangiogenic effect against tumors

10. RAD001 reduces microvessel density (B16/BL6) Vehicle Controls RAD001 Significant reduction in microvessel density following RAD001 treatment in primary tumor and cranial lymphnode metastases (shown)

11. RAD001: Combination potential Although RAD001 has antiproliferative activity as a monotherapy, its potential may be better realized in combination with other therapeutic agents • Chemotherapeutics • DNA-damaging agents (i.e. cisplatin, temozolomide) • Topoisomerase inhibitors (i.e. doxorubicin) • MT active agents (i.e. Taxol) • Targeted therapeutics • ErbB inhibitors (i.e. AEE788; trastuzumab) • Estrogen antagonists - aromatase inhibitors (i.e. letrozole) • BCR-ABL, Kit inhibitors (i.e. imatinib) • VEGFR inhibitors (i.e. PTK787) • IGF-1R inhibitors (i.e. AEW541) • Radiotherapy

12. Combinations with cisplatin(in RAD001-sensitive H596 NSCLC xenografts) Tumor Volumes Body Weights Combinations of RAD001 and low-dose cisplatin elicit a more potent antitumor effect than either agent alone (also in model derived from resistant line) * P < 0.05 versus controls by the Dunnett test.

13. Cisplatin combinations: Potentiate cell death(A549 cells: cell death with sub-optimal cisplatin concentrations) *Significant fold induction with P < 0.05, t-tests; two-way ANOVA indicates highlysignificant interaction between RAD001 and cisplatin P < 0.001.

14. Cell death is dependent on p53 status * Significant fold induction with P < 0.05, t-tests.

15. The p53/p21 response • DNA damage (i.e cisplatin treatment) activates p53. • In the presence of extensive DNA damage, p53 initiates a cell death program. • In the presence of sub-optimal DNA damage, p53 induces p21 expression, a cell cycle inhibitor, allows DNA repair (and cell survival). • RAD001 inhibits p21 expression forcing tumor cell death even at suboptimal cisplatin concentrations (non-lethal DNA damage) Beuvink et al. Cell. 2005;120:747-759.

16. Rationale for combination with letrozole • Akt activation predicts a worse outcome for breast cancer patients treated with endocrine therapy. • Activated Akt mediates resistance to antiestrogen therapy (related to HER2 overexpression). • mTOR inhibition restores responses to tamoxifen in breast cancer models with high levels of Akt activity. • Synergistic in vitro and in vivo effects have been seen with combined antiestrogen therapy and mTOR inhibition.

17. Inhibition of estrogen-induced proliferation(aromatase-expressing MCF7 cells) Highly significant interactions ( p < 0.001; two-way ANOVA) Synergistic effects (isobologram analysis) Also in aromatase-expressing T47D cells

18. Potential RADIANT Trials TUNEL YOPRO Decreased cell viability as compared to single agents (YOPRO:  p < 0.01; two-way ANOVA). Defined as apoptosis (TUNEL:  p < 0.05; Friedman Test). Also in aromatase-expressing T47D cells

19. Summary • mTOR acts as a central regulator of tumor cell growth and survival, and activation of the PI3K/AKT pathway may predict tumor response to mTOR inhibition. • RAD001 exhibits a broad antitumor activity, and inhibits elements of the angiogenic process. • Combination therapy targeting mTOR and other targets/processes deregulated in human cancer may provide enhanced anticancer activity. Targeting deregulated pathways has been a successful clinical strategy in cancer.