Unmet medical needs in

NSCLC treatment

R Rosell Catalan Institute of Oncology, Badalona, Barcelona

Molecular Oncology Research Foundation (MORe) Barcelona Cancer Therapeutics Innovation Group (CTIG), New York

XIV Congreso Nacional de Oncología Médica Salamanca

23 October 2013

• Coactivation of PI3K/Akt/mTOR and Ras signaling pathways occurs frequently in advanced cancer and is associated with adverse patient outcome (well established)

• Crosstalk between oncogenic pathways induces continual MEK-ERK signaling which maintains a reconfigured pattern inducing EMT-TF reprogramming (well established)

• Therefore, almost all patients with advanced cancer eventually develop resistance to current available cytotoxic and targeted therapies (well established)

• Genotyping is not enough for the patient’s expectations and deciphering crosstalk could be crucial for synthetic lethal therapies

Rosell et al. NEJM 2013 (MED12) Rosell, Bivona & Karachaliou. The Lancet 2013 (BIM) Rosell & Karachaliou. Nat Rev Clin Oncol. 2013 (mTOR)

Contributions: Bivona et al. Nature 2011 (NFKBIA - I) Zhang et al. Nature Genetics 2012 (AXL) Rosell. Lancet Oncology 2012 (EURTAC)

BIOMARKERS POTENTIAL THERAPEUTIC COMBINATIONS

Intrinsic apoptopic pathway

MCL1, FBW7, Noxa, BIM Vorinostat plus ABT-737; gefitinib plus ABT-737

KRAS-mutant tumours: Bcl-XL, BIM ABT-263 (navitoclax) plus selumetinib

Extrinsic apoptopic pathway

Trail, BIM, FOXO3, miR-494 TIC10 plus EGFR TKIs

GALNT14, FUT3/6, SIX1 Dulanermin, drozitumab

DR4/DR5, PEA15 Dulanermin, Drozitumab

MAPK/AKT/AMPK

FOXO3a, BIM, MED23 EGFR TKIs +Taxanes

VEGF-A, T790M Erlotinib+Bevacizumab (BELIEF)

KRAS-mutant tumours: FOXO3, LKB1, IGF1R

Selumetinib/trametinib; selumetinib/trametinib plus triciribine; selumetinib/trametinib plus paclitaxel; phenformin/metformin; IGF1R inhibitors plus selumetinib/trametinib

NOTCH3: HES1, DUSP1, NUMB EGFR TKIs plus γ-secretase inhibitor

TP53: MDM2, iASPP BRAF inhibitors plus MDM2 inhibitors plus iASPP inhibitors

AXL:AXL, GAS6, RELA, NFκB EGFR TKIs plus AXL inhibitors

IL-6:IL-6, JAK, STAT3 EGFR TKIs plus AXL inhibitors; EGFR TKIs plus pan-JAK inhibitors

TGF-β:TGF-βR2, MED12 EMT phenotype

EGFR TKIs plus TGF-β inhibitors

HEDGEHOG: GLI1, SOX2, SOX9, CXCR4, FGF19, aPKCl/λ,

EGFR TKIs plus aPKCl/λ inhibitors

mTOR/S6K1 EGFR TKIs plus mTOR inhibitors

cAMP–PKA: PKA, PDE4A/D EGFR TKIs plus PDE4 inhibitors

Rosell, Bivona, Karachaliou. Lancet. 2013

Stromal HGF confers EGFR TKI resistance and induces interreceptor crosstalk with integrin-4, Eph2, CDCP1, AXL and JAK1 (Gusenbauer, Vlaicu & Ullrich. Oncogene 2013) Also, HGF is present in melanoma stromal cells and correlates with poor response. A similar resistance mechanism was shown in a subset of BRAF mutant colorectal and glioblastoma cell lines (Straussman et al. Nature 2012)

Minimum 8,8 mm2 tumor and stroma tissue for the RNA analysis

Cuts of tissue mandatory 4 µm or more (5 cuts if block cannot be sent)

Further tissue can be cut as required from the FFPE block following pathology assessment if the whole block is sent

BELIEF trial. N Karachaliou, C Teixido

Rosell. NEJM 2013

Shin et al. JNCI 2013

Kanda et al. Cancer Res 2013

MAPK activity and EMT-TF reprogramming in the time course of tumor progression. A late EMT-TF reprogramming is dependent on continual MAPK signaling

Caramel Cancer Cell 2013

EGFRvIII induces secretion of IL-6 which activates gp130, generating a paracrin loop which promotes activation of EGFR in neighboring cells (Inda et al. Genes Dev 2010)

Wang et al. PNAS 2013

Gao et al. J Clin Investigation 2007

Zadeh et al. Cancer Cell 2013

Activated WT EGFR phosphorylates EGFRvIII triggering nuclear transport of EGFRvIII, and enhanced phosphorylation of STAT3 (Fan et al. Cancer Cell 2013)

Steder et al. Cancer Cell 2013

Bredel et al. NEJM 2010 Bivona et al. Nature 2011

Erlotinib treated EGFR mutant NSCLC

NFKBIA encoding IB the major negative regulator of NFB

BIM

IKK

α

IKK

β

Rosell, Bivona, Karachaliou. Lancet. 2013

• Nuclear factor of -light polypeptide gene enhancer in B-cells (NFB) a transcription factor activated by EGFR mutations (including EGFRvIII) and other oncogenes

• AXL/GAS6, mTORC2 and the non-cannonical NOTCH pathway activate NFB

• AXL also activates IL-6 and STAT3

• MYC-driven tumors can activate NFB (PDK1-PLK1-MYC)

- PLK1 inhibitor blocks mTOR inhibition-induced MYC activation (Tan et al. Cancer Cell 2010)

• EGFR T790M mutation-mediated acquired resistance – an unmet medical need

• Crosstalk of mTORC1 with Beclin, Bcl2 and BIM

• Cross-regulation DGK-PDE4A/D-PKA-cAMP-mTORC1

• Genomic fluidity (Wip1/PPM1D-LINE-1/APOBEC)

EURTAC

BREC/SCAT

BELIEF

GOAL

- Serial rebiopsy study

- AXL inhibitor trial

EUCROSS

NVALT

Platelets/plasma (EML4-ALK)

EGFR mutants (cDNA)

CTCs

EURTAC – cutoff January 2013 Rosell et al. Lancet Oncology 2012

• T790M mutation is present in up to 62-82% of cases at time of clinical

progression to erlotinib (Arcila CCR 2011; Su et al. JCO 2012)

• Allelic dilution makes it difficult to identify T790M by direct sequencing. A

low frequency of expression of the mutant allele HER2 T798M (3%) was

sufficient to confer drug resistance (Rexer et al. CCR 2013)

• Pre-existing T790M mutation found in 27% of EGFR-mutant patients by

massively parallel sequencing (Querings et al. PLoS ONE 2011), in 31.5% by MALDI-TOF

MS (Su et al. JCO 2012), in 35% by PCR-PNA assay (Rosell, Molina et al. CCR 2011), in 38% by

SARMS (Maheswaran et al. NEJM 2008), and related to shorter PFS to gefitinib or

erlotinib (Maheswaran et al. NEJM 2008; Rosell et al. CCR 2011; Su et al. JCO 2012)

• Pre-existing EGFR T790M found in 65% of EURTAC samples . Also, 50%

found in the BELIEF and GOAL studies

G3: Chemotherapy and T790M present (n=28)

G1: Erlotinib and T790M present (n=34)

G4:Chemotherapy and T790M absent (n=17)

G2: Erlotinib and T790M absent (n=16)

5·1 9·7 15·8 6·0

Patients at risk

G2

G4

G3 G1

EURTAC – cutoff January 2013

BIM

TRAIL

TRAIL

BIM

AC

GPCR

DR4/DR5

Rosell, Bivona, Karachaliou. Lancet. 2013

EURTAC – cutoff January 2013

EGFR mutant NSCLC patients

DGKa/PDE4/PKA/ERK/mTORC1

H1650 (ABT-263/gefitinib) Cragg et al. PLoS MED 2007

ABT-263/Erlotinib TORCi/SOCE agonists (IGR1)Corcoran et al. Science Transl Med 2013

Rosell & Karachaliou. Nat.Rev.Clin.Oncol. 2013

Maes et al. Trends in Molecular Medicine 2013 Wirawan et al. Cell Death and Disease. 2010

ULK1/2

FIP200

Atg13

mTORC1

Beclin1 Ambra1

CIII-PI3K/Vps34

Atg101 1) Autophagosome initiation

Atg14L p150

2) Nucleation

Atg5

Atg7

Atg10

Atg12 Atg16L

LC3 LC3-II

3) Elongation

Bcl-XL Bcl2

p62 U NBR1

U

LC3-II

4) Delivery & Degradation

BIM

ABT-737/ABT-263

AMPK

LKB1

TSC1/TSC2 Rheb

Metformin

KRAS/LKB1 (A549)

PDE4A/D DGKa

ERK

H1650

PI3K/AKT

L858R/T790M (H1975)

EGFR

AKT

Rubicon

Courtesy of Niki Karachaliou

Wei et al. Cell 2013

In the TKI-sensitive HCC827 cells (but not in TKI-resistant H1975 cells), erlotinib led to EGFR dephosphorylation, disruption of EGFR/Beclin 1 binding, disruption of Beclin 1/Rubicon binding, increased Beclin 1/VPS34 binding, decreased Beclin 1/Bcl-2 binding, and increased Beclin 1-associated VPS34 kinase activity. Also HCC827 cells but not H1975 cells show LC3-II conversion and P62 degradation after erlotinib treatment.

Beclin1 Ambra1 CIII-PI3K/Vps34

LC3 I LC3 II

BIM-EL

ABT-737/ABT-263

LKB1

TSC1/TSC2

Rheb ERK

(H1650)

Akt

Vasculogenic

mimicry

TAK-1

↑Ca++

CaMKKα/β

Soluble form Lipidated form

Autophagosome

RAS

DGKa

(H1975, A549)

p62 U

NBR1

U

LC3-II

(↑LC3 & Beclin1 mRNA & IHC)

autophagy

apoptosis

Enobosarm

Bcl-XL Bcl2 Mcl-1 Rubicon

VEGFA/VEGFR1

Hypoxia

ULK1/2 FIP200

Atg13 Atg101

AR SARMs

Atg5 Atg7

Atg10

Atg12 Atg16L

Atg9

mTORC1

Akt

FKBP5 PI3K

RAF

Amino-acid

starvation

Sirolimus/SOCE

agonists

PTEN PHLPP

AMPK

PDE4A/D

autophagy

Modified from Rosell, Bivona, Karachaliou. Lancet 2013

L858R/T790M (H1975)

Afatinib

STIM1

SOCE

Rap1 GTPase DOCK4 PRKA2

• PLK1 inhibitor blocks mTOR inhibitor-induced MYC activation (Tan et al. Cancer Cell 2010)

• A number of genes implicated in ESC are upregulated in HEK-PDK1 cells and HEK-MYC cells: SOX2, LIN28B, SALL4, EZH2. Others such as EPCAM, ALDHIA, S100A4 are also upregulated and considered markers of CSCs (Tan et al. Cancer Discovery 2013)

Cunningham & Ruggero. Cancer Discovery 2013

Well-characterized panel of cell lines for determination of clinically relevant markers

• H1975(EGFR del /T790M) rolipram sensitive PDE4D Our findings

(DGKa/PDE4/PKA/↑ERK/mTORC1)

• A549 (KRAS/LKB1) rolipram sensitive PDE4D Our findings

DGKa/PDE4/PKA/ERK

• H460 (KRAS/LKB1) rolipram resistant PDE4D intermediate / PDE4A (N Karachaliou)

NOTCH3/HES1/ERK1. Sensitive to erlotinib/secretase inhibitors

• H1650 (EGFR del/↓BIM) sensitive to ABT-263/gefitinib

DGKa/PDE4/PKA/↑ERK/mTORC1, rolipram partly sensitive

• IGR1 (BRAF melanoma) ↑BIM resistant to VEM (↓ERK/ ↑mTORC1 ) Sensitive to VEM/ABT-263, or a TORC inhibitor (Corcoran et al. Science Trans Med 2013)

Pullamsetti et al. Oncogene 2013 Dominguez et al. Cancer Discovery 2013

Rolipram reduces proliferation in H1975 cell line Serum-starved (0%, 0.1% and 0.5% FBS, normoxia)

0

0,2

0,4

0,6

0,8

1

1,2

0 1 50 250

Viability

Rolipram μM

H1975 0% FBS

0

0,2

0,4

0,6

0,8

1

1,2

0 1 50 250

Viability

Rolipram μM

H1975 0.1% FBS

0

0,2

0,4

0,6

0,8

1

1,2

0 1 50 250

Viability

Rolipram μM

H1975 0.5%

Pullamseti et al. Oncogene 2013

Molina & Bertran-Alamillo

BIM high/mTOR low-intermediate: mOS=35.8m BIM high/mTOR high: mOS=20.3m

BIM low/mTOR low-intermediate: mOS=17.7m BIM low/mTOR high: mOS=25.1m

N. Karachaliou and A. Droz

erlotinb gefitinib afatinib

erlotinb gefitinib afatinib

High

Low

High

T790M present

mTOR high

T790M absent

mTOR low

T790M model

mTOR model

+ mTORi+SOCE agonists or PLK i metformin or 2DG

BIM BIM

BIM mRNA expression

erlotinb gefitinib afatinib

+ Bcl2i PDE4i Taxanes

erlotinib gefitinib afatinib

A potential algorithm for directing EGFR TKI therapy in EGFR mutant NSCLC

Courtesty N Karachaliou

afatinib/pemetrexed Afatinib/bevacizumab

Conclusions

• Only BIM mRNA expression is an independent marker of both PFS and OS in

the EURTAC study

• mTORC1 modulates survival in NSCLC expressing high BIM (Karachaliou et al.)

• BIM and mTOR essential markers for adequate treatment of NSCLC patients

with EGFR mutations, and probably other subclasses of lung cancer

Acknowledgements

Ana Drozdowskyj, Miguel Angel Molina, Andres Felipe Cardona, Ana Giménez-Capitán,

Jordi Bertran-Alamillo, Clara Mayo, Jordi Codony, Radj Gervais,Teresa Moran,

Margarita Majem, Enriqueta Felip, Enric Carcereny, Felipe Cardenal, Ramon Palmero, Ruth Porta, Joaquim Bosch,

Santiago Ponce-Aix, Ana Estival, Rosario Garcia-Campelo, Santiago Viteri,

Amaya Gasco, Daniela Morales-Espinosa, Cristina Teixido,

Jose Luis Ramirez, Miquel Taron, Carlos Camps, Manuel Cobo, Manuel Domine, Isabel Bover, Mariano Provencio,

Guillermo Lopéz-Vivanco, Dolores Isla, Bartomeu Massuti, Alain Vergnenegre, Solange Peters,

Rolf Stahel, Jia Wei, Baroui Liu, Silvia Garcia-Roman, Roger Estrada, Trever Bivona, Niki Karachaliou

Spanish Lung Cancer Group (SLCG)

French Lung Cancer Group

European Thoracic Oncology Group (ETOP)

Comprehensive Cancer Center of Drum Tower Hospital Nanjing

Cancer Therapeutics Innovation Group (CTIG)

Pangaea Biotech S.L, Quirón Dexeus University Hospital

Pivotal, Madrid

Institut Químic de Sarrià (IQS) / Grup d’Enginyeria Molecular (GEM)