FGF401, a first-in-class highly selective and potent FGFR4 inhibitor for the treatment of FGF19-driven hepatocellular cancer

1Novartis Institutes for Biomedical Research, Oncology Disease Area, 4002 Basel, Switzerland
2Novartis Institutes for Biomedical Research, Global Discovery Chemistry, 4002 Basel, Switzerland
3Novartis Institutes for Biomedical Research, PK Sciences, 4002 Basel, Switzerland
4Novartis Institutes for Biomedical Research, Preclinical Safety, East Hanover NJ, USA
5Novartis Institutes for Biomedical Research, Preclinical Safety, 4002 Basel, Switzerland
6Novartis Institutes for Biomedical Research, Oncology Disease Area, Cambridge MA, USA
7Novartis Institutes for Biomedical Research, Chemical Biology and Therapeutics, 4002 Basel,
Switzerland
¤Current address: InSphero, CSO, Schlieren, Switzerland
#Current address: Daiichi Sankyo Co., Ltd, Tokyo, Japan
¥Current address: Broad Institute, Cambridge, MA 02139, USA
Conflict of interest
Andreas Weiss, Flavia Adler, Alexandra Buhles, Christelle Stamm, Robin A. Fairhurst, Thomas Knoepfel,
Nicole Buschmann, Catherine Leblanc, Robert Mah, Pascal Furet, Michael Kiffe, Jacqueline Kinyamu￾Akunda, Philippe Couttet, Heiko S. Schadt, Dario Sterker, Mario Centeleghe, Markus Wartmann,
Youzhen Wang, Patrizia Barzaghi-Rinaudo, Audrey Kauffmann, Jutta Blank, Francesco Hofmann and
Diana Graus Porta are employees of Novartis Institutes for BioMedical Research. Armin Wolf was an
employee of Novartis Institutes for BioMedical Research and is now an employee of InSphero. Masato
Murakami was an employee of Novartis Institutes for BioMedical Research and is now an employee of
Daiichi Sankyo Co., Ltd. William R. Sellers was an employee of Novartis Institutes for BioMedical
Research. W.R.S. is a Board member and Scientific Advisory Board member of Peloton Therapeutics,
Scientific Advisory Board member for Ideaya Biosciences and Epidarex Capital, and has consulted for
Array Pharmaceuticals, Astex Pharmaceuticals, Ipsen, Servier, and Sanofi.
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Abstract
Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and it is the third
leading cause of cancer-related deaths worldwide. Recently, aberrant signaling through the fibroblast
growth factor 19 (FGF19) / fibroblast growth factor receptor 4 (FGFR4) axis has been implicated in HCC.
Here, we describe the development of FGF401, a highly potent and selective, first in class, reversible￾covalent small-molecule inhibitor of the kinase activity of FGFR4. FGF401 is exquisitely selective for
FGFR4 versus the other FGFR paralogues FGFR1, FGFR2, FGFR3 and all other kinases in the kinome.
FGF401 has excellent drug-like properties showing a robust pharmacokinetic / pharmacodynamics /
efficacy relationship, driven by a fraction of time above the phospho-FGFR4 IC90 value. FGF401 has
remarkable anti-tumor activity in mice bearing HCC tumor xenografts and patient-derived xenograft
models that are positive for FGF19, FGFR4 and KLB. FGF401 is the first FGFR4 inhibitor to enter clinical
trials, and a PhI/II study is currently ongoing in HCC and other solid malignancies.
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Introduction
Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide (1). After
sorafenib, other multi-kinase inhibitors targeting angiogenesis have recently been approved for patients
with advanced disease, but mOS remains ~1 year. Advances in the IO field have also led to the approval
of nivolumab but only 20% of the overall population benefits from it (2). Thus, additional therapeutic
breakthroughs are still needed.
Genomic and functional studies have highlighted FGF19 as a potential driver gene in subsets of HCCs
(3). FGF19 is an ileum-derived postprandial hormone that has a fundamental role in the enterohepatic
bile acid/cholesterol system. In order for FGF19 to bind and activate its unique receptor FGFR4 it requires
the co-receptor KLB, a transmembrane glycoprotein without enzymatic activity. FGFR4 and KLB are co￾expressed in hepatocytes and mediate FGF19 transcriptional suppression of CYP7A1, the rate-limiting
enzyme in the bile acids synthesis pathway, thereby lowering the bile acids pool (4, 5). Work from various
laboratories including ours suggests that FGF19 is also an oncogene in HCC. First, FGF19 amplification
and aberrant expression occurs in some HCCs and HCC cell lines, where it likely activates FGFR4 in an
autocrine fashion (6, 7, 8). Second, transgenic mice expressing FGF19 in the skeletal muscle develop
hepatocellular carcinoma, which is abolished in an FGFR4 null background and by FGFR4 and FGF19
neutralizing antibodies (9, 10, 11).
Since FGF19 has unique specificity for FGFR4 (12), we hypothesized that selective FGFR4 kinase
inhibitors would offer a novel therapeutic modality to target FGF19-driven HCC with potentially better
tolerability than the current chemotherapies or multi-targeted agents. FGFR4 has a cysteine at position
552 (Cys552) in the middle-hinge region of the ATP binding site which is poorly conserved across the
kinome and provides an excellent opportunity to achieve selectivity. Recently, BLU-99331, an acrylamidecontaining irreversible covalent FGFR4 inhibitor targeting Cys552, has been described (13) and its
successor BLU-554 entered clinical trials (NCT02508467). Following a similar approach, a second
irreversible FGFR4 inhibitor, H3B-6527 (14) has also entered clinical studies (NCT02834780). Due to the
short re-synthesis rate of FGFR4 in HCC cell lines, we considered a reversible-covalent strategy a more
promising one as compared to an irreversible binder (15).
Our approach resulted in the identification of FGF401, a highly potent and selective, first in class,
reversible-covalent small-molecule inhibitor of the kinase activity of FGFR4 (16, 17). Given its selective
activity in a subset of HCC cells, positive for FGF19/FGFR4/KLB and robust oral PK properties, FGF401
holds promise as a new treatment option in FGF19-dependent HCC.
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Materials and Methods
Compound profiling in biochemical and cell-based assays
FGF401 was prepared within Novartis Pharmaceuticals following the procedures described in the
international application WO 2015/059668 (example 83) published under the patent cooperation treaty,
and the characterizing data are provided as supplementary file FGF401 characterizing data.
For in vitro profiling assays, FGF401 was dissolved at 10 mM in 100% DMSO. Biochemical kinase assays
were conducted as described (16, 17). The KINOMEscan®
profiling was performed at DiscoverRx (18).
Cellular FGFR auto-phosphorylation assays were performed using HEK293 cells transiently
overexpressing each of the FGFR paralogs. FGFR tyrosine-phosphorylation was quantified by ELISA
assay as described (19).
BLU-554 and H3B-6527 were acquired from ABCR GmbH&Co and ChemShuttle Inc respectively, and
prepared as 10 mM solutions in 100% DMSO.
Automated cell proliferation assays were conducted with an ultra-high throughput screening system as
described (20). For manual cell proliferation assays, cells were seeded in 96-well plates in triplicates and
treated with the indicated drugs in 8 point dose-response assays starting at 10 µM or 3 µM and DMSO for
72 h. Cell viability was determined with methylene blue staining.
Cell lines were obtained from ATCC, DSMZ and HSSRB and cultured in RPMI or DMEM with 10% FBS
(Invitrogen) at 37°C, 5% CO2. The cell line identity was confirmed by SNP genotyping. Their genomic
characterization has been described elsewhere (20), https://portals.broadinstitute.org/ccle. The RNAseq
data for FGF19, KLB, FGFR4 and gene copy number for FGF19 are depicted in Supplementary Table
S2. To analyse gene promoter methylation profiles we used standard RRBS.
Inducible knockdown of KLB and FGF19 in HCC cells
We cloned hairpin shRNAs targeting FGF19 and KLB in pLKO-Tet-On vector to produce replication￾incompetent lentiviruses. Upon lentivirus infection of HUH7 cells, stable cell lines were generated by
selection with puromycin (1.5 µg/mL) for 5 days. For cell proliferation and colony formation assays we
seeded cells in 96-well plates and 6-well plates respectively, and induced shRNA expression with 50
ng/mL doxycycline. Cell proliferation and colony growth were evaluated by methylene blue staining.
shRNA sequences are listed in the supplementary section.
In vivo studies in rodents
The experimental procedures involving animal studies strictly adhered to the Association for Assessment
and Accreditation of Laboratory Animal Care International guidelines as published in the Guide for the
Care and Use of Laboratory Animals, and to Novartis Corporate Animal Welfare policies. Studies with cell
lines-derived xenografts were performed at Novartis facilities and were conducted under licenses BS￾Downloaded from mct.aacrjournals.org on August 20, 2019. © 2019 American Association for Cancer Research.
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2498 and BS-2499 approved by the Cantonal Veterinary Office Basel-Stadt. Subcutaneous tumors were
induced by injecting cells in HBSS containing 50% BD Matrigel in the flank of athymic nude mice (RH30
Most of the studies with patient-derived tumor
xenografts (PDXs) were contracted at GenenDesign in China, and some conducted at Novartis facilities.
The details of PK/PD studies, anti-tumor efficacy studies and pharmacodynamics analyses are described
in the supplementary section.
Results
FGF401 is a potent, selective, first in class, reversible covalent inhibitor of FGFR4 kinase
FGF401 is a potent and highly selective FGFR4 kinase inhibitor optimized for oral delivery, the structure
of which is shown in Fig. 1A (16). Selective inhibition of FGFR4 is achieved through a reversible-covalent
binding interaction in which the 2-formyl tetrahydronaphthyridine moiety of FGF401 reacts with a cysteine
residue at position 552 in the kinase domain of FGFR4, to form a hemithioacetal addition product. Fig. 1B
depicts the X-ray structure of the addition product resulting from the reaction between FGFR4 and
FGF401. This unusual mode of kinase inhibition was revealed by an unbiased high throughput screening
approach aimed at identifying inhibitors of FGFR4 showing selectivity over FGFR2 (16). Cys552, a
residue situated two positions beyond the gatekeeper (GK+2) in the middle-hinge region of the ATP￾pocket, is poorly conserved across the human kinome. Only four other kinases have a cysteine residue at
this position, and the other FGFR family members contain a larger tyrosine residue at the GK+2 position
(Fig. 1C) (21). In biochemical kinase assays FGF401 inhibited FGFR4 with an IC50 of 2 nM and displayed
>2900 fold selectivity against the other 64 kinases tested (Supplementary Table S1). In FGFR
biochemical and mechanistic cellular assays, FGF401 inhibited only FGFR4 (Fig. 1D) and in a
KINOMEscan®
(456 kinases) FGF401 at 3 µM displayed binding affinity only for FGFR4 (Fig. 1E) (22).
Thus, FGF401 shows excellent selectivity for the inhibition of FGFR4 versus the other FGFR paralogs
and across the human kinome.
FGF401 activity in cancer cells
To define the pattern of cellular activity for FGF401 we conducted a high throughput proliferation screen
across 436 molecularly characterized cancer cell lines from the cancer cell line encyclopedia (CCLE) (20).
We generated dose response curves and derived IC50, EC50 and Amax for each cell line (Supplementary
Table S2).
These data revealed a pattern dominated by FGF401-insensitive cell lines with EC50>30 µM and/or
Amax>-50%. A small number of drug-sensitive outliers unveiled four HCC cell lines (JHH7, HUH7,
HEP3B217, SNU878), one gastric cancer cell line (FU97) and one breast cancer cell line (MDAMB453)
(Fig. 2A). As revealed by RNAseq expression, the four HCC cell lines and the gastric cancer cell line were
the highest co-expressers of FGF19 and KLB across 933 cancer cell lines (Fig. 2B). Among the HCC cell
lines for which RNAseq was available (n=24), JHH7, HUH7, HEP3B217, SNU878 were the highest
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expressers of FGF19, and all but SNU878 were outlier expressers for KLB (Supplementary Fig S1A).
FGFR4 expression was generally not distinct except for being highly overexpressed in the breast cancer
MDAMB453 cell line (Fig 2B and Supplementary Fig S1A), likely contributing to its known dependency on
FGFR4 (23) and thus, sensitivity to FGF401.
We inspected a genome-scale CRISPR-Cas9 screen performed in over 400 cancer cell lines
(https://depmap.org/portal/) (24), which included four of the FGF401-sensitive ones (HUH7, JHH7, FU97,
MDAMB453). Consistent with the FGF401 screen, we identified FGFR4 and KLB to be selectively
essential to the growth of HUH7, JHH7 and FU97, and FGFR4 to also be essential for MDAMB453 and 2
additional breast cancer lines (Fig. 2C, 2D) which were resistant to FGF401 (Supplementary Table S2). A
greater impact of knocking-out FGFR4 versus inhibiting its kinase could account for this difference. Unlike
FGFR4 and KLB, the depletion of FGF19 did not reveal a selective outlier profile, maybe due to less
optimal performance of the sgRNAs (https://depmap.org/portal/).
FGF401 inhibits proliferation and downstream signaling of FGF19/KLB positive cell lines
To verify the activity of FGF401 in HCC cell lines that are FGF19/KLB/FGFR4 positive, first we analysed
protein expression of the three markers and found a good correlation with RNAseq in the three instances
(Supplementary Fig S1B). Next, we performed one-by-one, manual proliferation assays with the HCC cell
lines from the CCLE (n=26) and included the gastric cancer FU97 cells. In keeping with the high
throughput screen, HUH7, Hep3B, JHH7, SNU878 and FU97 were the only FGF401-sensitive cell lines
(Fig. 2E, Supplementary Table S2). We confirmed co-expression of FGF19, FGFR4 and KLB protein in
lysates of these cell lines, and detected FGF19 in the media supernatants (Fig 2F, 2G). As FGF401, BLU-
554 and H3B-6527, the two additional clinical FGFR4 inhibitors were also active against the FGF401-
sensitive HCC cell lines, with slightly higher GI50 (Supplementary Table S3).
All the other HCC cell lines were insensitive to FGF401 with EC50 values >10’000 nM and/or Amax values
>-10% and most of them had undetectable FGF19 and/or KLB protein. HUH1 and SNU761 expressed
FGF19 and KLB but lower levels than the sensitive cell lines, potentially not reaching a threshold of
expression needed to sufficiently activate the FGFR4 pathway and confer dependency. SNU886 scored
slightly positive for FGF19 by RNAseq (Supplementary Fig S1C) however, the protein was only found in
the supernatant. KLB levels in this cell line were near the background levels of the assay, suggesting that
KLB expression might be too low to allow FGF19 binding to FGFR4 thus, providing a plausible
explanation for the cell lysate being FGF19 negative. Within the HCC lineage, FGF19 expression was the
best correlate with response to FGF401 (Supplementary Fig S1D). FGF19 lies within the 11q13 amplicon.
Some publications suggested that FGF19 is overexpressed in HCC because of gene amplification and
that this amplicon could be a response biomarker to anti-FGF19 therapies (6). In our analysis we found
that only JHH7 and SNU878 cells have FGF19 copy number gain above five copies. Conversely, other
HCC cell lines with FGF19 copy number >5 were insensitive to FGF401 and with the exception of
SNU886, FGF19 protein was below LOD or very low, in line with low RNAseq reads. In one of these cell
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lines, Li, high methylation content at the FGF19 promoter revealed by RRBS may explain the absence of
FGF19 mRNA and protein despite gene amplification (Supplementary Fig. S1C).
We analyzed FGF401 mediated inhibition of FGFR4 and downstream signaling in Hep3B, JHH7 and
HUH7 cells. As shown in Fig. 2H and 2I, FGF401 inhibited FGFR4 tyrosine phosphorylation at compound
concentrations needed to inhibit cell proliferation. Consistent with this result, in cells treated with FGF401
less FGFR4 was pulled down in anti-phosphotyrosine immunuprecipitation assays, though total FGFR4
levels were not significantly impacted by treatment. In agreement with FGFR4 inhibition, pFRS2 and
pERK were also inhibited. Modulation of pAKT was generally minor, with some reduction observed only in
Hep3B cells. The low baseline pAKT levels in JHH7 cells precluded further conclusions. A differential
gene expression analysis revealed downregulation of MAPK target genes including DUSP5, DUSP6,
SPRY4, ETV4, ETV5 after 18h treatment with FGF401 (Supplementary Fig. 1F), as well as upregulation
of components of the bile acids synthesis pathway including CYP7A1, and glucose metabolism. Thus, our
data suggests that HCC cells have retained some properties of hepatocytes and can in part regulate the
bile acids and glucose pathways. While FGF19 and FGFR4 were unaffected, KLB showed a statistically
significant increase of 2.5 fold (adj. P val. 1.5×10-11), potentially as a compensatory mechanism to FGFR4
inhibition.
Proliferation of FGF19/FGFR4/KLB positive HCC cell lines depends on each of the three markers
We have shown that FGF401 almost exclusively inhibits proliferation of cell lines positive for FGF19 when
co-expressed with FGFR4 and KLB (Fig. 2A-C). The genome-wide CRISPR screen suggested that triple
positive cell lines also depend on KLB. To investigate FGF19-dependency and verify KLB-dependency,
we engineered HuH7 cell lines to express inducible shRNAs targeting FGF19, KLB, non-targeting control
shRNAs (NT-1, NT-2) and PLK1-targeting shRNA as a positive control for profound cell growth inhibition.
Effective doxycycline-induced knockdown of FGF19 by four different shRNAs (F-2, F-4, F-5, F-6) led to
inhibition of the FGFR4 pathway as exemplified by reduced pFRS2, and to inhibition of cell proliferation.
shRNAs F-1 and F-3 were less efficient in knocking down FGF19 and thus, pFRS2 and cell proliferation
were less impacted. Non-targeting shRNAs did not affect pFRS2 nor cell proliferation (Fig. 3A, 3B).
Similarly, efficient KLB protein knockdown by shRNAs K-1 and K-4 led to a reduction of pFRS2,
proliferation and colony formation, whereas ineffective anti-KLB shRNAs and non-targeting controls did
not (Fig. 3C, 3D, 3E). Owing to optimal KLB knockdown achieved with K-1, we utilized the HUH7/K-1 cells
to examine FGFR4 activity in the absence of KLB. Doxycycline-induced KLB knockdown caused profound
reduction of baseline pFGFR4 and prevented FGF19-induced FGFR4 phosphorylation as compared to
non-doxycycline treated cells and cells expressing non-targeting shRNA NT-1, which displayed higher
pFGFR4 and remained sensitive to exogenous FGF19 treatment (Fig. 3F).
Knockdown of KLB also led to reduction of FGF19 in cells (Fig. 3C). To investigate whether FGF19
decrease was due to FGFR4 inhibition (as measured by reduction in pFGFR4, Fig 2F) we treated
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HUH7/K-1 cells with doxycycline or FGF401. As before, KLB knockdown resulted in reduced pFRS2 and
FGF19, while FGFR4 was unaffected. Instead, FGF401 inhibited pFRS2 but did not affect FGF19 levels
(Fig 3G). We also measured FGF19 by ELISA assay in cell lysates and supernatant. In line with
experiment in Fig 2A, HUH7/F-2 and HUH7/F-4 cells and supernatants thereof were negative for FGF19
upon doxycycline treatment. FGF19 was detected in the supernatants of HUH7/K-1 cells after doxycycline
treatment but not in the cell lysates. These data favor a model in which loss of KLB expression prevents
FGF19 from binding to FGFR4 resulting in its accumulation in the media.
In summary, our data confirm that FGF401-sensitive HCC cell lines are also dependent on FGF19 and
KLB for proliferation, and suggest that disruption of the signaling complex by targeting any of these
proteins may inhibit tumorigenesis.
Single dose pharmacokinetics and pharmacodynamics in RH30 tumor-bearing nude
We evaluated the pharmacokinetic properties of FGF401 in mice following a single dose of 1 mg/kg i.v. or
3 mg/kg p.o. (Supplementary Fig. S2A). Although clearance in mice is low, FGF401 shows a relatively
short half-life of 1.4 hours. The corresponding oral bioavailability was 21%.
To study the pharmacokinetic/pharmacodynamics (PK/PD) relationship of FGF401, we used the RH30
rhabdomyosarcoma (RMS) xenograft, which constitutively overexpresses activated
Supplementary Fig. S2B shows the means (n=2) of total drug concentration in plasma and phospho￾FGFR4 inhibition in tumor over 24 hours post single oral administration of FGF401 at 1 up to 100 mg/kg.
FGF401 exhibited a quick absorption at all doses with Cmax achieved at 2h (Tmax). FGF401 plasma
exposure was linear over the five dose levels with normalized AUCs of 0.33-0.52 µM*h and a dose
proportional increase in Cmax (Supplementary Fig. S2C). Tumor phospho-FGFR4 normalized to total
FGFR4 (p/tPFGFR4) was inhibited in a dose-dependent manner (Supplementary Fig. S2B).
Total drug concentration in plasma versus p/tFGFR4 in tumor was plotted (Fig. 4A) to calculate the in vivo
PD IC50 (2.1 nM) and IC90 (52.1 nM), which are highly consistent with the in vitro PD IC50 (10 nM) and IC90
(25 nM). Increasing doses of FGF401 led to more sustained p/tFGFR4 inhibition where doses ≥10 mg/kg
resulted in plasma drug concentration above IC50 for 12h, and doses ≥30 mg/kg in plasma drug
concentration above the IC90 for 8h (Fig. 4B).
Overall, FGF401 has good pharmacokinetic properties and based on the murine PK data we predicted it
would require a twice daily (bid) schedule to achieve sustained target inhibition, which is key for maximal
efficacy.
FGF401 shows antitumor activity in HCC xenografts
We addressed whether the FGF19/FGFR4/KLB positive HCC xenografts HUH7 and Hep3B were
sensitive to FGF401 in vivo. Based on the PK profile and PK/PD relationship (Fig. 4A, B and
Supplementary Fig. S2), doses of FGF401 of 10 mg/kg, 30 mg/kg and 100 mg/kg in an oral, bid schedule
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were chosen to assess its anti-tumor effect. FGF401 induced approximate tumor stasis at 10 mg/kg bid
and tumor regression at 30 and 100 mg/kg bid, and was well tolerated based on body weight increase
(Fig 4C, 4D, 4E). The antitumor effect correlated with the time of FGF401 plasma exposure over the in
vivo PD IC90 (Fig. 4B). Notably, the antitumor effect of FGF401 in HUH7 xenografts was superior to that of
sorafenib, first line SOC, dosed at 30 mg/kg qd (Supplementary Fig. S3A) which has been shown to
induce tumor stasis in a variety of xenograft models and to be similar to 60 mg/kg qd (25, 26).
To investigate PD biomarkers for FGF401 first we monitored pERK levels by immunohistochemistry in
HUH7 (Fig. 4F) and Hep3B (Supplementary Fig. S3B) tumors. pERK was almost completely abolished at
2 h post-dose and recovered to almost vehicle levels at 12h post-dose irrespective of whether FGF401
was administered as a single dose or as multiple treatments. Since FGF19 signaling impacts CYP7A1
transcription and bile acids synthesis, we investigated CYP7A1 modulation and 7alpha-hydroxy-4-
cholesten-3-one (C4) levels, a downstream product of CYP7A1 and precursor of bile acids (4, 5). We
detected an increase of CYP7A1 mRNA in both HUH7 tumors (Fig. 4G) and mouse liver (Supplementary
Fig. S3C) at 2h and 12h post-dose. Correspondingly, C4 was also elevated in HUH7 tumors (Fig. 4H)
treated with 100 mg/kg FGF401 at 2h and 12h post-dose, in mouse liver (Supplementary Fig. S3D) at all
doses at 2 h post-dose and to a lesser extend at 12h post-dose, and in plasma (Supplementary Fig.
S3E). Notably, CYP7A1 mRNA and C4 levels appeared significantly lower in HUH7 tumors as compared
to mouse liver, likely reflecting its suppression induced by the pathologically activated FGFR4 pathway in
the tumor. Finally, we used lysates of HUH7 tumors treated with FGF401 at 100 mg/kg bid to measure
CYP7A1 activity in a liver microsome assay. Total CYP7A1 activity was increased at 4h and 8h post-dose
compared to vehicle (Fig. 4I), which likely accounts for the increased C4 levels.
These results are concordant with the observed suppression of MAPK pathway genes and CYP7A1
induction in HCC cell lines treated with FGF401 in vitro (Supplementary Fig. S1E).
Activity of FGF401 against mutationally activated FGFR4
FGFR4 kinase domain mutations V550E and N535K occur in RMS, transform NIH3T3 cells and enhance
the metastatic phenotype of RMS cells (27). Since no preclinical models of RMS with FGFR4 activating
mutations are known, we examined the activity of FGF401 against NIH3T3 xenografts engineered to
constitutively express FGFR4/V550E and FGFR4/N535K mutants. Tumor-bearing nude mice were
treated with FGF401 at 30 mg/kg or 100 mg/kg p.o. bid. Whereas FGF401 at both dose levels could only
moderately delay the growth of tumors harboring the V550E mutation, tumors harboring the N535K
mutation regressed (Fig. 4J, K). This data suggest that FGF401 might be a therapeutic option for certain
forms of FGFR4-mutated cancers like RMS with FGFR4-N535K.
FGF401 efficacy is driven by a fraction of time above the p/tFGFR4 IC90
The dose-dependent sensitivity of HUH7 and Hep3B xenografts to treatment with FGF401 (Fig. 4C, E)
suggested a requirement for FGF401 plasma trough (Cmin) exposure near the IC90 (Fig. 4B) for most of
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the dosing interval to achieve strong antitumor activity (i.e. Cmin ~ IC90). We addressed this hypothesis in
rat using the HUH7 xenograft model, since the FGF401 half-life in rat is significantly longer than in mouse
(4.4 h in rat versus 1.4h in mouse, Supplementary Fig S2A and S4A).
We first conducted a dose-range efficacy study to identify the minimal efficacious FGF401 dose. We
treated HUH7 tumor-bearing nude rats with FGF401 in an oral bid schedule at doses of 1 mg/kg, 3 mg/kg,
10 mg/kg and 30 mg/kg. FGF401 induced approximate tumor stasis at 1 mg/kg bid (T/C = 18%), and
tumor regression at -52%, -82%, and -96% for the 3, 10 and 30 mg/kg bid doses, respectively
(Supplementary Fig S4B). In keeping with the mouse efficacy studies, the antitumor effect in rats
correlated with FGF401 blood exposure over time near the IC90 (Supplementary Fig. S4C).
As 3 mg/kg bid was the lowest dose tested resulting in tumor regression, we conducted a dose
fractionation study where the total FGF401 dose was kept constant and divided into either 3 mg/kg bid, 6
mg/kg qd or 12 mg/kg q2d dosing regimens in the HuH7 xenograft model. The 3 mg/kg bid dose was
found to result in the best tumor growth inhibition (regression = -84%) compared to the same daily dose of
6 mg/kg given in a once daily schedule (6 mg/kg qd; T/C = 1%) or in an every other day schedule (12
mg/kg q2d; T/C = 23%) (Fig. 5A, C). The efficacy correlated well with the fraction of time of FGF401
exposure over the IC90 (52 nM, total concentration in blood) during the dosing interval: 60% for 3 mg/kg
bid, 42 % for 60 mg/kg qd and 28% for 12 mg/kg q2d (Fig. 5B, C). Taken together, these data suggest
that the fraction of time above the p/tFGFR4 IC90 value is a key determinant associated
Antitumor activity of FGF401 in patient-derived tumor xenografts (PDXs)
The in vitro and in vivo data show that FGF401 inhibits the growth of cell lines and xenografts thereof that
co-express FGF19, its receptor FGFR4 and co-receptor KLB. To corroborate these findings, we assessed
the efficacy of FGF401 in 35 HCC and 2 gastric cancer PDX’s that included 4 HCC and 1 gastric cancer
models that were positive for FGF19, KLB and FGFR4 mRNA, and 1 gastric PDX with FGF19 gene
amplification but KLB negative. The other HCC PDX’s were FGF19 negative (Fig 6A, 6B, Supplementary
Table S4). FGF401 administered at 100 mg/kg bid to PDX-bearing nude mice caused a statistically
significant anti-tumor activity only in the 5 FGF19/KLB/FGFR4 positive PDX’s, with -55% regression, -
24% regression, 7% T/C and 3% T/C in the 4 HCC’s and 28% T/C in the gastric cancer PDX. The gastric
cancer model with FGF19 gene amplification and overexpression but KLB negative, was refractory to
FGF401, with 87% T/C (Fig. 6C, Supplementary Table S4). Further exploration of the FGF19, FGFR4 and
KLB RNA profiles showed that within the HCC lineage, FGF19 expression was the best correlate with
anti-tumor efficacy (Fig. 6D). As observed in cell lines, FGF19 copy number was not associated neither
with FGF19 expression nor with FGF401 efficacy (Fig 6B, 6D).
Discussion
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In this study, we describe the identification of FGF401, a first in class, highly selective and potent FGFR4
inhibitor, which might provide benefit to HCC patients whose tumors present with aberrant FGF19
expression. FGF401 has been the first FGFR4 inhibitor to enter clinical trials, and it is currently in a PhI/II
study (NCT02325739). FGF401 covalently binds in a reversible manner to FGFR4 utilizing a unique
Cys552 in the middle-hinge region of the ATP binding site and enabling a high level of kinase selectivity.
The selective nature of FGF401 translated also across safety pharmacology assays, it was well tolerated
in pharmacological studies at efficacious doses, and accordingly it is demonstrating good tolerability in
patients (28, 29)
Several studies have implicated FGFR4 in various cancer types besides HCC with FGF19 deregulation
and RMS with FGFR4 mutations (30, 31, 32, 33). In order to examine these hypotheses and to identify
clinically relevant patient selection biomarkers, we tested FGF401 in a high-throughput cell viability in
vitro screen consisting of 436 cancer cell lines that are part of our CCLE collection. Only 6 cell lines were
sensitive to FGF401, 1 breast cancer cell line, 4 HCC and 1 gastric. The breast cancer cell line
MDAMB453 is an outlier for FGFR4 over-expression and harbors an FGFR4 activating mutation (23).
However, the analysis by next generation sequencing of 4,853 tumors including 522 cases of breast
cancer did not identify this mutation (34), which questions the validity of the concept in the clinic. None of
the additional breast cancer cell lines, outliers for FGFR4 expression and for which we had response data
(n=8) were sensitive to FGF401. RMS/ARMS characterized by the oncogenic PAX3/FOXO1 fusion
leading to FGFR4 overexpression was also not identified as an indication for FGF401 since all the ARMS
cell lines with PAX3/FOXO1 fusion in the CCLE (n=7) were refractory to FGF401 despite high FGFR4
levels. Indeed, Marshall et al showed a lack of any contribution from FGFR4 to PAX3/FOXO1-driven
tumorigenesis (35). The more recent FGFR4 inhibitor, H3B-6527, has activity in two PAX3/FOXO1
positive ARMS cell lines. This may be because H3B-6527 is less selective than FGF401 and accordingly
it shows activity in additional cell lines and PDX models for which the mechanism is unclear (15).
Oncogenic mutant forms of FGFR4 have been detected only in some cases of rhabdomysarcomas (27).
To evaluate the potential of FGF401 in this disease, we used mechanistic NIH3T3 xenograft models
expressing FGFR4/V550E and FGFR4/N535K mutants found in RMS. While FGF401 only slightly
inhibited V550E tumors, N535K tumors responded to treatment and completely regressed. The impact of
these mutations on the activity of FGF401 is consistent with what is anticipated from the interaction of
FGF401 within the ATP binding site of FGFR4. Mutating the gatekeeper residue (V550) to the much
larger glutamic acid residue is predicted to hinder FGF401 binding due to unfavorable steric interactions
(36). Mutating a key residue of the molecular brake (N535) leads to FGFR4 being displaced towards a
higher occupancy of active-kinase conformations (37), and FGF401 retaining equivalent activity is
consistent with FGF401 being equally active against all activity states of FGFR4 (16, 17). These data
supports further exploration of FGF401 in rhabdomyosarcomas that harbor specific FGFR4 oncogenic
mutations.
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In contrast to previous reports that describe increased FGF19 levels by focal amplification of 11q13 as a
driver in HCC (6), our data strongly suggest that rather than FGF19 copy number, FGF19 expression in
the presence of FGFR4 and KLB is the determining factor. These results are well aligned with findings
published in the context of the two other FGFR4 selective inhibitors, BLU-99331 and H3B-6527 (13, 14).
Besides HCC, we identified two gastric cancer models, FU97 cell line and GA180 PDX, that were
sensitive to FGF401 and remarkably recapitulated the same biomarker profile as the HCC models with
FGF19 high expression in the absence of gene amplification, FGFR4 and KLB co-expression.
Interestingly, while this manuscript was being reviewed, Gao et al. have shown that instead, in HNSCC
FGF19 amplification is associated with FGF19 overexpression, poor patient outcome and dependency on
FGF19/FGFR4/KLB (38). Thus, their work reveals a potential new opportunity for FGF19/FGFR4
targeting therapies in HNSCC with FGF19 amplification.
FGF401 showed favorable PK properties and a consistent PK/PD/efficacy relationship in xenograft animal
models with p/tFGFR4 levels in the tumor robustly inhibited in a dose-dependent manner. pERK was also
strongly reduced at 2 h post-dose but unlike pFGFR4 it recovered to almost vehicle levels at 12 h post￾dose. This is likely due to feedback loops in the MAPK pathway that result in pathway reactivation for
instance by activating other RTKs or downstream kinases (39). The inhibition and recovery of pERK was
the same after single-dose and repeat dosing since FGF401 does not accumulate over time and
therefore, the PK/PD relationship remains the same throughout the dosing period. Further, in line with the
role of FGF19 as a regulator of bile acids synthesis and similar to BLU-99331 and H3B-6527, FGF401
treatment led to increased CYP7A1 mRNA and C4 levels in tumor and liver, and increased circulating
levels of C4. These findings highlight C4 as an alternative biomarker for FGF401 activity that can be
measured in blood and of value to also monitor potential on-target toxicity caused by bile acids elevation.
In conclusion, we provide evidence that FGF401 is a potent and highly selective FGFR4 inhibitor with
excellent drug-like properties that shows robust antitumor activity in FGFR4-dependent tumor models like
hepatocellular carcinomas with aberrant FGF19 overexpression. This prompted us to test its efficacy in
relevant cancer patients. FGF401 has been the first FGFR4 inhibitor to enter clinical trials, and a PhI/II
study is currently ongoing in HCC (NCT02325739). Since then, two additional selective FGFR4 inhibitors
BLU-554 and H3B-6527 have also started Phase I studies in advanced HCC (NCT02508467,
NCT02834780), and clinical data for BLU-554 was presented at ESMO 2017 (40). Until now, it is unclear
which of the three drugs is superior. Whereas FGF401 was slightly more potent than BLU-554 and H3B-
6527 in inhibiting proliferation of FGF19-driven HCC cell lines, multiple factors beyond its intrinsic
potency, including human PK properties and tolerability, will influence the clinical outcome. Indeed, BLU-
554 shows similar clinical activity to FGF401 in advanced HCC patients.
Our preclinical analyses of biomarkers for patient selection as well as the results from Hagel et al. and
Joshi et al. (12, 13) implicate that in HCC, a patient selection approach based on tumor FGF19
expression along with FGFR4 and KLB co-expression will likely enrich for responders to FGFR4 selective
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13
inhibitors. The results however, do not exclude the possibility that tumors that are only positive for KLB
and FGFR4 could be driven in a paracrine manner by FGF19 produced by non-tumor cells and
consequently respond to FGFR4 inhibitors (9). These considerations altogether prompted us to
implement a molecular pre-screening approach in the clinical study to enroll HCC patients whose tumors
are FGFR4 and KLB positive by RT-qPCR, while we monitor FGF19 expression in the tumor in a
retrospective manner by both RT-qPCR and IHC. A manuscript describing the design and results of the
Phase I/II clinical trial of FGF401 will be prepared shortly.
In summary, three novel FGFR4 selective inhibitors, FGF401 described in this manuscript, BLU-554 and
H3B-6527 described elsewhere (40, 14) are being evaluated for the treatment of advanced HCC patients
whose tumors display de-regulated FGF19/FGFR4 pathway. Since both FGF401 and BLU-554 exhibit
promising clinical responses, and given the underlying complexity and molecular heterogeneity of the
disease, it will be important to promptly identify the optimal drug combinations to ultimately achieve cures
in this patient population.
Acknowledgements
We thank Richard Ducray for his excellent project management, Joerg Trappe and Inga Galupa for
conducting the biochemical kinase profiling, the bioinformatics team at C-NIBR for their support with the
genomic data of the PDX models, and the FGF401 clinical team first led by Luigi Manenti and afterwards
by Andrea Myers, for enabling the clinical PoC of FGF401 in HCC.
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Figure Legends
Figure 1. FGF401: chemical structure, X-ray structure in complex with FGFR4 and kinase profile.
A. Chemical structure of FGF401. B. X-ray structure of FGF401 in complex with the tyrosine kinase
domain of FGFR4 at 2.13 Å resolution. C. amino acid sequence alignment of the middle-hinge region of
the ATP-pocket of FGFR1, FGFR2, FGFR3, FGFR4 highlighting the differentiating cysteine at the GK+2
position (FGFR4 numbering). D. Activity of FGF401 in biochemical FGFR kinase assays and cellular
FGFR auto-phosphorylation assays. IC50’s in nM. E. Kinome profiling of FGF401 at 3 µM against 456
kinases showing FGFR4 as the only kinase exhibiting > 35% inhibition of control binding (red dot, 0.65%
control binding; selectivity score 0.003).
Figure 2. FGF401 activity in cancer cells and comparison to CRISPR-Cas9 screen. A. Scatter plot
showing EC50 and Amax values of FGF401 in high-throughput cell viability assays for 436 cancer cell
lines across multiple lineages. B. Scatter plot of 933 cancer cell lines distributed according to FGF19 and
KLB transcript expression (RNAseq) and highlighting the FGF401-sensitive cell lines. Symbol size reflects
FGFR4 transcript levels (RNAseq). Circles: cell lines included in the proliferation screen. Squares: cell
lines that were not included in the screen. C, D. Waterfall plots of FGFR4 and KLB dependencies in 426
cancer cell lines from the genome-scale CRISPR-Cas9 screen. Outlier cell line drop-outs for each gene
are colored in red and ID is provided. E. Scatter plot of 26 HCC cell lines and one gastric cancer cell line
analyzed in manual proliferation assays and distributed according to FGF401 EC50 and Amax. F, G.
Scatter plots illustrating the distribution of cell lines according to KLB and FGF19 protein levels in cell
lysates (F), and in the supernatants (G). Horizontal and vertical dotted lines indicate the FGF19 LOD and
the KLB LOQ, respectively. Color coding in A, B, E, F and G indicates primary site of the cell line. H,I.
HUH7, JHH7, Hep3B2.1-7 cells treated with FGF401 at the indicated concentrations or DMSO (-) for 1 h
and 72 h. Bar plots show the ratio of Tyr-phosphorylated (pFGFR4) over total FGFR4 (tFGFR4)
measured by ELISA assay (H). Western blots show phosphorylated FGFR4 upon total pTyr pull down
and IgG-HC as loading control; total FGFR4, pFRS2, pERK and pAKT with ERK, AKT and α-actinin as
loading controls (I)..
Figure 3: FGF19 and KLB knockdown in HUH7 HCC cells stably transduced with lentiviruses
expressing doxycycline-inducible shRNAs. A, C. Western blots showing FGF19, KLB and pFRS2
levels in HUH7 cells harboring non-targeting shRNAs (NT-1, NT-2), FGF19-targeting shRNAs (F-1 to F-6)
or KLB-targeting shRNAs (K-1 to K-4), with and without doxycycline induction for 4 days. β-tubulin and β-
actin Western blot confirm equal loading. B, D, E. Cell viability assays with HUH7 cells harboring nontargeting shRNAs, FGF19 shRNAs, KLB shRNAs and PLK1 shRNA with and without doxycycline
induction. B. Cells were plated on 6-well plates and cell growth was monitored using methylene blue
staining after 10 days of doxycycline induction. D. Cells were plated on 96-well plates in quadruplicates
and stained with methylene blue 10 days post-doxycycline induction. Data are expressed as percentage
of control, no dox-treated cells and each bar is the average of 4 replicates +/- SD. E. Cells were seeded
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on 6-well plates and induced with doxycycline for 21 days. Colony formation was monitored with
methylene blue staining. F. HUH7 cells with NT-1 shRNA or K-1 shRNA were treated with doxycycline for
6 days and/or FGF19 (50 ng/mL) for 1 hour. Tyr-phosphorylated (pFGFR4) and total FGFR4 (tFGFR4)
were quantified by ELISA assay and expressed as a ratio. G. Cells were treated with doxycycline or
FGF401 50 nM for the indicated time and cell lysates analyzed by Western blot using β-tubulin as a
loading control. F. Cells were treated with doxycycline for 4 days and FGF19 levels in cells as well as in
supernatant were quantified by ELISA assay.
Figure 4. PK/PD and efficacy of FGF401. A. PK/PD relationship of FGF401 after one single p.o.
treatment of RH30 tumor-bearing nude mice. Total drug concentration in plasma was plotted vs.
p/tFGFR4 in tumor for each individual animal to show the correlation between PK and PD. In vivo IC50
and IC90 values were calculated from the curve. B. Time was plotted vs. total drug concentration in
plasma. The IC50 and IC90 concentrations of FGF401 are indicated with dotted lines. C and D. Female
nude mice bearing Hep3B subcutaneous xenografts were treated with FGF401 or vehicle control p.o. bid.
Values are mean ± SEM, n=5 mice per group. *p<0.05 vs vehicle using Kruskal-Wallis (Dunn’s post hoc)
on ΔTVol (C) and one-way ANOVA (Dunnett’s) on ΔBW (D). E. Female nude mice bearing HuH7
subcutaneous xenografts were treated with FGF401 or vehicle control p.o. bid. Values are mean ± SEM,
n=6 mice per group. *p<0.05 vs vehicle using Kruskal-Wallis (Dunn’s post hoc). F, G, H, I.
Pharmacodynamic analyses of FGF401 in HUH7 tumors. F. Tumor-bearing mice were treated with one
single dose of FGF401 or 6 repeated doses and sacrificed at 2 and 12 h (vehicle 2 h only) post-dose to
analyze tumors by pERK immunohistochemistry. G. Analysis of Cyp7a1 mRNA transcript at 2h and 12 h
post-last dose (n=3). H. 7α-hydroxy-4-cholesten-3-one (C4) measured by LC-MS in tumor (n=3). (I)
Cyp7A1 activity measured in tumor at 4h and 8h post-last dose (n=3). J and K. Female nude mice
bearing subcutaneous NIH3T3 xenografts constitutively expressing the mutant forms of FGFR4,
FGFR4/V550E (J) and FGFR4/N535K (K), were treated with vehicle or FGF401 p.o. bid. Values are mean
± SEM, n=6 mice per group. *p<0.05 vs vehicle using one-way ANOVA (Dunnett’s post hoc) (J) or
Kruskal-Wallis (Dunn’s post hoc) (K) on ΔTVol.
Figure 5. Anti-tumor activity of FGF401 is driven by a fraction of time above the p/tFGFR4 IC90. A.
Female nude rats bearing Huh7 subcutaneous xenografts were treated with FGF401 or vehicle control
p.o. at the indicated dose schedules. Values are mean ± SEM, n=8 mice per group. *p<0.05 vs vehicle
using Kruskal-Wallis (Dunn’s post hoc). B. Blood samples were collected at indicated time points post 8
days treatment to determine drug concentrations. These were plotted vs. time. Data show mean values ±
SD (n=2) for total drug concentration. IC90 (52.1 nM) level from the in vivo PD model is indicated with a
dotted line. C. Efficacy and PK parameters. Anti-tumor efficacy of FGF401 as % T/C or % regression.
Drug exposure is calculated as mean AUCs0-48h (area under the curve) (n=2); Cmax is the maximal
achieved drug concentration; Ctrough is the drug concentration before the next dose. For exposure at day
8, fraction of time above the IC90 (0.052 µM) is provided.
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Figure 6. Anti-tumor activity of FGF401 in a panel of 38 PDX’s. A, B. Scatter plots showing the
distribution of PDX samples according to KLB and FGF19 transcript expression (A) and FGF19 CN vs.
FGF19 transcript (B). Color-coding in A and B indicates primary site. Symbol size in A indicates FGFR4
transcript expression. C. Response to FGF401 (100 mg/kg p.o. bid) over time of four HCC PDX’s and two
gastric cancer PDX’s (Li: liver lineage; GA: gastric lineage). Values are mean ± SEM, n=3-4 mice per
group. *p<0.05 (unpaired t-test). D. Scatter plots illustrating the relationship between FGF401 response in
HCC PDX’s (expressed as % T/C or % regression) and FGF19 expression, KLB expression, FGFR4
expression or FGF19 copy number. The HCC PDX’s that respond to FGF401 are highlighted.
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