Futibatinib Is a Novel Irreversible FGFR 1-4 Inhibitor That Shows Selective Antitumor Activity Against FGFR-Deregulated Tumors

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Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Futibatinib is a novel irreversible FGFR 1-4 inhibitor that shows selective antitumor activity against FGFR-deregulated tumors

Hiroshi Sootome1, Hidenori Fujita1, Kenjiro Ito1, Hiroaki Ochiiwa1, Yayoi Fujioka1, Kimihiro Ito1, Akihiro Miura1, Takeshi Sagara1, Satoru Ito1, Hirokazu Ohsawa1, Sachie Otsuki1 , Kaoru Funabashi1, Masakazu Yashiro2, Kenichi Matsuo1, Kazuhiko Yonekura3, Hiroshi Hirai1

1Discovery and Preclinical Research Division, Taiho Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan. 2Department of Surgical Oncology Molecular Oncology and Therapeutics, Osaka City University Graduate School of Medicine, Osaka, Japan. 3Early Development Strategy & Planning, Taiho Pharmaceutical Co., Ltd., Kandanishiki-cho, Tokyo, Japan.

Running title: Irreversible FGFR1–4 inhibitor

Key words: Futibatinib, TAS-120, FGFR, covalent inhibitor, acquired resistance

Financial support: This study was funded by Taiho Pharmaceutical Co., Ltd.

Correspondence to: Hiroshi Sootome Discovery and Preclinical Research Division, Taiho Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, 300-2611, Japan Email: ([email protected]) Phone: +81-29-865-4527 Fax: +81-29-865-2170

Disclosure of Potential Conflicts of Interest: M. Yashiro reports research funding from Chiome Bioscience, Daiichi Sankyo, Eisai, Eli Lilly Japan, Five Prime, and Hayashi Kasei. All other authors have no conflicts of interest to disclose.

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Fibroblast growth factor receptor (FGFR) signaling is deregulated in many human cancers and

FGFR is considered a valid target in FGFR-deregulated tumors. Here we examine the preclinical profile of futibatinib (TAS-120; 1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-

pyrazolo[3,4-d] pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one), a structurally novel,

irreversible FGFR1-4 inhibitor. Among a panel of 296 human kinases, futibatinib selectively

inhibited FGFR1-4 with half-maximal inhibitory concentration (IC50) values of 1.4-3.7 nmol/L.

Futibatinib covalently bound the FGFR kinase domain, inhibiting FGFR phosphorylation and, in

turn, downstream signaling in FGFR-deregulated tumor cell lines. Futibatinib exhibited potent,

selective growth inhibition of several tumor cell lines (gastric, lung, multiple myeloma, bladder,

endometrial, and breast) harboring various FGFR genomic aberrations. Oral administration of

futibatinib led to significant dose-dependent tumor reduction in various FGFR-driven human

tumor xenograft models and tumor reduction was associated with sustained FGFR inhibition,

which was proportional to the administered dose. The frequency of appearance of drug-resistant

clones was lower with futibatinib than a reversible ATP-competitive FGFR inhibitor, and

futibatinib inhibited several drug-resistant FGFR2 mutants, including the FGFR2 V565I/L

gatekeeper mutants, with greater potency than any reversible FGFR inhibitors tested (IC50, 1.3-

50.6 nmol/L). These results indicate that futibatinib is a novel orally available, potent, selective,

and irreversible inhibitor of FGFR1-4 with a broad spectrum of antitumor activity in cell lines

and xenograft models. These findings provide a strong rationale for testing futibatinib in patients

with tumors oncogenically driven by FGFR genomic aberrations, with phase 1-3 trials ongoing.

Statement of Significance

Preclinical characterization of futibatinib, an irreversible FGFR1-4 inhibitor, demonstrates selective and potent antitumor activity against FGFR-deregulated cancer cell lines and xenograft models, supporting clinical evaluation in patients with FGFR-driven tumors.

Introduction

The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling axis is involved in many cellular processes that include proliferation, differentiation, migration, and

survival (1, 2). Deregulated FGFR signaling is associated with developmental disorders and

several cancers (2–4). FGFR genomic aberrations, such as gene amplifications,

fusions/rearrangements, activating mutations, and altered splicing have been reported in a range

of tumor types, including cholangiocarcinoma, breast, lung, gastric, bladder, hematologic, and

other malignancies; these aberrations have been tied to oncogenesis and to tyrosine kinase

inhibitor (TKI) resistance in these cancers (5–10). As a result of TKI resistance, cancers

characterized by FGF/FGFR aberrations are typically difficult to treat and have a poor prognosis

(11, 12). FGFR has emerged as a promising target in these FGFR-deregulated cancers that have a

substantial unmet need for novel therapies (1, 13, 14).

Several selective small-molecule FGFR inhibitors entered clinical development in

the past few years and have shown responses in patients with FGFR signaling pathway

aberrations in phase 1/2 clinical studies (14–20). However, the majority of these agents bind

reversibly to the adenosine triphosphate (ATP)-binding pocket of FGFRs. As has been observed

previously with epidermal growth factor receptor (EGFR) TKIs, the occurrence of drug

resistance caused by mutations within the drug-binding site is fairly common with reversible

ATP-competitive kinase inhibitors (12, 21). Consistent with this, reports of drug-resistant mutations with reversible FGFR inhibitors are emerging (22, 23).

Covalently binding kinase inhibitors inhibit kinase activity irreversibly and may

achieve superior potency along with a longer duration of action compared with conventional

reversible ATP-competitive inhibitors (21, 24, 25). Despite these potential advantages, the

development of covalently binding selective small-molecule pan-FGFR inhibitors has been slow, with PRN1371 among the few currently under clinical investigation (26).

The objective of this study was to describe the preclinical profile of the

investigational anticancer agent futibatinib (TAS-120; FBN). These data demonstrate that

futibatinib is a structurally novel, irreversible, highly selective FGFR1–4 inhibitor that potently

inhibits the growth of FGFR-deregulated cell lines and tumor xenografts. Consequently, these

findings support the clinical investigation of futibatinib in FGFR-driven tumors.

Materials and Methods

Chemical properties of futibatinib

Futibatinib (1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3,4-d] pyrimidin-

1-yl]-1-pyrrolidinyl]-2-propen-1-one) was synthesized using the procedure described in

International Patent Application WO 2013/108809 (27), in which futibatinib is described as

example 2 (Fig. 1A). The covalent binding of futibatinib to FGFR was assessed using liquid

chromatography mass spectrometric (LC-MS) analysis of the kinase domain of recombinant

FGFR2, which was incubated in the presence or absence of futibatinib (described in

Supplementary Methods).

Cell lines and reagents

The cell lines used in this study and their sources are listed in Supplementary Table S1. Cell lines

were authenticated by short tandem repeat profiling (Bio-Synthesis Inc.) or were used within 5

passages of original purchased stocks. The cells were confirmed to be free of mycoplasma by

PCR (CLEA Japan, Inc.). AZD4547 was synthesized at Taiho Pharmaceutical Co., Ltd.,

infigratinib and pemigatinib were purchased from MedChemexpress Co., Ltd., and erdafitinib

was purchased from Namiki Shoji Co., Ltd.

Kinase inhibition

The kinase inhibitory properties of futibatinib were investigated in a FGFR1–4 enzymatic assay.

As futibatinib binds to the FGFR ATP-binding pocket in the cytoplasmic kinase domain,

recombinant FGFR cytoplasmic domains were used in this assay. Phosphorylation of peptide

substrate was quantified by the off-chip mobility shift assay (MSA) using the LabChip3000

System (Caliper Life Science, USA). The ATP concentration used in each assay was similar to

the Km (Michaelis constant) for the respective kinase. Futibatinib kinase selectivity was assessed

against a panel of 296 kinases (Carna Biosciences, Inc., Japan); phosphorylation of each peptide

was quantified using the MSA or immobilized metal ion affinity-based fluorescence polarization

technologies.

Futibatinib-mediated FGFR kinase inhibition in FGFR-deregulated cell lines was

examined using immunoblotting and enzyme-linked immunosorbent assay (ELISA) with

antibodies against phosphorylated FGFR2 (detailed in Supplementary Methods).

Cell proliferation assay

Tumor cells were seeded into 96-well plates and treated with futibatinib or vehicle

(dimethylsulfoxide [DMSO]) for 72 hours. Tumor cell viability was determined by using the

CellTiter-Glo Luminescent Cell Viability Assay Reagent® (Promega, USA), and a multimode

plate reader was used to detect luminescence. The cell count at time zero (time at which the

compound was added) was used to determine the concentration of futibatinib that elicited 50%

growth inhibition (GI50).

Xenograft models

The antiproliferative activity of futibatinib was further evaluated in murine and rat xenograft

models. Animal studies were performed according to prescribed guidelines with the approval of

the institutional animal care and use committee of Taiho Pharmaceutical Co., Ltd. Tumor

xenografts were established in 6–12-week-old athymic nude (nu/nu) mice, severe combined

immunodeficient (NOD-SCID) mice, and nude rats (CLEA Japan, Inc., Japan) by subcutaneous

implantation of tumor cells (1 x 107 each of OCUM-2MD3 gastric cancer, KMS-11 multiple

myeloma, or RT-112/84 bladder cancer cells; 3.8 x 106 of MFM-223 breast cancer cells; or 5 x

106 of H1581 lung cancer cells) mixed 1:1 with Matrigel (BD Biosciences, USA) into the side

flank. Treatment with futibatinib or vehicle control (0.5 w/v% hydroxypropyl methylcellulose)

was initiated when the transplanted tumor reached a predetermined size of more than 0.2 cm3.

Futibatinib or vehicle control was administrated orally by gavage; each dose was administered to

multiple animals (mice, n=6; rats, n=5). Futibatinib was administered for 14 days, except for the

KMS-11 multiple myeloma model, in which futibatinib was dosed for 27 days. Futibatinib was

dosed once daily in OCUM-2M, KMS-11, and MFM-223 models; administration every other day

or twice per week was also tested. Animals were euthanized immediately at the end of treatment.

Tumor volume (measured by caliper) and animal body weight were monitored over the course of

the treatment. Statistical analysis was performed using 1-way ANOVA. Differences in mean

tumor volume between each dose group and control group were analyzed using Dunnett’s test.

Pharmacodynamic analysis

Tumor samples were collected at 4, 8, 12, 18, and 24 hours after a single dose of futibatinib or

vehicle. Frozen tumors from xenograft models were lysed in standard cell lysis buffer or reagents

according to the manufacturer’s instructions (DuoSet IC Kit, R&D Systems Inc., USA) and

phosphorylated FGFR quantified by ELISA or Western blotting (described in Supplementary

Methods).

FGFR inhibitor-resistant clones

Resistant clones were generated by continuously exposing OCUM-2MD3 cells to futibatinib or

AZD4547. The futibatinib dose was increased incrementally to 20 nmol/L over 10 weeks, and

the AZD4547 dose was increased to 400 nmol/L over 11 weeks. Resistant cells were maintained

at these final doses.

Expression vectors and transfections

Mutant FGFR2 expression vectors were constructed using site-directed mutagenesis; mutant and

wild-type constructs were transfected into HEK293T cells using Lipofectamine 2000 according to the manufacturer’s protocol. Transfected cells were treated with futibatinib, stimulated with

FGF-7 and heparin, and lysates were analyzed for FGFR2 phosphorylation. Additional details

are provided in Supplementary Methods.

Results

Futibatinib, a potent covalent (irreversible) small-molecule inhibitor, exhibited high

selectivity for FGFR1–4 among a panel of 296 kinases resulting in inhibition of FGFR

phosphorylation and downstream signaling pathways

Futibatinib potently inhibited the kinase activities of recombinant FGFR1, 2, 3, and 4 in a dose-

dependent manner, with half-maximal inhibitory concentrations (IC50 ± standard deviation [SD])

values of 1.8 ± 0.4, 1.4 ± 0.3, 1.6 ± 0.1, and 3.7 ± 0.4 nmol/L, respectively (Fig. 1A). The kinase

selectivity of futibatinib was assessed against a panel of 296 human kinases using 100 nmol/L

futibatinib, which is 50 times the IC50 for FGFR2/3 (Supplementary Table S2). Futibatinib

showed strong selectivity for FGFR1–4 in this panel; only 3 non-FGFR kinases showed more

than 50% inhibition by futibatinib: mutant RET (S891A; 85.7%), MAPKAPK2 (54.3%), and

CK1 (50.7%). These results highlighted the high selectivity of futibatinib inhibition for

FGFR1–4, with very low off-target activity against other kinases.

The covalent binding of futibatinib to FGFR was assessed by LC-MS analysis of the

recombinant FGFR2 kinase domain incubated in the presence or absence of futibatinib. In the

presence of futibatinib, the observed mass of the FGFR2 kinase domain was 418.5 ± 0.2 Da

higher than that in the absence of futibatinib (Fig. 1B). This mass difference almost exactly

corresponded to the molecular weight of futibatinib (418.45 Da), which indicated covalent

binding of futibatinib to the FGFR2 kinase domain.

As futibatinib bound to the FGFR2 kinase domain in vitro, the effect of futibatinib on

FGFR phosphorylation and signaling was examined in FGFR-deregulated cell lines. Treatment

of the FGFR2-amplified gastric cancer cell lines OCUM-2MD3 and OCUM-2M with increasing

doses of futibatinib for 30 minutes resulted in dose-dependent inhibition of FGFR

phosphorylation as demonstrated by Western blotting (Fig. 2A) and ELISA (Fig. 2B), with an

IC50 value of 4.9 ± 0.1 nmol/L obtained by ELISA. Futibatinib treatment also resulted in similar

reductions in Akt and extracellular-signal-regulated kinase (ERK) phosphorylation levels in

proportion to that of FGFR phosphorylation (Fig 2A). Similar results were observed with SNU-

16, another gastric cancer cell line with FGFR2 amplification, and OPM-2 and KMS-11, both

multiple myeloma cell lines with FGFR3 translocations (Supplementary Fig. S1A–C). These

results suggest that futibatinib potently inhibits FGFR phosphorylation and downstream

signaling via mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase

(PI3K)/Akt pathways.

Futibatinib demonstrated potent antiproliferative activity in diverse cancer cell lines

harboring FGFR genomic aberrations and antitumor activity in a number of FGFR-

deregulated tumor xenograft models

The antiproliferative activity of futibatinib was assessed in cancer cell lines from diverse tissue

origins (gastric, breast, lung, endometrial, and bladder cancer, as well as multiple myeloma) with

or without FGFR genomic aberrations by examining cell growth after 3 days of exposure to

futibatinib. Across all tumor types studied, futibatinib inhibited the growth of cell lines with

various FGFR genomic aberrations, but not of cell lines that did not harbor such aberrations

(Table 1; Fig. 3A). These FGFR aberrations included FGFR1/2 amplifications (breast cancer),

FGFR1 amplifications (lung cancer), FGFR2 amplifications (gastric cancer), FGFR2 point

mutations (endometrial cancer), FGFR3 fusions (bladder cancer), and FGFR3 translocations

(multiple myeloma; Table 1) (5, 6, 28–30). Among cell lines with FGFR genomic aberrations,

the potency of futibatinib inhibition ranged between GI50 values of ~1 and 50 nmol/L for most

cell lines. At least 3 cell lines with FGFR aberrations did not show sensitivity to futibatinib;

these included the FGFR3 fusion-expressing SW780 bladder cancer cell line (GI50 4800 nmol/L)

and multiple myeloma cell lines H929 (GI50 1000 nmol/L) and LP-1 (GI50 >3000 nmol/L), both

of which harbor FGFR3-t(4;14) translocations. Together, these results indicate that futibatinib

demonstrated antiproliferative activity against FGFR-deregulated cancer cell lines of diverse

tissue origins. Antiproliferative activity was specific to tumor cell lines harboring FGFR

genomic aberrations, and inhibition was seen regardless of FGFR subtype or the nature of the genetic alteration (amplification, fusion, or point mutation).

The antitumor activity of futibatinib was further evaluated in murine or nude rat

xenograft models having tumors expressing FGFR genomic aberrations. Mice were administered

oral futibatinib once daily for 2 weeks (except for KMS-11, as explained below). In the FGFR2- amplified OCUM-2MD3 gastric cancer model, once-daily administration of futibatinib at 0.15

mg/mL resulted in statistically significant tumor growth inhibition, and dosing at 0.5, 1.5, and 5

mg/kg resulted in dose-dependent tumor reduction (Fig. 3B). There was no early evidence of

resistance during the treatment period, even in mice treated with lower futibatinib doses (eg, 0.5

mg/kg). In the FGFR3-fusion–positive KMS-11 multiple myeloma model, significant tumor

regression was observed at 5 mg/kg futibatinib (Fig. 3C). Because tumor regression in this model

was observed beginning on day 12 with definite tumor regression on day 15, dosing was

continued until day 27 in this model. In the FGFR1/2-amplified MFM-223 breast cancer

xenograft model, robust growth inhibition was noted with daily futibatinib doses ranging from

12.5 to 50 mg/kg (Fig. 3D).

Preliminary experiments were also performed to examine intermittent futibatinib

dosing schedules (every other day or twice weekly) in FGFR-deregulated xenograft mouse

(OCUM-2MD3) or nude-rat (RT-112 and H1581) models. Tumor reduction was observed with

intermittent dosing in the OCUM-2MD3 gastric cancer and RT-112 bladder carcinoma models

(Supplementary Fig. S2A–C), but not in the H1581 non-small cell lung cancer (NSCLC) model

(Supplementary Fig. S2D). Tumor growth curves for individual mice or rats are shown in

Supplementary Fig. S3A–C (once-daily dosing) and Supplementary Fig. S4A–D (intermittent

dosing). Collectively, these results highlight potent and broad-spectrum antitumor efficacy of

futibatinib against mouse or rat xenograft models harboring various FGFR aberrations.

All once-daily dosing regimens of futibatinib were well tolerated in mice and did not

induce body weight reduction during the experiments. In our experiments in the RT-112 and

H1581 nude-rat xenograft models, hyperphosphatemia was observed in rats after futibatinib

dosing, an effect that has been previously reported with other FGFR inhibitors and was expected

given the mechanism of action of futibatinib (31). The hyperphosphatemia observed was

transient, and phosphate levels returned to normal 48 hours after futibatinib dosing.

Futibatinib demonstrated an inhibitory effect on FGFR phosphorylation and tumor

growth that was sustained after short drug exposures and proportional to the administered

dose

Because futibatinib binds covalently (irreversibly) to a unique cysteine residue in the ATP- binding pocket of FGFRs (32), the inhibitory effect of futibatinib on FGFR kinase activity was

expected to persist after even short drug exposures. The association between futibatinib FGFR

kinase inhibition and antitumor activity was assessed both in cell lines and in xenograft models.

Duration of FGFR phosphorylation inhibition and antiproliferative effect in cancer cell lines

To assess the duration of FGFR kinase inhibition, OCUM-2M and SNU-16 cells were treated with futibatinib for 1 hour, followed by drug washout and quantification of FGFR

phosphorylation. Without washout, phosphorylation of FGFR2 in both OCUM-2M and SNU-16

cell lines remained inhibited with 10 nmol/L futibatinib; however, with washout of futibatinib,

FGFR phosphorylation levels reversed to that of control within 6 hours (Fig. 2B; Supplementary

Fig. S1A). Similar results were obtained in the OPM-2 and KMS-11 cell lines (Supplementary

Fig. S1B–C). In these cell lines, similar recovery of FGFR phosphorylation levels was observed

after washout experiments with AZD4547 (Supplementary Fig. S5A–B). Experiments with

brefeldin A (which suppresses new protein synthesis) indicated that only 20% of phosphorylated

FGFR protein remained after 4 to 8 hours of treatment (detailed in the Supplement;

Supplementary Fig. S5C–E), which suggested a high turnover of FGFR in the cell lines studied.

This high turnover explains why a short exposure of futibatinib did not cause sustained inhibition

of FGFR in these cell lines.

To determine the optimal exposure time of futibatinib for antitumor activity in

FGFR-deregulated cell lines, the FGFR2-amplified OCUM-2M gastric cancer cell line was treated with futibatinib for 4, 8, 24, 48, or 72 hours, followed by drug washout. Cell viability was then monitored at 72 hours. Maximal growth inhibition was observed with 24 hours of treatment

proportional to the dose administered, and no additional antiproliferative effect was observed

when treatment was extended beyond 24 hours (Fig. 4A). These results indicated that continuous

futibatinib exposure up to 24 hours was essential for maximal efficacy in futibatinib-sensitive

FGFR-deregulated cells. This was consistent with the high turnover of FGFR in these cells as

observed above. However, futibatinib treatment for longer than 24 hours did not result in

additional antiproliferative activity.

Duration of inhibition of FGFR phosphorylation in xenograft models

The correlation between tumor regression and FGFR kinase inhibition with futibatinib treatment

was investigated in xenograft models. Futibatinib was orally administered to OCUM-2MD3- tumor–bearing nude mice at doses of 0.5, 1.5, and 5 mg/kg; xenografts were harvested 4 hours

after a single dose of futibatinib, and phosphorylation of FGFR2, ERK, and Akt was measured

by Western blotting. As shown in Fig. 4B, futibatinib exhibited dose-dependent inhibition of

FGFR2 phosphorylation in the OCUM-2MD3 tumors. In addition, dose-dependent inhibition of

ERK phosphorylation (which is downstream of FGFR) was also observed, although Akt

phosphorylation was only marginally inhibited. This indicated that FGFR phosphorylation and

downstream signaling were inhibited in xenograft models.

To understand the relationship between FGFR kinase inhibition and antitumor

activity in the preclinical models, the duration of FGFR inhibition was determined at once-daily

doses of 1.5, 5, and 15 mg/kg. The serum half-life of futibatinib was between 0.25 and 1 hour in

these models (Supplementary Fig. S6). FGFR2 phosphorylation was potently inhibited (≥80%) at

4 hours after single doses of either 1.5 or 5 mg/kg of futibatinib (Fig. 4C). The inhibition of

FGFR2 phosphorylation was partially sustained at 8 hours at these doses (~50%), with a return to

control levels at 12 hours. In the 15-mg/kg dosing group, ~90% inhibition of FGFR

phosphorylation was observed 8 hours after initial drug administration, and at 12 hours, FGFR

inhibition remained at ~50%. These data showed sustained futibatinib-mediated inhibition of

FGFR phosphorylation in the xenografted tumors and confirmed that the in vivo antitumor

activity of futibatinib was associated with dose- and time-proportional pharmacodynamic modulation of FGFR2 phosphorylation, even though the futibatinib serum half-life was found to

be short.

Futibatinib was associated with a low risk of drug resistance and showed potent inhibition

of FGFR mutants resistant to reversible ATP-competitive FGFR inhibitors

To assess the risk of drug resistance with futibatinib treatment, FGFR2-amplified OCUM-2MD3

gastric cancer cells were propagated in increasing concentrations of futibatinib or the reversible

ATP-competitive FGFR inhibitor AZD4547 for comparison, until maximum concentrations for

viability were reached (20 nmol/L for futibatinib and 400 nmol/L for AZD4547). A total of 12

resistant clones survived at 20 nmol/L futibatinib (a 13-fold higher concentration than the IC50 value for wild-type FGFR2) compared with 170 resistant clones that were obtained through

exposure to 400 nmol/L AZD4547 (45-fold higher than the IC50 for wild-type FGFR2; Fig. 5A;

Supplementary Table S3). This suggested that tumors with FGFR2 genomic aberrations exposed

to futibatinib were less prone to the development of resistant clones than those exposed to a

reversible ATP-competitive inhibitor such as AZD4547.

To characterize the mutations in the resistant clones, cDNA encoding the kinase domain

of FGFR2 was sequenced. In 61.2% (104/170) of clones resistant to AZD4547, the K660N

mutation was identified in the kinase domain (Fig. 5B), but this mutation was not present in futibatinib-resistant clones. Futibatinib potently inhibited FGFR phosphorylation and growth of

AZD4547-resistant OCUM-2MD3 clones, with IC50 values of 3.1 nmol/L and 4.8 nmol/L,

respectively (Supplementary Tables S4 and S5). On the other hand, the mean AZD4547 IC50 for growth increased from 6.1 nmol/L in the parental cells to 158.6 nmol/L in resistant cells.

Infigratinib (another reversible ATP-competitive FGFR inhibitor) also showed cross-resistance

toward AZD4547-resistant clones, with the IC50 for growth increasing from 7.6 nmol/L in

parental cells to 55.6 nmol/L in resistant cells (Supplementary Table S5). These results indicated

potent activity of futibatinib against tumor cells resistant to treatment with reversible ATP-

competitive FGFR inhibitors.

We then determined whether futibatinib was effective against other mutations resistant to

reversible ATP-competitive FGFR inhibitor treatment. The N550H and E566G mutations in the

FGFR2 hinge region have been reported to cause resistance to dovitinib (33). K660M within the

FGFR2 activation loop is an activating mutation reported in breast, endometrial, and cervical

cancer (29), and V565I is a gatekeeper mutation, frequently seen in drug-resistant tumors (33).

We generated 4 mutant FGFR2 constructs with these individual mutations and transfected them

into HEK293T cells along with a wild-type FGFR2 construct as a control. The activity of the constructs was confirmed by detection of autophosphorylation activity, and the effect of

futibatinib and reversible ATP-competitive inhibitors AZD4547 and infigratinib on FGFR2

autophosphorylation was determined using phosphorylated FGFR2 ELISA. The activity of

AZD4547 and infigratinib is shown in Fig. 6A: robust inhibition of wild-type FGFR was

observed as expected. Futibatinib showed potent inhibitory activity against all mutants, with IC50

values ranging from 1.3 nmol/L (against V565I) to 5.2 nmol/L (against K660M), which was

similar to its inhibitory potency against wild-type FGFR2 (0.9 nmol/L). In contrast, the activity

of both reversible ATP-competitive FGFR inhibitors was significantly lower with the 4 mutants

than with wild-type.

Very recently, 2 FGFR inhibitors, erdafitinib and pemigatinib, were approved for the

treatment of patients with advanced/metastatic FGFR-aberrant urothelial cancer or cholangiocarcinoma, respectively (34, 35). The activity of futibatinib and these agents was

evaluated with 3 representative FGFR2 mutants: N550K (hinge region), K660M (activation

loop), and V565L (gate-keeper) (Fig. 6B). All 3 inhibitors showed similar inhibitory activity

against wild-type FGFR2. Futibatinib demonstrated the strongest inhibition of N550K and

V565L, with IC50 values only 3- and 14-fold higher than that with wild type, respectively. In

contrast, erdafitinib and pemigatinib demonstrated limited activity against these 2 mutations:

relative to wild type, IC50 values for erdafitinib were 10- and 34-fold higher and those for

pemigatinib were 83- and 236-fold higher with the N550K and V565L mutants, respectively.

Together, these results suggested that the risk of drug resistance with futibatinib was low

and demonstrated potent inhibitory activity of futibatinib against FGFR mutants that are resistant

to reversible ATP-competitive inhibitors.

Discussion

In this manuscript, we report the identification of futibatinib, a potent, selective, and covalent

(irreversible) small-molecule inhibitor of FGFR1–4, and its first pharmacologic characterization

as an orally bioavailable antitumor agent. Futibatinib demonstrated remarkable selectivity for

FGFR among 296 kinases and was found to bind covalently to the FGFR kinase domain. The

binding mode of futibatinib to FGFR was shown to be quite different from known reversible

ATP-competitive FGFR inhibitors, such as AZD4547, infigratinib, erdafitinib, or pemigatinib, or

irreversible FGFR inhibitors such as PRN1371 (26, 32, 36, 37). Recent structural analysis of the

futibatinib–FGFR complex revealed that futibatinib targets the P-loop in the ATP-binding pocket

of the FGFR tyrosine kinase domain, forming a rapid covalent adduct with a unique cysteine

upon contact (32). Futibatinib is distinct in its ability to capture a number of conformations of the

highly flexible FGFR P-loop. Futibatinib showed potent inhibition of all 4 FGFR isoforms

(FGFR1–4) at nearly equivalent single-digit nanomolar IC50 values, in contrast to both AZD4547

and infigratinib, which showed strong inhibition of FGFR1–3, but only marginal inhibition of

FGFR4 in earlier studies (36, 38, 39). Similarly, PRN1371 has been reported to exhibit a 50-fold

lower affinity for FGFR4 than for FGFR1–3 (26).

Futibatinib selectively suppressed cell growth of cancer cell lines harboring FGFR

genomic aberrations, regardless of tumor type or type of FGFR aberration, which included

activating mutations, gene amplifications, or translocations in FGFR1–3. The extent of growth

inhibition was similar in cell lines with FGFR1, FGFR2, or FGFR3 genomic aberrations (GI50

ranging from 1 to ~50 nmol/L), which is consistent with the finding that futibatinib showed

inhibition of all FGFR isoforms at nearly equivalent potency in biochemical experiments. Cell

lines without FGFR genomic aberrations were insensitive to futibatinib. Although certain FGFR-

deregulated cell lines (H929 and LP-1 [multiple myeloma] and SW780 [bladder cancer]) were

also insensitive to futibatinib, further characterization of these cell lines provided possible

explanations. H929 cells were found to have an activating NRAS mutation, the FGFR3 mutation

in LP-1 cells was determined not to be an activating mutation (40), and the relative expression

level of FGFR3 was low in the SW780 bladder cancer cell line compared with the other FGFR3-

deregulated bladder cancer cell lines, RT4 and RT112/84 (30). Overall, the antiproliferative

effect of futibatinib in cancer cells was consistent with its highly selective biochemical profile.

Futibatinib also demonstrated potent antitumor activity in multiple xenograft models when administered orally on a once-daily schedule. Dose-dependent tumor regression was

observed across multiple tumor types, consistent with observations in the cancer cell lines.

Because futibatinib is a covalent inhibitor, it was expected that its inhibitory activity

would persist even after short periods of exposure in the cells, as has been observed with other

covalent inhibitors (26, 41). However, as FGFR turnover was determined to be rapid (half-life

~4 hours) in the cell lines used in our study, the duration of futibatinib inhibition could not be

accurately estimated. Nevertheless, evaluation of the duration of futibatinib exposure necessary

for antiproliferative effects indicated that 24 hours was sufficient to achieve maximal growth

inhibition in cancer cell lines. The sustained inhibitory effect of futibatinib was confirmed in

xenograft models, even though the serum half-life of futibatinib was found to be short.

Antitumor efficacy was noted beginning at 5 mg/kg in the OCUM-2MD3 model, and this

correlated with inhibition of FGFR2 phosphorylation, which lasted for up to 8 hours after the

first dose. At doses of 15 mg/kg, significant FGFR2 inhibition (up to 50%) was observed for up

to 12 hours after initial dose administration. The short serum half-life of futibatinib could

provide a possible explanation for the loss of FGFR2 inhibition beyond 18 hours of dosing.

Given the sustained inhibition of FGFR kinase activity with futibatinib, intermittent

futibatinib dosing schedules (every other day or twice weekly) were examined in FGFR-

deregulated xenograft models. Although robust antitumor efficacy was observed with

intermittent dosing in some tumor types (gastric cancer [OCUM-2MD3] and bladder carcinoma

[RT-112]), the antitumor effect was not optimal in other tumor types (NSCLC [H1581]). On this

basis, a continuous dosing regimen was chosen to maximize the efficacy of futibatinib in clinical

studies. In initial results of a phase 1 dose-escalation study with futibatinib in patients with

advanced solid tumors, responses were achieved in patients who received once-daily futibatinib

(42–44). In these patients, maximum plasma concentrations of futibatinib were achieved 2.2

hours after dosing, and the plasma half-life was determined to be 3.3 hours (42). Efficacy has

also been observed with once-daily futibatinib in patients with advanced/metastatic intrahepatic

cholangiocarcinoma in an ongoing phase 2 study (objective response rate, 34.3%) (45).

The appearance of resistance mutations is a major concern in the use of molecularly targeted kinase inhibitors. Consistent with this, secondary FGFR kinase domain mutations were

reported to be an important mechanism of clinically acquired resistance to the reversible ATP-

competitive FGFR inhibitor infigratinib (23). Although encouraging antitumor activity was

observed in infigratinib-treated patients with FGFR2 fusion-positive cholangiocarcinoma in a

phase 2 trial, acquired resistance inevitably developed, limiting the durability of response (15).

Genomic analysis revealed a number of point mutations in the FGFR2 kinase domain (N549H,

N549K, E565A, K641R, or K659M), as well as the gate-keeper mutations V565F and V565I

(23). Similarly, AZD4547 has been shown to be susceptible to acquired resistance (46). Data on

acquired resistance with the newer inhibitors erdafitinib and pemigatinib (which have shown

efficacy in patients with advanced cancers [17, 20]) have yet to be reported. The results reported

here, however, revealed reduced in vitro activity of these 2 inhibitors against FGFR2 mutants

associated with drug resistance.

Our experiments in FGFR-deregulated gastric cancer cell lines indicated that in

contrast to the reversible ATP-competitive inhibitor AZD4547, very few resistant clones

appeared with prolonged futibatinib treatment, and no mutations were observed in the FGFR2

kinase domain in futibatinib-resistant clones. Futibatinib also demonstrated robust activity

against FGFR mutations known to be resistant to reversible ATP-competitive FGFR inhibitors.

Specifically, potent inhibition of the V565I and V565L gatekeeper mutants of FGFR2 was

observed, in addition to other drug-resistant mutants; IC50 values with the mutants assayed in this

study were generally comparable to that obtained with wild-type FGFR2.

There are at least 2 possible explanations for the observed activity of futibatinib

against resistant FGFR mutations. The first is related to the covalent futibatinib–FGFR binding

interaction: the rapid covalent bond formation between the cysteine in the ATP-binding pocket

and futibatinib is less likely to be affected by mutations in the ATP-binding pocket that reduce

the binding affinity of reversible ATP-competitive FGFR inhibitors. As covalent complexes are

generally stable, this would allow effective target inhibition. Other irreversible FGFR inhibitors

such as FINN-2, FINN-3, and PRN1371 have similarly shown activity against drug-resistant

FGFR2 mutations, including gatekeeper mutations (26, 47). This provides support for the use of

irreversible FGFR inhibitors in overcoming the resistance associated with reversible ATP-

competitive FGFR inhibitors. Secondly, the amino acid residues within the FGFR ATP-binding

pocket involved in binding interactions with futibatinib are different than those binding reversible ATP-competitive FGFR inhibitors. The binding of these inhibitors occurs primarily

within the hinge region of the FGFR ATP-binding pocket (where drug-resistant mutations often

occur), whereas futibatinib interacts with a reactive cysteine in the P-loop (32, 36). These results

support the use of futibatinib in patients with resistance to prior TKI regimens. Indeed, in a

preliminary report, clinical responses were observed with futibatinib in patients with

cholangiocarcinoma that was resistant to reversible ATP-competitive inhibitor treatment (48).

Future research with all of these agents is warranted to gain insight into resistance mechanisms

with FGFR inhibitors and to clarify the role of futibatinib within the changing FGFR inhibitor

landscape.

There are several irreversible FGFR inhibitors currently under investigation.

PRN1371, which demonstrated robust FGFR1–3 inhibitory activity in preclinical experiments,

has also been reported to potently inhibit several FGFR2 and FGFR3 drug-resistant mutants, but

was shown to have reduced activity against the common drug-resistant gatekeeper mutant

(V561M) of FGFR1 (26). A phase 1 trial (NCT02608125) of this drug is ongoing in patients

with advanced tumors. FINN-2 and FINN-3 are irreversible FGFR inhibitors that have

demonstrated in vitro inhibition of FGFR1 and FGFR2 gatekeeper mutants but have not yet been

tested in the clinic (47). Other irreversible inhibitors in development include BLU9931 and

fisogatinib, which target Cys522 in the hinge region of FGFR4 and selectively inhibit FGFR4,

but have weak inhibitory activity against FGFR1–3. These compounds are only active in cell

lines with FGFR4-pathway deregulation (49). Fisogatinib has shown antitumor activity in

patients with advanced hepatocellular carcinoma with aberrant FGFR4-pathway signaling in a

phase 1 study (NCT02508467) (50). Among all irreversible inhibitors, futibatinib remains the

most advanced in clinical development, with preliminary results of phase 1 and 2 trials reported

and a phase 3 trial ongoing.

In summary, the results of this study demonstrate that futibatinib is a potent,

irreversible, highly selective inhibitor of FGFR1–4 that exhibits broad antiproliferative activity

in FGFR-deregulated cancer lines and animal tumor models. Sustained FGFR kinase inhibition

was noted in preclinical models. Futibatinib was also associated with a low risk of drug

resistance and exhibited activity against drug-resistant FGFR mutants. The preclinical and

pharmacologic profiles of futibatinib provide strong support for clinical testing of futibatinib in

patients with advanced FGFR-driven tumors and form the basis for ongoing phase 1/2

(NCT02052778), phase 2 (NCT04024436; NCT04189445), and phase 3 (NCT04093362) trials

being conducted in patients with advanced tumors harboring FGFR aberrations.

Authors’ Contributions

Conceptualization: Hiroshi Sootome, Takeshi Sagara, Satoru Ito, Sachie Otsuki, Hiroshi Hirai

Resources: Hiroshi Sootome, Hidenori Fujita, Kenjiro Ito, Hiroaki Ochiiwa, Yayoi Fujioka,

Kimihiro Ito, Akihiro Miura, Hirokazu Ohsawa, Kaoru Funabashi, Masakazu Yashiro, Kenichi

Matsuo

Data curation:

Formal analysis: Hiroshi Sootome, Hidenori Fujita, Takeshi Sagara, Satoru Ito, Sachie Otsuki

Supervision: Hiroshi Sootome, Kazuhiko Yonekura, Hiroshi Hirai

Funding acquisition:

Validation:

Investigation: Hiroshi Sootome, Hidenori Fujita, Kenjiro Ito, Hiroaki Ochiiwa, Yayoi Fujioka,

Kimihiro Ito, Akihiro Miura, Hirokazu Ohsawa, Kaoru Funabashi, Masakazu Yashiro, Kenichi

Matsuo

Visualization: Hiroshi Sootome, Akihiro Miura, Takeshi Sagara, Sachie Otsuki, Masakazu

Yashiro, Hiroshi Hirai

Methodology: Hiroshi Sootome, Hirokazu Ohsawa

Writing—original draft: Akihiro Miura, Takeshi Sagara, Sachie Otsuki, Masakazu Yashiro,

Hiroshi Hirai

Project administration:

Writing—review and editing: Hiroshi Sootome, Hidenori Fujita, Kenjiro Ito, Hiroaki Ochiiwa,

Yayoi Fujioka, Kimihiro Ito, Akihiro Miura, Takeshi Sagara, Satoru Ito, Hirokazu Ohsawa,

Sachie Otsuki, Kaoru Funabashi, Masakazu Yashiro, Kenichi Matsuo, Kazuhiko Yonekura,

Hiroshi Hirai

Acknowledgements:

The authors thank TAS-120 project members in Tsukuba and Tokushima Research institute, and

Taiho Pharmaceutical Co., Ltd. for the support they provided for this work.

This study was sponsored by Taiho Oncology, Inc. and Taiho Pharmaceutical Co., Ltd. Medical

writing and editorial assistance was provided by Vasupradha Vethantham, PhD, and Anne

Cooper of Ashfield Healthcare Communications (Lyndhurst, NJ, USA) and funded by Taiho

Oncology, Inc.

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Table 1. Antiproliferative activity of futibatinib in cancer cell lines

Cancer type Cell line FGFR genomic aberration GI50 (µmol/L) Mean ± SD Gastric OCUM-2M FGFR2 amplification 0.00075 ± 0.00004 OCUM-2MD3 FGFR2 amplification 0.00089 ± 0.00005 SNU-16 FGFR2 amplification 0.00091 ± 0.00011 AGS No FGFR aberrations 11 ± 2 SNU-1 No FGFR aberrations ≥11 MKN45 No FGFR aberrations 13 ± 2 Breast MFM-223 FGFR1 amplification 0.00078 ± 0.00012 FGFR2 amplification MDA-MB-134-VI FGFR1 amplification 0.056 ± 0.029 SK-BR-3 No FGFR aberrations 6.7 ± 3.3 MCF-7 No FGFR aberrations 9.1 ± 3.4 Lung NCI-H1581 FGFR1 amplification 0.00093 ± .00020 DMS-114 FGFR1 amplification 0.062 ± 0.094 LK-2 FGFR1 amplification 0.55 ± 0.74 NCI-H1975 No FGFR aberrations 9.9 ± 3.9 A549 No FGFR aberrations 11 ± 2 Endometrial AN3 CA FGFR2 (K310R +N549K) 0.011 ±0.004 MFE-280 FGFR2 (B252W) 0.016 ± 0.009 MFE-296 FGFR2 (N549K) 0.31 ± 0.13 KLE No FGFR aberrations 3.3 ± 1.1 HEC-1-B No FGFR aberrations ≥5.6 HEC-1-A No FGFR aberrations ≥6.4 Bladder RT4 FGFR3 overexpression; 0.004 ± 0.002 FGFR3-TACC3 fusion RT1 12/84 FGFR3 overexpression; 0.013 ± 0.005 FGFR3-TACC3 fusion SW780 FGFR3 overexpression; 4.8 ± 1.5 FGFR3-BAIAP2L1 fusion UM-UC-3 No FGFR aberrations ≥9.7 T24 No FGFR aberrations >10 Multiple myeloma OPM-2 FGFR3 (K650E) translocation 0.011 KMS-11 FGFR3 (Y373C) translocation 0.012 H929 FGFR3 translocation 1.0 LP-1 FGFR3 (F384L) translocation >3.0 KMS-20 No FGFR aberrations >3.0 RPMI8226 No FGFR aberrations >3.0 U266 No FGFR aberrations >3.0 NOTE: GI50 values shown are averaged from 3 independent experiments.

Figure Legends

Figure 1. Target inhibition and selectivity of futibatinib. (A) Chemical structure of futibatinib

and inhibition of FGFR kinase activity in biochemical assays. The average IC50 (nmol/L) from

three independent experiments is shown. Data shown are mean ± SD (n=6). (B) Target

engagement of recombinant FGFR2 enzyme by futibatinib via covalent binding. Relative

molecular mass (Da) was determined by mass spectrometry of 4 different phosphorylation states

of the FGFR2 kinase domain (peaks a, b, c, and d indicate the fragment having a di-, tri-, tetra-,

or penta-phosphorylated site, respectively). Asterisks indicate the peaks in the presence of

futibatinib. Mass differences were determined by subtracting the molecular mass of the FGFR2

kinase domain in the DMSO control (upper graph) from that in the presence of futibatinib (lower

graph). FGFR2cat, FGFR2 catalytic (kinase) domain.

Figure 2. Inhibition of FGFR signaling by futibatinib in FGFR-deregulated tumor cell lines.

(A) Effect of futibatinib on FGFR signaling. FGFR2-amplified OCUM-2MD3 gastric cancer cells were treated with futibatinib for 1 hour, and lysates were analyzed by Western blotting for

phosphorylation of FGFR, Akt and ERK (downstream signaling components of the FGFR

pathway). GAPDH was used as an equal loading control. (B) Sustainability of FGFR inhibition

after futibatinib washout was determined by pFGFR ELISA in FGFR2-amplified OCUM-2M

cell lines. In washout experiments, cells were treated with varying concentrations of futibatinib

for 1 hour, after which, the cells were washed using PBS and cultured in fresh medium for a

further 6 hours. In the no-washout control, cells remained exposed to futibatinib for the same

time period. Prior to cell lysis, cells were stimulated with FGF-7 for 15 minutes. As a control,

cells were stimulated for 15 minutes with FGF-7 after futibatinib treatment for 1 hour and lysates

were analyzed. Data are shown as mean ± SD (n=3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pFGFR, phosphorylated FGFR.

Figure 3. Futibatinib-mediated inhibition of growth of FGFR-deregulated cancer cell lines

and tumors in xenograft models. (A) The GI50 of futibatinib in cell lines with different types of

FGFR genomic aberrations derived from various tumor types was assessed after exposure of

cells to futibatinib for 3 days. GI50 values were categorized by the type of cancer and FGFR gene aberration. Data shown are mean ± SD (n=3). (B) OCUM-2MD3-tumor–bearing mice were

administered vehicle or the indicated doses of futibatinib orally once daily for 14 days. (C)

KMS-11-tumor bearing mice were administered vehicle or 5 mg/kg of futibatinib orally once

daily for 27 days. (D) MFM-223-bearing mice were administered vehicle or the indicated doses

of futibatinib orally once daily for 14 days. Data shown for B, C, and D, are mean ± SD (n=6).

* P < 0.05 compared with vehicle-treated mice (Dunnett’s test).

Figure 4. Duration of futibatinib inhibitory effect on cell proliferation and FGFR

phosphorylation in vivo. (A) OCUM-2M cells were seeded on day 1 and treated with the

indicated concentrations of futibatinib for 4, 8, 24, 48, or 72 hours starting on day 2, after which

the drug was washed out. Cell viability was assessed on day 5. Data shown are representative of

3 independent experiments. (B) OCUM-2MD3–bearing mice were treated with futibatinib for 4

hours at the indicated doses (control refers to untreated mice). Inhibition of FGFR

phosphorylation and phosphorylation of downstream signaling components (Akt and ERK) were

analyzed by Western blotting. Data shown are representative of 3 independent experiments. (C)

Duration of FGFR inhibition by futibatinib was assessed by determining the phosphorylation

level of FGFR2 by ELISA at the indicated time points. The Y axis shows the pFGFR signal as a

percentage of untreated control (100%). Data shown are mean ± SD of 3 independent

experiments (n=3). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; pFGFR, phosphorylated FGFR.

Figure 5. Drug resistance with futibatinib treatment. (A) Drug-resistant clones were

established by continuous exposure of OCUM-2MD3 cells to increasing concentrations of

futibatinib or AZD4547. Following clonal selection, the frequency of appearance of resistant

clones was determined in two independent experiments. (B) Sequencing of the FGFR2 kinase

domain cDNA identified the pK660N mutation in the AZD4547-resistant clones, but not in the

futibatinib-resistant clones.

Figure 6. Activity of futibatinib on FGFR mutants resistant to reversible ATP-competitive

inhibitors. Inhibition of mutated FGFR2 phosphorylation by futibatinib and reversible ATP-

competitive FGFR inhibitors was investigated at the cellular level. HEK293T cells were

transfected with wild-type or mutated FGFR2 expression constructs, and FGFR2

phosphorylation was detected by ELISA. IC50 values of each inhibitor are shown in the graphs.

(A) Reversible ATP-competitive FGFR inhibitors: AZD4547, infigratinib; FGFR2 mutants:

N550H, E566G, K660M, V565I; n=3 each. (B) Reversible ATP-competitive FGFR inhibitors: erdafitinib, pemigatinib; FGFR2 mutants: N550K, K660M, or V565L; n=4 each.

A Figure 1 O O

Enzyme inhibition NH2 FGFR (IC50, nmol/L) N FGFR1 1.8 ± 0.4 N FGFR2 1.4 ± 0.3 N N FGFR3 1.6 ± 0.1 FGFR4 3.7 ± 0.4 N O

FGFR2 + DMSO B 36164.5 cat 100 36084.7 b c

a 36244.2 36004.6 d

36324.1 35924.7 Relative signal intensity (%) intensity Relativesignal 36404.0 0 35600 35800 36000 36200 36400 36600 36800 37000 37200

FGFR2cat + futibatinib

100 36582.9 36503.0 c* b*

ΔMr = 418.5±0.2 Da

a* 36662.9 d* 36423.1

36742.7

Relative signal intensity (%) intensity Relativesignal 36140.3 36343.1 36082.3 36820.3 0 35600 35800 36000 36200 36400 36600 36800 37000 37200 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Molecular mass (Da) Research. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A Futibatinib (µmol/L) B Figure 2

OCUM-2M/FGF-7 plus Without washout Control Control 0.00041 0.0012 0.0037 0.011 0.033 0.10 KDa With washout 3.0 Phospho-FGFR 145 2.5 FGFR 145

2.0 Phospho-Akt 60 1.5 Akt 60 1.0 Phospho-ERK1/2 42/44 pFGFR2 signal signal pFGFR2 0.5 ERK1/2 42/44 0.0 FGF-7 – FGF-7 + 1.25 2.5 5 10 20 GAPDH 37 Downloaded from cancerres.aacrjournals.org on OctoberDMSO 2, 2021. © 2020 AmericanFutibatinib Association (nmol/L) for Cancer Research. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Figure 3 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A Breast Lung Gastric Endometrial Bladder Multiple Myeloma FGFR1/2 FGFR1 FGFR2 FGFR2 FGFR3 FGFR3 amplification amplification amplification mutation amp/fusion translocation 10

0.1

(µmol/L) (µmol/L) 0.01 50 GI 0.001

0.0001 T24 RT4 RT4 KLE AGS AGS LP-1 LK-2 A549 U266 H929 SNU-1 MCF-7 OPM-2 OPM-2 SW780 MKN45 KMS-11 KMS-11 SNU-16 AN3 CA AN3CA KMS-20 HEC-1-A HEC-1-A SK-BR-3 HEC-1-B MFE-280 MFE-296 DMS-114 DMS-114 UM-UC-3 MFM-223 RT112/84 RT112/84 RPMI8226 OCUM-2M NCI-H1581 NCI-H1975 OCUM-2MD3 MDA-MB-134-VI

With FGFR genomic aberrations Without FGFR genomic aberrations

B OCUM-2MD3: once-daily dosing C KMS-11: once-daily dosing

2.5

Vehicle 2.0 Futibatinib; 0.15 mg/kg Futibatinib; 1.5 0.5 mg/kg 1.0 Vehicle Futibatinib; Futibatinib; 1.5 mg/kg 5 mg/kg 1.0 * Futibatinib; 5 mg/kg * *

Relative volume tumor 0.5 Relative tumor volume (log scale) (log Relative volume tumor * 0.0 * 0.1 0 2 4 6 8 10 12 14 16 0 5 10 15 20 25 30 Days Days

D MFM-223: once-daily dosing

3.0

2.5 Vehicle Futibatinib; 6.3 mg/kg 2.0 Futibatinib; 12.5 mg/kg 1.5 Futibatinib; * 25 mg/kg Futibatinib; 1.0 * 50 mg/kg Relative volume tumor * 0.5

0.0 0 2 4 6 8 10121416 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer DaysResearch. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A 140 Figure 4

120

100

80 4 h

60 8 h 24 h 40 48 h 20 Cell viability (%) viability Cell 72 h 0 1 10 100 –20 Log [futibatinib] (nmol/L) –40

Day 1 2 3 4 5

Seed Futibatinib Cell viability

4 h washout 8 h washout 24 h washout 48 h washout 72 h washout

B Futibatinib (mg/kg) 4 h Control 0.5 1.5 5.0 KDa

pFGFR 145 pERK 42/4442/4 pAkt 60

GAPDH 37

C 120

100

20 Relative pFGFR2 signal (%) Relativesignal pFGFR2

0 Control 1.5 5 1.5 5 15 1.5 5 15 5 15 15

4 h 8 h 12 h 18 h 24 h Futibatinib (mg/kg/day) Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A Figure 5

AZD4547 Experiment 1

Experiment 2

Futibatinib

0 20 40 60 80 100 Number of resistant clones

B Parent AZD4547 resistant

Lys Lys Thr Thr Lys Asn Thr Thr C AAAAAAAA A A AG ACCCCCCC A A C AAAAAAAT CCCC 439 442 445 448 436 439 442 445 448

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer G g T Research. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A Figure 6 Futibatinib AZD4547 Infigratinib 120 120 120

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0 50 100 150200 250 300 350 pFGFR2 signal (% of control) of (% signal pFGFR2 pFGFR2 signal (% of control) of (% signal pFGFR2

pFGFR2 signal (% of control) of (% signal pFGFR2 50 100 150 200 250 300 350 50 100 150 200 250 300 350 Concentration (nmol/L) Concentration (nmol/L) Concentration (nmol/L)

pFGFR2 inhibition (IC50, nmol/L) Futibatinib AZD4547 Infigratinib Wild type 0.9 11.1 6.0 N550H 3.6 >300 >300 E566G 2.4 189 33.6 K660M 5.2 167 119 V565I 1.3 93.8 >300

B Futibatinib Erdafitinib Pemigatinib 120 120 120

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0 50 100 150 200 250 300 350 50 100 150 200 250 300 350 50 100 150 200 250 300 350 pFGFR2 signal (% of control) of (% signal pFGFR2 pFGFR2 signal (% of control) of (% signal pFGFR2 –20 control) of (% signal pFGFR2 –20 –20 Concentration (nmol/L) Concentration (nmol/L) Concentration (nmol/L)

pFGFR2 inhibition (IC50, nmol/L) Futibatinib Erdafitinib Pemigatinib Wild type 3.5 4.6 4.0 N550K 11.9 45.6 330.8 V565L 50.6 158.5 950.0 Downloaded3.4 from cancerres.aacrjournals.org on October 2, 2021. © 2020 American7.5 Association for Cancer 10.3 K660M Research. Author Manuscript Published OnlineFirst on September 24, 2020; DOI: 10.1158/0008-5472.CAN-19-2568 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Irreversible covalent bond

Cell proliferation, angiogenesis, differentiation, anti-apoptosis

FGFR-deregulated tumor

Futibatinib, a highly speciﬁc, potent, irreversible FGFR1–4 inhibitor, shows selective activity against tumors harboring various FGFR aberrations, including mutations resistant to ATP-competitive FGFR inhibitors.

Futibatinib is a novel irreversible FGFR 1-4 inhibitor that shows selective antitumor activity against FGFR-deregulated tumors

Hiroshi Sootome, Hidenori Fujita, Kenjiro Ito, et al.

Cancer Res Published OnlineFirst September 24, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2568

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/09/24/0008-5472.CAN-19-2568.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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