The PI3K/AKT axis. Dr. Lucio Crinò Medical Oncology Division Azienda Ospedaliera-Perugia. Introduction

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1 The PI3K/AKT axis Dr. Lucio Crinò Medical Oncology Division Azienda Ospedaliera-Perugia Introduction Phosphoinositide 3-kinase (PI3K) pathway are a family of lipid kinases discovered in 1980s. They have been found to have key regulatory roles in many cellular processes, including cell survival, proliferation, differentiation and angiogenesis. They are major effectors downstream of receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). The tumor suppressor gene PTEN (Phosphatase and tensin homologue) is the most important negative regulator of the PI3K signaling pathway. 1

2 PI3K pathway activation Growth factor stimulation and subsequent activation of receptor tyrosine kinases. PI3Ks are recruited to the membrane by direct interaction of the p85 subunit with the activated receptors. The activated p110 catalytic subunit converts PtdIns(4,5)P2 to PtdIns(3,4,5)P3 at the membrane. This provides docking sites for signaling proteins including PDPK1 and AKT This results in conformational change in AKT exposing two crucial amino-acid residues for phosphorylation. PDPK1 phosphorylates and activates AKT, which activates multiple downstream signaling Nature Reviews Drug Discovery 8, (August 2009). Liu P et al 2

Two of the most frequent PIK3CA mutations, E545K and H1047R, have been shown to increase PtdIns(3,4,5)P3 levels, activate t AKT signaling and induce cellular l transformation.

3 Mutations of class IA PI3Ks PI3K catalytic subunit alpha-isoform gene (PIK3CA), which encodes p110α, is frequently mutated in various tumors. PIK3CA mutations consist of somatic missense mutations clustered in two hotspots regions in exon 9 and 20, corresponding to the helical and kinase domains of p110α, respectively. Two of the most frequent PIK3CA mutations, E545K and H1047R, have been shown to increase PtdIns(3,4,5)P3 levels, activate t AKT signaling and induce cellular l transformation. ti Recent analysis have also shown mutations in PIK3R1 gene which encodes p85α in many cancers. PI3K inhibitors in NSCLC: few patients have an activated PI3K pathway PTEN Histotype PIK3CA Negative mutation 1 Mutant 2 (<10% protein expression by IHC Squamous 10% 10% appr % Non-squamous 2-5% 1.7% appr % 1 Gain-of-function mutations; 2 Loss-of-function mutations Marsit et al. Hum Pathol 2005 Guang et al. Lung Cancer

Current targeting of PI3K pathway Nature Reviews Drug Discovery 8, 627-644 (August 2009).

Wortmannin binds irreversibly to PI3k enzymes by covalent bonding LY29400 was the first synthetic drug-like small molecule inhibitor capable of reversibly

Numerous PI3K-targeted compounds are being introduced into clinical trials, some of them are dual PI3K-mTOR inhibitors, some inhibit multiple class I PI3K

4 Current targeting of PI3K pathway Nature Reviews Drug Discovery 8, (August 2009). Liu P et al Targeting PI3K Wortmannin and LY are the two well known, first generation PI3K inhibitors. Wortmannin binds irreversibly to PI3k enzymes by covalent bonding LY29400 was the first synthetic drug-like small molecule inhibitor capable of reversibly targeting PI3K family members at concentrations in the micromolar range. Both Wortmannin and LY showed little or no selectivity for individual PI3K isoforms. Numerous PI3K-targeted compounds are being introduced into clinical trials, some of them are dual PI3K-mTOR inhibitors, some inhibit multiple class I PI3K isoforms, some of them inhibit the mtor kinase activity by binding to the ATP-binding pock. Preclinical data with some of these agents have shown strong antiproliferative activity against tumour xenografts that have abnormal PI3K signaling (loss of PTEN function or gain-of-function PI3K mutations). 4

5 An open label two-stage study of orally administered BKM120 in patients with metastatic NSCLC with activated PI3K pathway Defined as having one of the following molecular alterations in tumor tissue: PIK3CA mutation (Central or local lab) PTEN mutation (Central or local lab) PTEN negative (<10% protein expression by IHC) (Central lab) At least 35% of the total enrolled population is required to carry a PIK3CA mutation NCT Patients with KNOWN acrtvated PI3K pathway Stage 1 Metastatic NSCLC patient identified Patients with UNKNOWN PI3Kpathway status General ICF Pre-screening ICF Screening (up to 21 days Eligibility worksheet approved by sponsor Enrollment Tumor tissue test locally or shipment Mutational analysis confirming PI3K Pathway activation Group 1: Squamous NSCLC pretreated with 1 chemo BKM 120 (40 patients) Group 2: Non-squamous NSCLC pretreated with 1 or 2 systemic therapies BKM 120 (30 patients) Only when either or both groups have a 12-week PFS rate 50 %, enrollment into Stage 2 will begin 5

Patients with KNOWN activated PI3K pathway Stage 2 Metastatic NSCLC patient identified Patients with UNKNOWN PI3K pathway status General

shipment Mutational analysis confirming PI3K Pathway activation Group 1: Squamous NSCLC pretreated with 1 chemo Group 2: Non-squamous

Docetaxel or Pemetrexed (20 patients) mtor mtor plays a crucial part in the regulation of cell growth and proliferation by monitoring

mtor exists in two distinct complexes: mtorc1 and mtorc2.

6 Patients with KNOWN activated PI3K pathway Stage 2 Metastatic NSCLC patient identified Patients with UNKNOWN PI3K pathway status General ICF Pre-screening ICF Screening (up to 21 days Eligibility worksheet approved by sponsor Randomization Tumor tissue test locally or shipment Mutational analysis confirming PI3K Pathway activation Group 1: Squamous NSCLC pretreated with 1 chemo Group 2: Non-squamous NSCLC pretreated with 1 or 2 systemic therapies Rand 2:1 Rand 2:1 BKM 120 (40 patients) Docetaxel (20 patients) BKM 120 (40 patients) Docetaxel or Pemetrexed (20 patients) mtor mtor plays a crucial part in the regulation of cell growth and proliferation by monitoring nutrient availability, cellular energy levels, oxygen levels and mitogenic signals. mtor exists in two distinct complexes: mtorc1 and mtorc2. AKT can activate mtor by phosphorylating both PRAS40 and tuberous sclerosis 2 protein (TSC2 or tuberin) to attenuate their inhibitory effects on mtorc1. When bound to rictor in the mtorc2 complex, mtor functions as a PDPK2 to phosphorylate AKT. Engelman JA. Nat Rev Cancer Aug;9(8):

7 Targeting mtor Rapamycin associates with its intracellular receptor, FK506- binding protein 12 (FKBP12), binds directly to mtorc1 and suppresses mtor-mediated phosphorylation of S6K and 4EBP1. Analogues of rapamycin (rapalogues), temsirolimus and everolimus inhibit mtor through the same mechanism as does rapamycin, but have better pharmacological properties for clinical use. Recent studies have described torkinibs and torin1 - potent and selective ATP-competitive inhibitors of mtor that inhibit both mtorc1 and mtorc2 complexes and dimpair i cell growth and proliferation more effectively Also notable is that several kinases in the PI3K pathway are client proteins for the heat shock protein 90. Phase II studies of single-agent mtor inhibitors in NSCLC Author Agent No. pts Prior therapy RR DCR Molina (2007) Temsirolimus 55 No 8% 30% Soria (2009) Everolimus 42 Soria (2009) Everolimus 43 2 lines of chemo 2 lines of chemo and an EGFR-TKI 7.1% 52.3% 2.3% 41.8% 7

KRAS wild-type cells Demonstrated in vivo antitumor activity in xenograft models dl of flung cancer Dancey, Nat Rev Clin

8 The novel mtor inhibitor Deforolimus Demonstrated preclinical antiproliferative activity in a broad range of human tumor cell lines In a large panel of more than 100 NSCLC cell lines, deforolimus activity was comparable against KRASmutant and KRAS wild-type cells Demonstrated in vivo antitumor activity in xenograft models dl of flung cancer Dancey, Nat Rev Clin Oncol 2010 A randomized discontinuation phase II trial of Deforolimus in NSCLC patients with KRAS mutations NCT

9 Future directions and challenges The presence of an activating mutation or other genomic alteration in the targeted kinase is probably the most accurate predictor of potential success. PI3K inhibitors will be effective in tumors that feature activating mutations in p110alpha, or loss of PTEN and AKT mutations will sensitize a tumor to AKT inhibitors. Other alterations in genes can alter the sensitivity of the tumor with PI3K inhibitors. Tumors with amplifications or mutations in various downstream kinases may block the effects of upstream inhibitors. Future directions and challenges Identification of new and potentially more robust biomarkers is a crucially important in the development of PI3K inhibitors and in the conduct and interpretation of clinical studies using these inhibitors. Use of readily obtainable tissues, such as skin, hair follicles and peripheral blood mononuclear cells as surrogate tissues to assess the effect of PI3K inhibitors. Non invasive monitoring of PI3K and AKT inhibition that could predict outcome include measuring blood insulin levels (disruption of insulin signaling), PET imaging (inhibition of glucose transport and metabolism) and dynamic contrast enhanced MRI (decreasing tumor vasculature permeability). As PI3K inhibitors progress through clinical trials, issues related to toxicity and resistance will be unraveled. 9

A particular set of insults induces apoptosis (part 1), which, if inhibited, can switch to autophagy. At least in some cellular settings, autophagy se

A particular set of insults induces apoptosis (part 1), which, if inhibited, can switch to autophagy. At least in some cellular settings, autophagy serves as a defence mechanism that prevents or retards