Sensitivity and resistance profiles over 240 cell lines

Resistant

Sensitive

IC50

GI50

PLX-4032 analog (BRAF inhibitor)

PD0325901 (MEK inhibitor) Sorafenib (RAF, PDGFR, cKIT, VEGFR inhibitor)

Erlotinib (EGFR inhibitor)Cl-1040 (MEK inhibitor)

BRAF And MEK inhiBitoR pRoFiling AcRoss 240 tuMoR cEll linEs to coRRElAtE with sEnsitivity And REsistAnt BioMARKERs

Karen Bernards, Yulia Ovechkina, Jushun Cheng, John Kushleika, Alison Angione, James Hnilo, Christine O’Day and Usha WarriorRicerca Biosciences, LLC – Bothell, WA, USA

intRoductionActivated kinases that induce cell proliferation have been attractive targets for targeted cancer therapy development. Cancer cells could become dependent on tumor-specific activated kinases and this tendency has been described as oncogene addiction. Here, we investigated several activated kinase inhibitors: BRAF inhibitor, PLX-4032 analog, Sorafenib and the MEK inhibitors, Cl1040 and PD0325901. We established a high throughput cellular approach to profile 240 human tumor cell lines identifying genotype-correlated sensitivity or resistance. Proliferative, apoptotic and cell cycle arrest responses were measured using multiplexed high content screening with automated fluorescence microscopy and image analysis based technology. Growth index was measured using nuclear dye. Activated caspase 3 antibodies were used for the apoptosis induction detection. Phospho-histone H3 antibodies were used to measure the cell cycle block. We generated cell line profiles to reveal drug sensitivity and resistance patterns that may correlate with the clinical genotype responses (Rodriguez et al., Journal of Laboratory Automation, 2007).

The dependence of transformed cells on the activation of survival signaling pathways has long been recognized. Two of the most important signaling cascades frequently dysregulated in cancer are the Ras/Raf/MEK/ERK and the PI3K/AKT/mTOR pathways. Activating mutations of Ras and Raf occur frequently in both solid tumor and hematologic malignancies, leading to activation of their downstream targets MEK1/2 and ERK1/2. Similarly, the PI3K/AKT/mTOR pathway is one of the most frequently mutated pathways in solid tumor malignancies. PLX4032, specific inhibitor of BRAFV600E kinase, is an inhibitor which controls the cell proliferation and arrests the growth of RAF mutated tumor cells. Erlotinib is a small molecule EGFR inhibitor that targets the ATP-binding site of EGFR, resulting in the inhibition of EGFR tyrosine kinase activity. Erlotinib is considered a standard therapy for patients with advanced non-small cell lung cancer (NSCLC). However, erlotinib has shown little benefit for NSCLC patients whose tumors have a mutated form of the gene KRAS. MEK inhibitors show a potent action within tumors which show mutation within KRAS gene. Thus, combination therapy of EGFR and MEK inhibitors may show enhanced efficacy (Diep et al., Clin Cancer Res, 2011).

conclusions1. BRAF mutations correlated with sensitivity of PLX-4032 inhibitor while RAS mutations may

confer resistance to both RAF inhibitors, PLX-4032 and Sorafenib.

2. BRAF and RAS mutations correlate with the sensitivity of both MEK inhibitors while PIK3CA confer resistance.

3. BRAF inhibitor sensitive cell lines are also sensitive to MEK inhibitors.

4. RAS mutations confer resistance to the BRAF inhibitor while associating with sensitivity to both MEK inhibitors.

5. PTEN and PIK3CA mutations correlate with resistance to Sorafenib and Erlotinib.

6. High levels of endogenous phospho ERK and phospho AKT correlate with erlotinib resistance.

7. High levels of endogenous phospho AKT correlate with RAF inhibitor resistance while most of RAF inhibitor sensitive cell lines show high levels of phospho ERK.

8. Combination analysis of EGFR and MEK inhibitors showed synergy in the HUPT4 pancreatic cell line.

REFEREncEs1. McDermott U, Sharma S, Dowell L, Greninger P, Montagut C, Lamb J, Archibald H, Raudales

R, Tam A, Lee D, Rothenberg S, Supko J, Sordella R, Ulkus L, A. John Iafrate A, Maheswaran S, Njauw C, Tsao H, Lisa Drew, Hanke J, Xiao-Jun Ma, Erlander M, Gray N, Haber D, Settleman J. Identification of genotype-correlated sensitivity to selective kinase inhibitors by using high-throughput tumor cell line profiling. PNAS, 2007, 104 (50), 19936-19941

2. Chou, T.-S. Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies. Pharmacological reviews, 2006, 58 (3), 621-680

3. Duska LR, Hamblin MR, Miller JL, Hasan T. Combination photoimmunotherapy and cisplatin: effects on human ovarian cancer ex vivo. J Natl Cancer Inst. 1999, 01(18), 1557-63

4. Roberts P, Der C . Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene, 2007, 26, 3291–3310

5. Diep C, Munoz R, Choudhary A, Von Hoff D, Han H. Synergistic Effect between Erlotinib and MEK Inhibitors in KRAS Wild-Type Human Pancreatic Cancer Cells. Clin Cancer Res, 2011, 17, 2744-2756

6. Rodriguez R, Alfonso J, O’Day C, Ovechkina Y, Ward C. 297 Distinct Cell Lines: A High-Content Analysis Assay and a Full-Automation Design Solely Using Noncontact Liquid Dispensing. Journal of Laboratory Automation, 2007, 12 (5), 318-326

Cancer therapeutic agents

RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways GI50 values across 240 cell line panel

BRAF and RAS mutations correlate with the sensitivity of both MEK inhibitors

BRAF mutations correlated with sensitivity of PLX-4032 inhibitor while RAS mutations may confer resistance

High levels of endogenous phospho ERK and phospho AKT correlate with Erlotinib resistance

BRAF inhibitor sensitive cell lines also show sensitivity to MEK inhibitors

High levels of endogenous phospho AKT correlate with RAF inhibitor resistance while most of RAF inhibitor sensitive cell lines show high levels of phospho ERK

BRAF and KRAS mutations confer resistance to EGFR inhibitor, Erlotinib

High levels of endogenous phospho ERK and phospho AKT do not show significant correlation with MEK inhibitor responses

PTEN and PIK3CA mutations may correlate with resistance to Sorafenib and Erlotinib

Both RAF and MEK inhibitors inhibit cell growth while inducing apoptosis and G1/S cell cycle arrest

RAF

Cell membrane

PLX-4032

CI-1040PD0325901

Erlotinib

RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways

PI3K

AKT

mTOR

RAS

MEK

ERK

Survival

Proliferation

EGFR overexpression:• Colorectal cancer (27-77%)• Pancreatic cancer (30-50%)• Lung cancer (40-80%)• Non-small cell lung cancer (14-91%)

RAS mutation:• Pancreatic cancer (90%)• Papillary thyroid cancer (60%)• Colon cancer (50%)• Non-small cell lung cancer (30%)

BRAF mutation:• Melanoma (70%)• Papillary thyroid cancer (50%)• Colon cancer (10%)

Roberts et al., Oncogene, 2007

Midpoint of the GI50 range was used to distinguish sensitive and resistant populations. The GI50 of the sensitive and resistant populations was then averaged to produce the “sensitive and resistant response mean GI50”.

240 Cell Lines

GI5

0, m

icro

M

PLX-4032 analog (BRAF inhibitor)

PD0325901 (MEK inhibitor)

Erlotinib (EGFR inhibitor)Cl-1040 (MEK inhibitor)

Sorafenib (RAF, PDGFR, cKIT, VEGFR inhibitor)

Resistant

Sensitive

240 Cell Lines

GI5

0, m

icro

M

Cl-1040 (MEK inhibitor)PD0325901 (MEK inhibitor)

BRAF mutations RAS mutations

Synergistic Activity of EGFR and MEK inhibitors in HUPT4 pancreatic cell line

Cel

l pro

lifer

atio

nA

popt

osis

Cel

l cyc

le

Cel

l pro

lifer

atio

nA

popt

osis

Cel

l cyc

le

Sorafenib treated KPL-1 cell linePLX-4032 analog treated Colo829 cell line Cl-1040 treated A375 cell line

Cel

l pro

lifer

atio

nA

popt

osis

Cel

l cyc

le

PD0325901 treated SW579 cell line

Cel

l pro

lifer

atio

nA

popt

osis

Cel

l cyc

le

240 Cell Lines

GI5

0, m

icro

M

BRAF mutations RAS mutations

PLX-4032 analog (BRAF inhibitor) Sorafenib (RAF, PDGFR, cKIT, VEGFR inhibitor)

240 Cell Lines

GI5

0, m

icro

M

Phospho ERK measured by AlphaScreen® SureFire® platform

Phospho AKTmeasured by AlphaScreen® SureFire® platform

Erlotinib (EGFR inhibitor) Erlotinib (EGFR inhibitor)

Combination index analysis

Erlotinib

PD

0325

901

Combination index >1 denotes antagonism; combination index = 1 denotes additivity: combination index <1 denotes synergy (Chou, T. S., 2006).

Isobologram analysis showing synergy of EGFR and MEK inhibitors in inhibiting cell proliferation. Points below the isobole indicate a synergistic interaction (http://www.neuroproof.com/eng/services/DrugInteractions.htm).

Isobolographic analysis

synergy

antagonism

PLX-4032 analog sensitive cell lines

240 Cell Lines

GI5

0, m

icro

M

PD0325901

Cl-1040 sensitive cell lines

PD0325901

PD0325901 sensitive cell lines associated with Cl-1040 MEK inhibitor sensitivity

PD0325901 sensitive cell lines associated with BRAF inhibitor sensitivity

240 Cell Lines

GI5

0, m

icro

M

Sorafenib (RAF, PDGFR, cKIT, VEGFR inhibitor)

PLX-4032 analog (BRAF inhibitor)

PLX-4032 analog (BRAF inhibitor)

Sorafenib (RAF, PDGFR, cKIT, VEGFR inhibitor)

Phospho ERK measured by AlphaScreen® SureFire® platform

Phospho AKTmeasured by AlphaScreen® SureFire® platform

GI5

0, m

icro

M

Erlotinib (EGFR inhibitor)

240 Cell Lines

BRAF mutations RAS mutations

240 Cell Lines

GI5

0, m

icro

M

Phospho ERK measured by AlphaScreen® SureFire® platform

Phospho AKTmeasured by AlphaScreen® SureFire® platform

PD0325901 (MEK inhibitor)

Cl-1040 (MEK inhibitor)

PD0325901 (MEK inhibitor)

Cl-1040 (MEK inhibitor)

GI5

0, m

icro

M

PLX-4032 analog (BRAF inhibitor)

Sorafenib (RAF, PDGFR, cKIT, VEGFR inhibitor)

Cl-1040 (MEK inhibitor)

PTEN mutations

PD0325901 (MEK inhibitor)

240 Cell Lines

Erlotinib (EGFR inhibitor)

PIK3CA mutations