1347, a Selective FGFR Inhibitor ERK Signal Suppression and Sensitivity

Author Manuscript Published OnlineFirst on October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ERK signal suppression and sensitivity to FGFR inhibitor

ERK signal suppression and sensitivity to CH5183284/Debio 1347, a selective FGFR

inhibitor

Yoshito Nakanishi, Hideaki Mizuno, Hitoshi Sase, Toshihiko Fujii, Kiyoaki Sakata,

Nukinori Akiyama, Yuko Aoki, Masahiro Aoki, Nobuya Ishii

Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa

247-8530, Japan

Running Title: ERK signal suppression and sensitivity to FGFR inhibitor

Keywords: CH5183284/Debio 1347, FGFR inhibitor, pharmacodynamic marker, ERK

signaling, DUSP6

Financial/grant information: None

Corresponding Author: Yoshito Nakanishi, Research Division, Chugai Pharmaceutical Co.,

Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan; Phone: 81-467-47-6262; Fax:

81-467-46-5320; E-mail: [email protected]

Disclosure of Potential Conflicts of Interest: All authors are employees of Chugai

Pharmaceutical Co., Ltd. The company was involved in the data analysis and the decision to

publish this manuscript.

Word count (from beginning of Introduction to the end of Discussion): 3202 words

Word count (Abstract): 220 words

Total number of figures and tables: 5 figures.

Downloaded from mct.aacrjournals.org on October 3, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ERK signal suppression and sensitivity to FGFR inhibitor

Abstract

Drugs that target specific gene alterations have proven beneficial in the treatment of cancer.

Because cancer cells have multiple resistance mechanisms, it is important to understand the

downstream pathways of the target genes and monitor the pharmacodynamic markers

associated with therapeutic efficacy. We performed a transcriptome analysis to characterize

the response of various cancer cell lines to a selective fibroblast growth factor receptor

(FGFR) inhibitor (CH5183284/Debio 1347), a mitogen-activated protein kinase kinase

(MEK) inhibitor, or a phosphoinositide 3-kinase (PI3K) inhibitor. FGFR and MEK inhibition

produced similar expression patterns, and the extracellular signal-regulated kinase (ERK)

gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with

these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR

inhibitor-sensitive cell line. Because the mitogen-activated protein kinase (MAPK) pathway

functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR

inhibitor efficacy in a collection of cell lines with the ERK signature and identified

dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Although a MEK inhibitor

suppressed the MAPK pathway, most FGFR inhibitor-sensitive cell lines are insensitive to

MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We

therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling

without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of

FGFR inhibitor efficacy in FGFR-addicted cancers.

ERK signal suppression and sensitivity to FGFR inhibitor

Introduction

Several tyrosine kinase-targeting agents have recently been developed. Each of these

agents has demonstrable efficacy when used in patient cohorts that are stratified based on the

genetic status of their respective molecular targets. The fibroblast growth factor receptors

(FGFRs) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic

alterations such as gene amplification, point mutation, or chromosomal

translocation/rearrangement (1, 2). Genetic alterations of FGFR may also be predictive

indicators of patient response to FGFR inhibitors (2, 3). For instance, dovitinib, a

multi-targeted kinase inhibitor that inhibits FGFRs, produced three unconfirmed partial

responses in breast cancer harboring FGFR1 gene amplification (4). Although genetic

alterations could predict drug efficacy, acquired genetic alterations confer resistance to

molecular-targeted drugs. Acquired mutations in target genes or downstream components are

major mechanisms of resistance (5-13). Although acquired FGFR mutations have not yet

been identified in patients, several FGFR mutations that confer resistance to FGFR inhibitors

have been reported (7, 14). Because cancer cells continue to utilize the pathway to which they

are originally addicted, they acquire some genetic alterations to re-activate the pathway.

Therefore, monitoring of changes in the pathways utilized by cancer cells could be used to

predict the efficacy of an inhibitor in tumors.

The FGFR family consists of FGFR1, FGFR2, FGFR3, and FGFR4, each of which

is bound by a subset of 22 fibroblast growth factor (FGF) ligands. FGFRs are activated by

ligand-dependent or ligand-independent dimerization that leads to intermolecular

phosphorylation. FGFR substrate 2 (FRS2) is a key adaptor protein that is phosphorylated by

FGFR. Phosphorylated FRS2 recruits several other adaptor proteins and activates the

mitogen-associated protein kinase (MAPK) or PI3K/AKT pathways (1). However, the

pathway associated with effective FGFR suppression in FGFR-addicted cancers has not yet

ERK signal suppression and sensitivity to FGFR inhibitor

been identified. Gaining an understanding of the FGFR pathway by studying its function in

the presence of FGFR inhibitors will enable identification of candidate pathways utilized by

FGFR-addicted cancers and the pharmacodynamic markers of these pathways.

In a previous study, we analyzed the signaling pathway of an FGFR fusion kinases,

FGFR3-BAIAP2L1 (15). A Rat-2 cell line stably expressing FGFR3-BAIAP2L1 exhibited

potent tumorigenic activity. Gene expression analysis revealed strong up-regulation of genes

downstream of the MAPK pathway, and up-regulation of the MAPK pathway was validated

by western blotting; in contrast, the PI3K/AKT pathway was not activated by

FGFR3-BAIAP2L1. Therefore, we suggested that MAPK pathway activation is essential to

the tumorigenic activity of FGFR3-BAIAP2L1. To generalize the MAPK pathway

dependency of FGFR, we characterized the pathway modulation by CH5183284/Debio 1347,

a selective FGFR inhibitor, in several FGFR genetically altered cancer cell lines. We

analyzed differential transcript expression by microarray analysis and found that MAPK

pathway modulation was associated with the efficacy of CH5183284/Debio 1347. We

identified the dual specificity phosphatase 6 (DUSP6) gene, which lies downstream of

MAPK, as a candidate pharmacodynamic marker of FGFR inhibitor efficacy. Finally, we

applied a MEK inhibitor to FGFR genetically altered cancer cells to validate the significance

of MAPK pathway modulation by FGFR inhibition.

ERK signal suppression and sensitivity to FGFR inhibitor

Materials and Methods

Reagents and cell lines

CH5183284/Debio 1347 (FGFR inhibitor), CH4987655 (MEK inhibitor),

CH5126766 (RAF-MEK inhibitor), and CH5132799 (PI3K inhibitor) were synthesized at

Chugai Pharmaceutical Co. Ltd., as previously described (7,16-18). AZD4547 was

synthesized at Chugai Pharmaceutical Co. Ltd. (patent publication WO2008075068).

PD173074, PD0325901, and selumetinib were purchased from Sigma-Aldrich and Selleck

Chemicals. The DMS-114, NCI-H520, NCI-H1581, NCI-H716, KATO-III, SNU-16, AN3

CA, RT-4, NCI-N87, ZR-75-1, and HCT116 cell lines were purchased from the American

Type Culture Collection (Manassas, VA, USA). The KMS-11 cell line was purchased from

the Health Science Research Resources Bank. MFE-296 and UM-UC-14 cell lines were

purchased from Health Protection Agency Culture Collections. The MFE-280 cell line was

purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. The

HSC-39 and MKN-45 cell lines were purchased from Immuno-Biological Laboratories, and

the SUM-52PE cell line was purchased from Asterand. All cell lines were obtained more than

one year prior to the experiments and were propagated for less than 6 months after thawing

and cultured according to supplier instructions.

Microarray

Cells were treated with 1 µM inhibitor doses and incubated for 24-h at 37°C. Total

RNA was purified with the RNeasy kit (Qiagen). Total RNA was reverse transcribed, labeled,

and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix) according to the

manufacturer’s instructions. The microarray data were deposited in the GEO database (GEO

number: GSE73024). The expression value for each probe was calculated using the

ERK signal suppression and sensitivity to FGFR inhibitor

guanine-cytosine robust multi-array analysis (GC-RMA) algorithm (19). To reduce noise

from the low-signal range, probes with a median signal of <10 across samples were filtered

out. The log-ratio of expression relative to dimethyl sulfoxide (DMSO)-treated samples was

calculated to identify down-regulated genes (<50% expression) and up-regulated genes

(>200% expression). Cluster 3.0 was used for clustering and Java Treeview was employed for

heatmap construction (20).

Signature analysis

The microarray dataset from human keratinocytes treated with extracellular

signal-regulated kinase 1/2 (ERK1/2) siRNA (GSE15417) (21) was obtained from the Gene

Expression Omnibus. This dataset consists of profiles from two independent siRNA

experiments (set A and B). Both profile sets and the log-transformed expression data for each

probe were analyzed by a t-test comparison against the control. Then, significantly

down-regulated probes with t-test p<0.01 in both comparisons were selected. We used 672 of

22,277 probes as an ERK1/2-inducible gene signature.

For the datasets in the “Microarray” section, the log-ratios for the replicated

experiments were averaged. Then, distributions of the log-ratios for probes in the signature

and not in the signature were plotted as a histogram. Differences in their distribution patterns

indicate a coordinated change in expression for the gene signature. R

(http://www.r-project.org/) was used for calculation.

Cell proliferation assay

All cell lines were cultured according to the supplier’s instructions. The cells were

seeded in 96-well plates and incubated at 37°C with inhibitors. After 4 days, Cell Counting

Kit-8 solution (Dojindo Laboratories) was added, and, after incubation for several more hours,

ERK signal suppression and sensitivity to FGFR inhibitor

the absorbance at 450 nm was measured with the iMark Microplate-Reader (Bio-Rad

Laboratories). Anti-proliferative activity was calculated using the formula (1 − T/C) × 100

(%), where T and C represent the absorbance at 450 nm of treated (T) and untreated controls

(C). The IC50 values were calculated using Microsoft Excel 2007.

Western blot analysis

Cells were treated with inhibitors or a solvent control (0.1% DMSO) for 2-h or 24-h

and lysed in Cell Lysis Buffer (Cell Signaling Technology) containing protease and

phosphatase inhibitors. For animal studies, xenograft tumors were obtained 4 h after the 11th

treatment of 100 mg/kg CH5183284/Debio 1347 and homogenized using a BioMasher (K.K.

Ashisuto) before lysis. All in vivo studies were approved by the Chugai Institutional Animal

Care and Use Committee. Cell lysates were denatured with Sample Buffer Solution with

Reducing Reagent for SDS-PAGE (Life Technologies) and resolved on precast 10% or 5–

20% SDS-PAGE gels (Wako Pure Chemical Industries). After electroblotting, western blot

analysis was performed as described previously (22). The following primary antibodies (Cell

Signaling Technology) were used: anti-phospho-FGF receptor (pFGFR-Tyr653/654, #3471),

anti-phospho-ERK1/2 (pERK-The202/Tyr204; #9101), anti-ERK1/2 (ERK; #9102),

anti-phospho-AKT (pAKT-Ser473; #9271), anti-AKT (AKT; #9272), anti-phospho-STAT3

(pSTAT3-Tyr705; #9145), anti-STAT3 (STAT3; #9139), anti-phospho-MET

(pMET-Tyr1234/1235; #3126), anti-MET (Met; #3127), anti-phospho-EGFR

(pEGFR-Tyr1068; #2234), anti-phospho-Src (pSrc-Tyr416; #2101), anti-DUSP6 (MKP3;

#3058), and anti-Cyclin D1 (Cyclin D1; #2926). The primary antibody against FGFR2

(F0300) was obtained from Sigma-Aldrich, the primary antibody against Src (OP07) was

obtained from EMD Millipore, and the primary antibody against EGFR (sc-03) was obtained

from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated secondary antibodies

ERK signal suppression and sensitivity to FGFR inhibitor

against rabbit IgG (NA934V) and mouse IgG (NA931V) were obtained from GE Healthcare

Life Sciences.

Results

FGFR inhibition affects gene expression via the MAPK pathway in an FGFR1

gene-amplified cancer

The MAPK and PI3K/AKT pathways are the main downstream effectors of FGFR

(1). We investigated the effects of CH5183284/Debio1347 and AZD4547 (both FGFR

inhibitors), CH4987655 (MEK inhibitor), and CH5132799 (PI3K inhibitor) on the

transcriptome of the NCI-H1581 lung cancer cell line harboring FGFR1 gene amplification.

We determined the effects of a 24-h exposure on gene expression. Expression values are

presented relative to the DMSO control and clustered by similarity (Figure 1A). We identified

2850 probes that were modulated by CH5183284/Debio 1347. Of these, 2273, 1675, and 158

were also affected by AZD4547, CH4987655, and CH5132799, respectively. Because FGFR

inhibition and MEK inhibition produced similar differential expression patterns, we analyzed

our data in the context of the gene set associated with ERK siRNA-mediated changes

(GSE15417). To analyze the coordinated change in expression of the gene signature, the

log-ratio to the control was calculated and distributions of log-ratios for probes in the

signature (blue) and not in the signature (red) were plotted as a histogram. The FGFR and

MEK inhibitors, but not the PI3K inhibitor, showed differential gene expression patterns

regulated by ERK (Figure 1B). These results suggest that the FGFR inhibitor mainly

modulates the MAPK pathway and not the PI3K pathway.

ERK signal suppression is associated with sensitivity to FGFR inhibition

We investigated the effect of CH5183284/Debio 1347 on the MAPK pathway in 14

ERK signal suppression and sensitivity to FGFR inhibitor

cancer cell lines harboring other FGFR alterations. Thirteen of the cell lines were sensitive to

CH5183284/Debio 1347 and harbored FGFR genetic alterations such as NCI-H716,

KATO-III, HSC-39, SNU-16, MFE-280, MFE-296, AN3CA, DMS-114, NCI-H520,

NCI-H1581, KMS-11, UM-UC-14, and RT-4; the other cell line, NCI-N87, was insensitive to

CH5183284/Debio 1347 and had no FGFR alterations. Data on genetic status and sensitivity

to CH5183284/Debio 1347 are available elsewhere (7) or in Supplementary Table S1. We

determined the effects of a 24-h exposure to 1 µM CH5183284/Debio 1347 on gene

expression relative to the DMSO control. We confirmed that the ERK (GSE15417) and MEK

signatures (23) were suppressed by CH5183284/Debio 1347 only in sensitive cell lines

harboring FGFR alterations (Figure 2A, Supplementary Figure S1). Consistent with this,

suppression of phospho-ERK by a 2-h treatment with CH5183284/Debio 1347 was observed

in seven cancer cell lines harboring FGFR alterations (Figure 2B). However, phospho-AKT

was suppressed only in four cell lines (NCI-H1581, SNU-16, KATO-III, and SUM-52PE),

suggesting that FGFRs likely depend on the MAPK pathway and that ERK signal

suppression is associated with sensitivity to FGFR inhibition.

Differential expression of DUSP6 and sensitivity to FGFR inhibition

Because FGFRs likely depend on the MAPK pathway, we searched for genes that

could be modulated by and associated with sensitivity to CH5183284/Debio 1347 among the

672 genes of the ERK signature (GSE15417). We calculated the ratio of the geometric mean

of each probe in 13 cell lines treated with DMSO and CH5183284/Debio 1347 (NCI-H716,

KATO-III, HSC-39, SNU-16, MFE-280, MFE-296, AN3 CA, DMS-114, NCI-H520,

NCI-H1581, KMS-11, UM-UC-14, and RT-4). We identified 47 genes (51 probes) that were

significantly modulated (p < 0.05; 2-fold change) by treatment (Figure 3A, Supplementary

Table S2). The most significantly modulated gene was the DUSP6 gene. Expression of

ERK signal suppression and sensitivity to FGFR inhibitor

DUSP6 was suppressed by CH5183284/Debio 1347 in every sensitive cancer cell line that we

tested but not in NCI-N87, which is insensitive to CH5183284/Debio 1347 (Figure 3B,

Supplementary Figure S2). PD173074, another FGFR inhibitor, also suppressed

phospho-ERK, resulting in decreased DUSP6 protein expression levels in the FGFR

inhibitor-sensitive cell lines after a 24-h treatment (Figure 3C). As expected, Phospho-AKT

level was not constantly suppressed. Notably, one FGFR1 gene-amplified but FGFR

inhibitor-insensitive cell line, ZR-75-1, did not show suppression of phospho-ERK and

DUSP6 expression. PD0325901, a MEK inhibitor, reduced phospho-ERK and DUSP6

protein levels in the FGFR inhibitor-sensitive and -insensitive cell lines. To show the

usefulness of DUSP6 as a pharmacodynamic marker of CH5183284/Debio 1347 in in vivo

model, we obtained the tumor tissues 4 h after the 11th daily administration of 100 mg/kg

CH5183284/Debio 1347. We utilized SNU-16 and DMS-114 as a CH5183284/Debio

1347-sensitive model and NCI-N87 and MKN-45 as an insensitive model. The tumor growth

inhibitory activities of CH5183284/Debio 1347 against those models are available

(Supplementary Table S3). Consistent with the in vitro findings, CH5183284/Debio 1347

suppressed phospho-ERK and decreased DUSP6 protein expression levels in the FGFR

inhibitor-sensitive xenograft models but not in the insensitive models (Figure 3D). These data

suggest that DUSP6 is the most reliable pharmacodynamic marker associated with the

efficacy of FGFR inhibitors in FGFR genetically altered cancers.

MAPK pathway suppression without feedback activation of bypass pathways could be

important for FGFR inhibitor activity in FGFR genetically altered cancers

To investigate the significance of MAPK pathway suppression by FGFR inhibitors,

we tested the sensitivity of FGFR-addicted cancer cell lines to MEK inhibitors, such as

CH4987655, CH5126766, or selumetinib. Although MEK inhibition suppressed DUSP6

ERK signal suppression and sensitivity to FGFR inhibitor

protein expression in FGFR genetically altered cancer cell lines, these cell lines were not

sensitive to the MEK inhibitor, whereas the B-RAF-mutated cell line (SK-MEL-1),

K-RAS-mutated cell line (HCT116), and NF-1-null cell line (MeWo) were sensitive to MEK

inhibitors (Figure 4). To clarify the mechanisms of innate resistance to MEK inhibitors, we

treated the SNU-16 cell line harboring FGFR2 gene amplification with CH5183284/Debio

1347, CH5126766 (RAF-MEK inhibitor), or CH5132799 (PI3K inhibitor), either alone or in

combination (Figure 5). CH5183284/Debio 1347 suppressed phospho-FGFR, phospho-AKT,

phospho-ERK, and DUSP6. In the presence of CH5126766-mediated suppression of

phospho-ERK and DUSP6, we observed elevated phosphorylation of FGFR, MET

proto-oncogene, receptor tyrosine kinase (MET), epidermal growth factor receptor (EGFR),

AKT, SRC proto-oncogene, non-receptor tyrosine kinase (SRC), and signal transducer and

activator of transcription 3 (STAT3). Feedback activation by MEK inhibition was abrogated

by CH5183284/Debio 1347. Thus, MAPK pathway suppression alone re-activated FGFR and

induced potent activation of other pathways. CH5132799 did not produce this result. DUSP6

and Cyclin D1 suppression, and thus MAPK pathway inhibition, was stronger with

CH5183284/Debio 1347 than with CH5126766. These results show that the FGFR inhibitor

suppressed the MAPK pathway more effectively than the MEK inhibitor alone without

feedback activation of other signals, thus leading to the efficacy of FGFR inhibitors in

FGFR-addicted cancers.

Discussion

Investigations of the signaling pathways associated with oncogenes provide

important information leading to combination therapies or increased understanding of

resistance mechanisms. For instance, in an endocrine therapy-resistant hormone

receptor-positive breast cancer, the PI3K/AKT pathway is aberrantly activated and a

ERK signal suppression and sensitivity to FGFR inhibitor

downstream kinase, S6 kinase, independently phosphorylates and activates the estrogen

receptor ligand (24). Therefore, combination of endocrine therapy with letrozole and PI3K

pathway inhibition by everolimus exhibits great clinical efficacy (25). The MAPK and

PI3K/AKT pathways are the main downstream effectors of FGFR (1). Every FGFR-altered

cancer cell line that was tested in the present study was dependent on the MAPK pathway but

not the PI3K/AKT pathway (Figure 1, 2). This is consistent with several genetic profiling

studies. FGFR and RAS mutations are mutually exclusive in bladder cancer and endometrial

cancer (26, 27); in contrast, FGFR mutations are frequently associated with mutations in the

PI3K/AKT pathway (26, 27). Conversely, when an FGFR-altered cancer also carries a KRAS

mutation, FGFR inhibitors are ineffective (28), suggesting that FGFR and RAS likely activate

the same pathway. Although MAPK is considered the main downstream effector of FGFR,

we have shown that MEK inhibitors are not always effective in FGFR-altered cancer because

of feedback activation (Figure 5). Similarly, PI3K inhibition enhanced HER2 signaling in

HER2 over-expressing breast cancer. In that context, the combined administration of PI3K

and HER2 inhibitors produced superior anti-tumor activity in comparison to monotherapy

(29). Therefore, the combination of FGFR and MEK inhibition could be another approach to

treat patients harboring FGFR alterations.

DUSP6 is a member of the dual-specificity protein phosphatase subfamily that

inactivates target kinases, such as ERK, by dephosphorylation. Over-expression of DUSP6

was observed in response to activated RAS or BRAF (30-34), representing an increase in

negative feedback of the MAPK pathway. Therefore, DUSP6 expression could reflect activity

of the MAPK pathway. In addition, a role of DUSP6 during early mouse development in

response to FGF signaling has been suggested (35, 36). In mouse embryos, DUSP6

contributes as a negative feedback regulator of FGF-stimulated ERK signaling. The DUSP6

loss-of-function mutant causes several symptoms, such as variably penetrant, dominant

ERK signal suppression and sensitivity to FGFR inhibitor

postnatal lethality, skeletal dwarfism, coronal craniosynostosis, and hearing loss. These

phenotypes are also characteristic of FGFR active mutations. Therefore, we suggest that

DUSP6 could be the most reliable pharmacodynamic marker of FGFR inhibition; the

responsiveness of DUSP6 expression could predict the efficacy of FGFR inhibitors.

Studying pharmacodynamic biomarkers will help accelerate the development of

molecular-targeted agents for the treatment of cancer (37, 38). In general, the gold-standard

pharmacodynamic assay is immunohistochemistry on a tumor sample. Several clinical studies

evaluated the phosphorylation status of the drug target or molecules on the downstream

pathway (39, 40). Although the evaluation of differential phosphorylation could be used to

assess inhibitor activity in the tumor, there are limitations to phospho-related assays. The loss

of immunoreactive phospho-AKT and phospho-ERK during sample procurement has been

reported (41). Therefore, we must consider multiple pre-analytical factors that influence the

stability of phosphorylated proteins during procurement and preservation of clinical samples

(42). Non-invasive samples such as circulating tumor cells and circulating tumor DNA

obtained from a liquid biopsy could be the next-generation source of pharmacodynamic

markers (43). In solid tumors, for instance, monitoring for EGFR-activating mutations

enables the prediction of anti-EGFR therapeutic efficacy in patients; identification of

acquired EGFR mutations in circulating DNA is also a way to guide therapeutic

decision-making (44, 45). The FGFR family incurs many types of genetic alterations in

multiple tumor types; therefore, assays must be developed to capture all genetic alterations in

circulating DNA. The detection of DUSP6 could overcome these issues, as assays would not

need to be developed for each FGFR alteration and the stability of phosphorylation would not

be an issue. Alternatively, DUSP6 mRNA could also be monitored in circulating tumor cells

or the exosome in liquid biopsy samples.

Monitoring of downstream molecules enables prediction of the efficacy of

ERK signal suppression and sensitivity to FGFR inhibitor

molecular-targeted drugs. Sensitivity to BYL719, a PI3K p110α selective inhibitor, in breast

cancer carrying a mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic

subunit alpha (PIK3CA) was associated with full inhibition of signaling through the CREB

regulated transcription coactivator 1 (TORC1) pathway. Conversely, cancer cells that were

not phospho-S6-suppressed were resistant to BYL719, although AKT phosphorylation was

inhibited (46). CH5126766, a RAF-MEK inhibitor, showed superior anti-tumor efficacy in

comparison to the MEK inhibitor alone (17). Although both inhibitors suppressed

phospho-ERK in tumors, CH5126766 more strongly suppressed downstream signaling targets

such as DUSPs and SPREADs (17). In FGFR-altered cancer cells, DUSP6 was more

significantly suppressed by CH5183284/Debio 1347 than by CH5126766 (Figure 5).

CH5126766 suppressed phospho-ERK but not the downstream ERK effector DUSP6,

suggesting that CH5126766 does not strongly suppress MAPK, resulting in the resistance of

FGFR-altered cancer cells to MEK inhibition. Therefore, monitoring of DUSP6 could be

more beneficial than that of phospho-ERK.

In summary, measurement of the status of downstream signaling could be used to

predict a molecule’s therapeutic efficacy. FGFR likely depends on the MAPK pathway, and

ERK signal suppression is associated with sensitivity to FGFR inhibition. Therefore, DUSP6,

which functions downstream of the ERK signal, is one of the most reliable pharmacodynamic

markers of the efficacy of an FGFR inhibitor. CH5183284/Debio 1347 is currently in phase I

clinical trials by Debiopharm International S.A. in patients harboring FGFR alterations

(NCT01948297).

Acknowledgments

ERK signal suppression and sensitivity to FGFR inhibitor

We thank Yasue Nagata for performing the pharmacological assays. We also thank

Anne Vaslin and Corinne Moulon from Debiopharm International S.A. for their helpful

discussions.

Grant Support

This study was funded by the Chugai Pharmaceutical Co., Ltd.

The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked as advertisement in accordance with 18

U.S.C. Section 1734 solely to indicate this fact.

ERK signal suppression and sensitivity to FGFR inhibitor

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ERK signal suppression and sensitivity to FGFR inhibitor

FIGURE LEGENDS

Figure 1. Differential expression in the presence of several pathway inhibitors in

NCI-H1581, an FGFR1-amplified cancer cell line

(A) Heatmap showing expression log-ratio to control (DMSO) for each probe after a 24-h

treatment with 1 µM CH5183284/Debio 1347, AZD4547 (FGFR inhibitor), CH4987655

(MEK inhibitor), or CH5132799 (PI3K inhibitor) in NCI-H1581 harboring FGFR1 gene

amplification (n = 2). Probes were clustered by similarity at the left side of the panel. (B)

Distributions of log-ratio to control (geometric mean of DMSO-treated samples) were plotted

as a histogram. The X-axis represents the log-ratio; the Y-axis represents the frequency. The

distribution pattern for probes in the ERK1/2 signature is blue; distributions of other probes

are red (n = 2)

Figure 2. MAPK pathway suppression by CH5183284/Debio 1347 in FGFR-altered

cancer cell lines

(A) Distributions of expression log-ratio to control (geometric mean of DMSO-treated

samples) were plotted as a histogram. The X-axis represents the log-ratio; the Y-axis

represents the frequency. Distribution patterns for probes in the ERK1/2 signature are blue;

other probes are red. Cells were treated with 1 µM CH5183284/Debio 1347 or DMSO for

24-h. Total RNA was analyzed by microarray. IC50 concentrations of CH5183284/Debio 1347

in anti-proliferative assay are provided. (B) Inhibition of ERK or AKT phosphorylation. After

a 2-h incubation with CH5183284/Debio 1347, cultures of DMS-114, NCI-H1581, SNU-16,

KATO-III, SUM-52PE, UM-UC-14, and KMS-11 cells were lysed and analyzed by western

blotting. Ampli: Amplification, Mut: Mutation

Figure 3. Modulation of DUSP6 expression in FGFR inhibitor-sensitive cancer cell lines

ERK signal suppression and sensitivity to FGFR inhibitor

(A) A volcano plot representation showing the magnitude (log2 ratio, x-axis) and significance

(log10 p-value, y-axis) of all genes in the ERK signature. The red circles represent the

significantly modulated probes and the white circles represent others. (B) Bar graphs show

the ratio of probes that recognize DUSP6 vs. DMSO in 14 cancer cell lines. The black bars

denote cell lines harboring FGFR alterations, and gray bars denote the FGFR wild-type cell

line. (C) Inhibition of DUSP6 expression or ERK phosphorylation. After a 24-h treatment

with PD173074 (FGFR inhibitor) at 3 µM or with PD0325901 (MEK inhibitor) at 1 µM,

cultures of NCI-H1581, DMS-114, AN3 CA, SNU-16, KMS-11, ZR-75-1, NCI-N87, and

HCT116 cells were lysed and analyzed by western blotting. (D) Pharmacodynamic response

in in vivo models. Mice bearing SNU-16, DMS114, NCI-N87, or MKN-45 cells were orally

administered CH5183284/Debio 1347 at 100 mg/kg, and the tumors were collected and lysed

at 4-h post-dosing (n=2). Lysates were then analyzed by western blotting. Ampli:

Amplification, Mut: Mutation

Figure 4. Sensitivity of FGFR-altered cancer cell lines to MEK inhibitors

Cells were seeded in 96-well plates and treated for 4 days with various concentrations of

CH4987655 (MEK inhibitor), CH5126766 (RAF-MEK inhibitor), or selumetinib (MEK

inhibitor) prior to viability assays.

Figure 5. Pathway biology upon MEK inhibitor treatment in FGFR2-amplified cancer

cells

After a 24-h treatment with CH5183284/Debio 1347, CH5132799 (PI3K inhibitor),

CH5126766 (RAF-MEK inhibitor), or combinations thereof, SNU-16 cancer cells harboring

FGFR2 gene amplification were lysed and analyzed by western blotting. The drug

concentrations were 0.3 or 1 µM.

Downloaded from mct.aacrjournals.org on October 3, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497 Figure 1. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A. B.

CH5183284 AZD4547 DMSO (FGFR inhibitor) (FGFR inhibitor) DMSO_1 DMSO_2 CH5183284_1 CH5183284_2 AZD4547_1 AZD4547_2 CH4987655_1 CH4987655_2 CH5132799_1 CH5132799_2

CH4987655 CH5132799 (MEK inhibitor) (PI3K inhibitor)

Frequency

x Log-ratio 2 0 -2 Color code

Downloaded from mct.aacrjournals.org on October 3, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497 Figure 2. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

FGFR2 altered cancers

NCI-H716 KATO-III HSC-39 SNU-16 MFE-280 MFE-296 AN3 CA (Amplification) (Amplification) (Amplification) (Amplification) (Mutation) (Mutation) (Mutation)

IC50: 0.013 μM IC50: 0.018 μM IC50: 0.050 μM IC50: 0.017 μM IC50: 0.41 μM IC50: 0.042 μM IC50: 0.054 μM

FGFR1 altered cancers FGFR3 altered cancers FGFR wild type DMS-114 NCI-H520 NCI-H1581 KMS-11 UM-UC-14 RT-4 NCI-N87

(Amplification) (Amplification) (Amplification) (t(4;14), Mutation) (Mutation) (Fusion) IC50: 10 μM IC50: 0.18 μM IC50: 0.30 μM IC50: 0.22 μM IC50: 0.14 μM IC50: 0.11 μM IC50: 0.35 μM y

Frequency x Log-ratio

CH5183284 (µM)

0 0.01 0.03 0.1 0.3 1 3 10 0 0.01 0.03 0.1 0.3 1 3 10 0 0.01 0.03 0.1 0.3 1 3 10 0 0.01 0.03 0.1 0.3 1 3 10 DMS-114 (FGFR1 ampli.) NCI-H1581 (FGFR1 ampli.) SNU-16 (FGFR2 ampli.) KATO-III (FGFR2 ampli.) SUM-52PE (FGFR2 ampli.) UM-UC-14 (FGFR3 mut) KMS-11 (t(4;14) FGFR3 mut) pERK Total ERK1/2 pAKT Total AKT

-6 SNU-16 DMS-114 NCI-N87 MKN45 (FGFR2 ampli.) (FGFR1 ampli.) DUSP6

p-value -4 (208892_s_at) 10 Log -2 DUSP6 (208891_at) Vehicle Vehicle Vehicle CH5183284 CH5183284 Vehicle Vehicle CH5183284 CH5183284 Vehicle Vehicle CH5183284 CH5183284 Vehicle Vehicle CH5183284 CH5183284 0 -6 -5 -4 -3 -2 -1 0 1 2 3 DUSP6 Log2 ratio B. pERK DUSP6: 208891_at DUSP6: 208892_s_at CH5183284 CH5183284 ERK1/2 DMSO DMSO 1.2 (1 µM) 1.2 (1 µM) 1 1 GAPDH

0.8 0.8

0.6 0.6

0.4 0.4

0.2 DMSO to Ratio 0.2 Ratio to DMSO to Ratio

0 0 RT-4 RT-4 RT-4 RT-4 AN3CA AN3CA AN3CA AN3CA SNU-16 HSC-39 SNU-16 HSC-39 KMS-11 KMS-11 SNU-16 HSC-39 SNU-16 HSC-39 KMS-11 KMS-11 KATO-III NCI-N87 KATO-III NCI-N87 KATO-III NCI-N87 KATO-III NCI-N87 MFE-280 MFE-296 MFE-280 MFE-296 MFE-280 MFE-296 MFE-280 MFE-296 DMS-114 DMS-114 DMS-114 DMS-114 NCI-H520 NCI-H716 NCI-H520 NCI-H716 NCI-H520 NCI-H716 NCI-H520 NCI-H716 UM-UC-14 UM-UC-14 UM-UC-14 UM-UC-14 NCI-H1581 NCI-H1581 NCI-H1581 NCI-H1581

C. Sensitive Insensitive NCI-H1581 DMS-114 AN3 CA SNU-16 KMS-11 ZR-75-1 (t(4;14), NCI-N87 HCT116 (FGFR1 ampli.) (FGFR1 ampli.) (FGFR2 mut) (FGFR2 ampli.) (FGFR1 ampli.) FGFR3 mut)

DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO PD173074 PD173074 PD173074 PD173074 PD173074 PD173074 PD173074 PD173074 PD0325901 PD0325901 PD0325901 PD0325901 PD0325901 PD0325901 PD0325901 PD0325901 DUSP6

pERK

ERK1/2

pAKT

AKT Downloaded from mct.aacrjournals.org on October 3, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497 Figure 4. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

CH4987655 CH5126766 selumetinib (MEK inhibitor) (RAF-MEK inhibitor) (MEK inhibitor)

0.2 1 2

0.8 0.15 1.5

0.6 (µM) (µM) (µM) 0.1 1 50 50 50

IC IC 0.4 IC

0.05 0.5 0.2

0 0 0 RT4 RT4 RT4 MeWo MeWo MeWo Kato III Kato III Kato III HSC-39 SNU-16 AN3 CA KMS-11 HSC-39 SNU-16 HSC-39 SNU-16 AN3 CA AN3 CA KMS-11 KMS-11 HCT-116 MFE-280 MFE-296 DMS 114 HCT-116 HCT-116 MFE-280 MFE-296 MFE-280 MFE-296 DMS 114 DMS 114 NCI-H520 NCI-H716 NCI-H520 NCI-H716 NCI-H520 NCI-H716 SK-MEL-1 SK-MEL-1 SK-MEL-1 UM-UC-14 UM-UC-14 UM-UC-14 NCI-H1581 NCI-H1581 NCI-H1581

1 2 3

1 2 3 1 2 3

1 1 1 BRAF NF KRAS FGFR FGFR FGFR BRAF NF KRAS FGFR FGFR FGFR BRAF NF KRAS FGFR FGFR FGFR

Downloaded from mct.aacrjournals.org on October 3, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2015; DOI: 10.1158/1535-7163.MCT-15-0497 Figure 5. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

(µM) CH5183284 - 0.3 1 - - - - 0.3 1 0.3 1 CH5183284 - 0.3 1 - - - - 0.3 1 0.3 1 (FGFR inhibitor) (FGFR inhibitor) CH5132799 - - - 0.3 1 - - 0.3 1 0.3 1 - - - - CH5132799 - - - 0.3 1 - - 0.3 1 0.3 1 - - - - (PI3K inhibitor) (PI3K inhibitor) CH5126766 - - - - - 0.3 1 - - - - 0.3 1 0.3 1 CH5126766 - - - - - 0.3 1 - - - - 0.3 1 0.3 1 (RAF/MEK inhibitor) (RAF/MEK inhibitor) pFGFR FGFR2

pMET MET

pEGFR EGFR pAKT AKT

pERK ERK pSrc Src

pSTAT3 STAT3

DUSP6

Cyclin D1

ERK signal suppression and sensitivity to CH5183284/Debio 1347, a selective FGFR inhibitor

Yoshito Nakanishi, Hideaki Mizuno, Hitoshi Sase, et al.

Mol Cancer Ther Published OnlineFirst October 5, 2015.

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