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Published OnlineFirst January 5, 2016; DOI: 10.1158/2159-8290.CD-15-0896
Research Article
Oncogenic BRAF Deletions That Function as
Homodimers and Are Sensitive to Inhibition
by RAF Dimer Inhibitor LY3009120
Shih-Hsun Chen1, Youyan Zhang1, Robert D. Van Horn1, Tinggui Yin1, Sean Buchanan1, Vipin Yadav1,
Igor Mochalkin2, Swee Seong Wong3, Yong Gang Yue3, Lysiane Huber1, Ilaria Conti4, James R. Henry2,
James J. Starling1, Gregory D. Plowman1, and Sheng-Bin Peng1
We have identified previously undiscovered BRAF in-frame deletions near the αChelix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers.
These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type
pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity
and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the
β3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation
that favors dimer formation. Expression of L485-P490–deleted BRAF is able to transform NIH/3T3
cells in a BRAF dimer–dependent manner. BRAF homodimer is confirmed to be the dominant RAF
dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor
vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions,
LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive.
Abstract
SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism
dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and
represents a potential treatment option for patients with cancer with these BRAF deletions, or other
atypical BRAF mutations where BRAF functions as a dimer. Cancer Discovery; 6(3); 1–16. ©2016 AACR.
INTRODUCTION
Somatic mutations in the BRAF gene were discovered
in 2002 in melanoma, where they behave as potent oncogenes and activate downstream MAPK signaling and cancer
cell growth (1–5). BRAF mutations have subsequently been
found in many other tumor types, including thyroid, ovarian,
colorectal, and non–small cell lung cancers, as well as in hairy
cell leukemia and Langerhans cell histiocytosis (1, 6–9). In
melanoma, the V600E hotspot mutation is particularly prevalent, but mutations affecting side chains other than valine
600 (non-V600 or atypical) have been described either adjacent to the activation segment (e.g., L597, G596, F595, and
1
Oncology Research, Eli Lilly and Company, Indianapolis, Indiana. 2Discovery
Chemistry Research and Technologies, Eli Lilly and Company, Indianapolis,
Indiana. 3Tailored Therapeutics, Eli Lilly and Company, Indianapolis, Indiana.
4
Oncology Business Unit, Eli Lilly and Company, Indianapolis, Indiana.
Corresponding Author: Sheng-Bin Peng, Lilly Research Laboratories, Eli
Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285. Phone:
317-433-4549; Fax: 317-276-1414; E-mail: [email protected]
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscovery.aacrjournals.org/).
©2016 American Association for Cancer Research.
doi: 10.1158/2159-8290.CD-15-0896
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E586) or within the glycine-rich GXGXXG motif (e.g., G464,
G466, and G469) of the kinase domain (1, 10). In patients
with melanoma, atypical BRAF mutations were detected in
37 out of 499 (7.4%) patient specimens (11). In colorectal
cancer samples with BRAF mutations, it has been reported
that approximately 50% of them are V600E, and the remaining 50% are atypical mutations, i.e., D594V and T599I (12).
BRAF mutations are found in 6.7% of lung adenocarcinomas,
and 80% of these are atypical BRAF mutations (13). In addition to missense mutations, oncogenic BRAF fusions were
also identified in cancers of the thyroid and prostate, melanoma, and astrocytomas (14–19). These fusions either encode
protein partners that contribute coiled-coil (CC) or zinc-finger dimerization motifs to produce constitutively activated
BRAF dimers, or remove at least the first eight exons of BRAF
that are known to promote BRAF dimerization.
Recent mechanistic studies suggest that BRAFV600E protein
functions as a monomer, whereas most of the atypical BRAFmutant proteins function as dimers (5, 20–23). The ectopic
expression of BRAFV600E induces constitutive activation of
downstream ERK1/2 signaling, including negative feedback
regulation of RTKs and RAS. Among BRAFV600E-expressing
cells, BRAF monomer remains the primary driver of MEK1/2
and ERK1/2 signaling with minimal contribution from RTKs
and RAS, thus making it an attractive anticancer target
(4, 24). This led to the identification and FDA approval of the
BRAF-selective inhibitors vemurafenib and dabrafenib. Both
inhibitors showed antitumor activities in BRAF-mutant xenograft models (25–27), and significant clinical benefit among
patients with BRAF-mutant melanoma (28–30). However, these
first-generation BRAF drugs are not effective inhibitors of
dimeric forms of RAF, including RAS-activated RAF dimers,
many atypical BRAF mutants, BRAF splice forms, and BRAF
fusions. Indeed, vemurafenib and dabrafenib have been
shown to induce the dimerization of RAF proteins and promote paradoxical pathway activation of the MAPK pathway
in BRAF wild-type (WT) cells (31–33). The paradoxical activation is thought to explain the promotion of tumor growth
and metastasis observed with BRAF inhibitors in BRAF WT
preclinical models (33, 34). Clinically, these compounds promote skin side effects including keratoacanthomas and squamous cell carcinomas (29, 30). We have recently developed
LY3009120, a pan-RAF and RAF dimer inhibitor currently in
clinical studies. LY3009120 is able to effectively inhibit active
RAF dimers with minimal paradoxical activation (35, 36). In
this study, we have discovered novel BRAF aberrant variants,
which have in-frame deletions within or adjacent to residues
L485-P490 of the αC-helix region in patient samples and cell
lines of pancreatic, lung, and ovarian cancers. Further analyses revealed that the L485-P490–deleted BRAF is an activating
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RESEARCH ARTICLE
BRAF mutation, functions as a BRAF homodimer, and is able
to transform the cells. Tumor cells with these BRAF deletions
are resistant to the BRAF selective inhibitor vemurafenib but
sensitive to the RAF dimer inhibitor LY3009120 in vitro and
in vivo. LY3009120 represents a potential opportunity for treatment of patients with cancer with BRAF deletions or other
atypical BRAF mutations where BRAF is activated as a dimer.
RESULTS
Identification and Confirmation of BRAF In-Frame
Deletions within or Adjacent to Residues
L485-P490 of the aC-Helix Region in Cancer
Cell Lines and Patient Samples
In an unbiased screen of a large panel of tumor cell lines
for their sensitivity to MAPK pathway inhibitors, we observed
that BxPC-3 cells were very sensitive to the RAF dimer inhibitor LY3009120, but not sensitive to the BRAF-selective inhibitors vemurafenib or dabrafenib (36). BxPC-3 cells, derived from
a pancreatic adenocarcinoma, are unusual for not having a
KRAS mutation. As part of our Lilly internal genomics effort,
we have genetically characterized the tumor cell line panel by
whole exome sequencing (WES). When we carefully evaluated
these data for mutations in RAS pathway genes in BxPC-3
cells, including small in-frame deletions which are easy to be
ignored by routine data analysis, we discovered that BxPC-3
cells have a 5-amino acid deletion near the αC-helix region of
the BRAF kinase domain (V487-P492>A). One other cell line,
NCI-H2405, also stood out in our tumor cell line profiling for
being sensitive to LY3009120, but insensitive to BRAF-selective
inhibitors, despite having no well-described mutations in RAS
pathway genes (36). Close inspection of WES data revealed an
in-frame deletion in H2405 affecting the same region of the
BRAF gene in BxPC-3 cells (Table S1). Further searching of
cell line databases identified one additional cell line, OV-90,
an ovarian adenocarcinoma with a similar BRAF N486-P490
deletion, and sensitive to LY3009120, but not vemurafenib.
These in-frame deletions were further verified and confirmed by Sanger sequencing (i.e., BxPC-3; Supplementary
Fig. S1A–S1C).
These results hinted that the small in-frame deletions
might explain the high sensitivity of these cells to LY3009120
and therefore would indicate a potential subset of patients
who could benefit from this drug. To evaluate if these inframe deletions occur in patients with cancer, we analyzed the
publicly available databases from The Cancer Genome Atlas
(TCGA) and The International Cancer Genome Consortium
(ICGC). Indeed, several variations of these in-frame deletions were discovered in pancreatic cancer patient samples
(T488-Q493>K, N486-Q490 deletion) and thyroid carcinoma
samples (P490-Q494, T488-P492, N486-P490 deletion) from
TCGA studies. Two pancreatic cancer patient samples harboring in-frame deletion variants (T488-Q493>K and N486-P940
deletion) were also found in ICGC studies (Supplementary
Table S1). Further analysis revealed that these BRAF in-frame
deletions are mutually exclusive from RAS and BRAFV600 mutations. In all above reported cases, no missense mutation in
KRAS (G12, G13, Q61), NRAS (Q61), or BRAF (V600) was
found. This is especially relevant in pancreatic cancers that
have a high prevalence of KRAS mutation. Overall, the rate of
Chen et al.
BRAF deletion in patients with KRAS WT pancreatic cancer is
4.21% based on TCGA and ICGC studies (Fig. 1A). For thyroid
carcinomas, 3 out of 506 patients (0.59%) were identified to
have the BRAF in-frame deletion. These incidence rates are
likely underestimated due to the technical challenge in detecting a deletion and the limitations of sequencing technologies.
BRAF In-Frame Deletions Activate MAPK Signaling
in Tumor Cells and Ectopically Expressed Cells
To evaluate if these in-frame BRAF deletions activate downstream signaling, we conducted RAF isoform–specific knockdown with siRNA in tumor cell lines H2405, BxPC-3, and
OV-90. As demonstrated in Fig. 1B with H2405 cells, siRNA
knockdown of BRAF alone, or combinations of BRAF and
other RAF isoforms, showed significant decreases in phosphoMEK and ERK. However, knockdown of ARAF or CRAF alone,
or their combination, had minimal effects on phospho-MEK
or ERK levels. Similar siRNA knockdown results were observed
in BxPC-3 (Fig. 1C) and OV-90 (Fig. 1D) cells, although the
degree of phospho-MEK and ERK inhibition was different
among these cells, likely due to differences in BRAF knockdown efficiency. These results suggest that BRAF is a major
isoform to activate MAPK signaling in these tumor cells. To
further confirm pathway activation, we transfected a representative BRAF L485-P490 deletion (∆BRAF) with or without the
RAF dimer–deficient mutation (BRAF R509H) into HEK293
cells. As shown in Fig. 1E, ∆BRAF caused significant elevation
of phospho-MEK and ERK. Interestingly, the R509H mutation
showed significantly reduced activation, suggesting that the
BRAF deletion may function as a RAF dimer. In addition to
activating MAPK signaling, BRAF in-frame deletions appear to
be important for tumor cell proliferation. siRNA knockdown
of BRAF alone showed significant inhibition of cell proliferation in H2405 (Fig. 1F), BxPC-3 (Fig. 1G), and OV-90 (Fig. 1H)
cells. However, knockdown of either ARAF or CRAF had no
statistically significant effect on cell proliferation as compared
with controls. Overall, the MAPK activation and cell proliferation data suggest that the identified BRAF deletions are activating alterations and potentially oncogenic.
BRAF Deletions Transform Cells in a BRAF ­
Dimer–Dependent Manner
To validate if BRAF deletions possess oncogenic transformation activities, mouse NIH/3T3 cells were stably transfected
with ∆BRAF and then grown in soft-agar culture. In three
independent studies, ectopic expression of ∆BRAF was able to
transform NIH/3T3 cells and promote colony formation in soft
agar (Fig. 2A and Supplementary Fig. S2), whereas expression
of WT BRAF revealed no transformation activity (Supplementary Fig. S2). As a positive control, the anchorage-independent
growth of NIH/3T3 cells was also observed with the expression
of BRAFV600E. Again, ∆BRAF with a R509H mutation did not
support anchorage-independent growth in soft-agar culture
(Fig. 2A and B and Supplementary Fig. S2), indicating that
∆BRAF-promoted anchorage-independent growth is dependent on BRAF dimerization. Additionally, the ectopic expression
of ∆BRAF in NIH/3T3 cells elevated MAPK signaling as evaluated by phospho-MEK and ERK levels, whereas a concomitant
R509H mutation reduced phospho-MEK and ERK (Fig. 2C),
consistent with the observation in HEK293 cells (Fig. 1E).
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Oncogenic BRAF Deletions Functioning as Homodimers
RESEARCH ARTICLE
A
Cancer type
Study
Sample
counts
BRAF
deletion cases
Overall
prevalence
Thyroid
TCGA
506
3
0.59%
Pancreas
TCGA
131
2
Pancreas
ICGC PACA-AU
392
Pancreas
ICGC PACA-CA
Pancreas
TCGA + ICGC
1.53%
38
5.25%
1
0.26%
43
2.33%
112
1
0.89%
14
7.14%
635
4
0.63%
95
4.21%
D
A & B & CRAF
B & CRAF
A & CRAF
A & BRAF
CRAF
BRAF
Control
ARAF
OV-90
A & B & CRAF
B & CRAF
A & CRAF
A & BRAF
CRAF
BRAF
BxPC-3
ARAF
A & B & CRAF
B & CRAF
A & CRAF
A & BRAF
CRAF
BRAF
Control
ARAF
Prevalence
in KRAS WT
C
H2405
Control
B
Sample count
(KRAS WT)
siRNA
ARAF
BRAF
CRAF
pMEK
pERK
β-Actin
F
G
HEK293
H
H2405
BxPC-3
pMEK
pERK
ERK
Relative luminescence
FLAG
Relative luminescence
∆RAFB
R509H
∆BRAF
Control
**
120
100
80
60
40
20
0
Control ARAF BRAF CRAF
120
OV-90
*
*
Relative luminescence
E
100
80
60
40
20
0
Control ARAF BRAF CRAF
120
100
80
60
40
20
0
Control ARAF BRAF CRAF
Figure 1. Prevalence of BRAF deletions in thyroid and pancreatic patient samples and BRAF dependency of MAPK activation in tumor cells and HEK293
cells harboring BRAF deletions. A, prevalence of BRAF deletions in thyroid and pancreatic patient samples. PACA-AU, pancreatic cancer Australia; PACACA, pancreatic cancer Canada. B–D, knockdown of BRAF, but not ARAF or CRAF, inhibits MEK and ERK phosphorylation (p) in BRAF deletion cells. H2405,
BxPC-3, and OV-90 cells with endogenous expression of BRAF deletions were transfected with control scramble siRNA or ARAF, BRAF, or CRAF siRNA
either individually or in combination, as indicated. Cell lysates were analyzed for MEK and ERK phosphorylation by Western blot analysis. E, ectopic expression of BRAF deletion activates MAPK signaling in HEK293 cells. MEK and ERK phosphorylation in HEK293 cells transfected with pcDNA3.1 vector control
or FLAG-tagged BRAF L485-P490 deletion (∆BRAF) with or without the RAF dimer disrupting mutation (BRAFR509H). F–H, knockdown of BRAF, not ARAF
or CRAF, inhibits cell proliferation in BRAF deletion cells. Cell viability (mean ± SEM, relative to control) of H2405, BxPC-3, and OV-90 transfected with
control, ARAF, BRAF, or CRAF siRNA were analyzed by the CellTiter-Glo assay (*, P < 0.05; **, P < 0.01, one-tailed t test).
Overall, these results strongly suggest that ΔBRAF is an activating and oncogenic alteration, because the anchorage-independent growth is one of the hallmarks of cell transformation.
To further evaluate the role of the in-frame BRAF deletions
in the transformation of tumor cells, H2405 and OV-90 were
transfected with ARAF, BRAF, or CRAF siRNA and grown in softagar culture. On the one hand, as demonstrated in Fig. 2D–F,
knockdown of BRAF resulted in a significant decrease in colony
formation. On the other hand, knockdown of either ARAF or
CRAF had minimal effect compared with the control. This
suggests that BRAF plays the most important role among RAF
isoforms in maintaining transformation activities in these
tumor cells. It is also noteworthy that knockdown of KRAS
showed minimal effects on transformation activities (Fig. 2G–I)
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B
∆BRAF R509H
C
BRAF V600E
50
Number of colonies
**
40
∆BRAF
∆BRAF
Control
Control
∆BRAF R509H
A
BRAF V600E
Chen et al.
RESEARCH ARTICLE
BRAF
***
30
MYC
20
FLAG
10
pMEK
pERK
0
Control ∆BRAF ∆BRAF BRAF
R509H V600E
D
E
BRAF
CRAF
F
60
60
H2405
OV-90
Number of OV-90 colonies
ARAF
Number of H2405 colonies
Control
β-Actin
50
40
30
20
**
10
50
40
30
20
10
Control ARAF BRAF CRAF
Control ARAF BRAF CRAF
H
KRAS
60
Number of H2405 colonies
OV-90
H2405
Control
I
50
40
30
20
10
J
60
n.s.
Number of OV-90 colonies
G
n.s.
50
40
Control KRAS
H2405 OV-90
−
+
+
−
−
+
+
−
KRAS siRNA
Ctrl siRNA
KRAS
30
pMEK
20
pERK
10
β-Actin
0
0
***
0
0
Control KRAS
Figure 2. ∆BRAF is an oncogenic alteration and transforms NIH/3T3 cells in a BRAF-dimer dependent manner. A, ∆BRAF is able to transform NIH/3T3
cells in a BRAF dimer–dependent manner. NIH/3T3 cells stably expressing FLAG-tagged ∆BRAF with or without R509H mutation, MYC-tagged BRAFV600E,
or empty vector were seeded in soft-agar plates for colony formation. Colonies were examined by light microscopy after 3 weeks, and representative
images from at least three independent studies were taken. B, quantification of colonies in soft-agar growth of A. The numbers of colonies are expressed
as mean ± SEM from three experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001, relative to control, one-tailed t test). C, ∆BRAF activates MAPK signaling in
a BRAF dimer–dependent manner in NIH/3T3 cells. Cell lysates from NIH/3T3 expressing the indicated BRAF proteins were examined for MEK and ERK
phosphorylation. D, BRAF, but not ARAF or CRAF, is important for cell proliferation in tumor cells with BRAF deletions. H2405 and OV-90 cells were seeded
in soft-agar plates 48 hours after transfection with indicated control, ARAF, BRAF, or CRAF siRNA. Representative images from at least two independent
studies were taken after 3 weeks. E and F, quantification of colonies in soft-agar growth of D. G, KRAS is not important in tumor cells harboring BRAF deletions. H2405 and OV-90 cells were seeded in soft agar 48 hours after transfection with control or KRAS siRNA. Representative images from at least two
independent studies were taken after 3 weeks. H and I, quantification of colonies in soft-agar growth of G. J, KRAS-independent MAPK activation in tumor
cells with BRAF deletions. Cell lysates from H2405 and OV-90 with KRAS knockdown were analyzed for MEK and ERK phosphorylation.
or MAPK signaling (Fig. 2J) in these BRAF deletion–expressing
tumor cells, suggesting that these BRAF deletions function as
activating mutations in a KRAS-independent manner.
BRAF In-Frame Deletions Mainly Function as
BRAF Homodimers
To understand if BRAF deletions mainly function as BRAF
homodimers or BRAF/CRAF heterodimers, we developed
in situ proximity ligation assays (PLA) as described previously
(37, 38). As a control for BRAF homodimers, we transfected
A375 cells with a construct encoding the p61BRAFV600E
splice variant. Consistent with previous observations that
p61BRAFV600E mainly functions as a BRAF homodimer (5),
the ectopic expression of p61BRAFV600E exhibits a strong
in situ PLA signal for BRAF homodimers but minimal detectable BRAF/CRAF heterodimer signal (Fig. 3A). HeLa cells
treated with EGF to induce BRAF/CRAF heterodimers served
as a positive control for the ability of our PLA system to
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Oncogenic BRAF Deletions Functioning as Homodimers
H2405
BxPC-3
B
OV-90
BRAF/CRAF
heterodimers
A375 p61V600E
BRAF/BRAF
homodimers
Signals per cell
12
BRAF/CRAF
BRAF/BRAF
10
8
***
***
***
***
6
4
2
0
A375 H2405 BxPC-3 OV-90
p61V600E
C
∆BRAF
BRAF E586K
∆BRAF R509H
BRAF/CRAF
heterodimers
Control
D
***
BRAF/CRAF
BRAF/BRAF
BRAF/BRAF
homodimers
Signals per cell
25
***
20
15
***
5
0
Control
+
−
+
∆BRAF-MYC
FLAG
Input
HEK293
BRAF ∆BRAF ∆BRAF
E586K
R509H
NIH/3T3
∆BRAF
−
F
∆BRAF R509H
FLAG
∆BRAF
∆BRAF
FLAG
Control
E
pCRAF S338
MYC
CRAF
β-Actin
BRAF
FLAG
IP: FLAG
MYC
FLAG
**
10
Control
A
RESEARCH ARTICLE
pMEK
pERK
ERK
β-Actin
IP: MYC
MYC
Figure 3. BRAF in-frame deletions mainly function as BRAF homodimers. A, detection of BRAF/CRAF heterodimers and BRAF homodimers in H2405,
BxPC-3, OV-90, and A375 cells ectopically expressing p61BRAFV600E using in situ PLA. B, quantification of in situ PLA signals of A (mean ± SEM). The
number of PLA signals per cell with at least 1,000 cells for all reactions in triplicate was quantified and analyzed by Cellomics ArrayScan VTI Reader and
HCS software (**, P < 0.01; ***, P < 0.001, one-tailed t test). C, detection of BRAF/CRAF heterodimers and BRAF homodimers in HEK293 cells ectopically
expressing BRAFE586K, BRAF L485-P490 deletion (∆BRAF) with or without R509H, or empty vector (control) using PLA. PLA signals (red spots) were
examined under a confocal microscope and representative images were shown from at least two or three independent experiments. D, quantification of
in situ PLA signals of C (mean ± SEM) in HEK293 expressing the indicated BRAF proteins. E, detection of BRAF homodimer by IP and Western blot
analysis. HEK293 cells stably expressing FLAG-tagged ∆BRAF or ∆BRAF R509H protein were transfected with vectors encoding MYC-tagged ∆BRAF
followed by IP using anti-FLAG or anti-MYC antibody–conjugated beads. The input and IP-prepared proteins were subjected to Western blot analysis with
anti-FLAG and anti-MYC antibodies. F, CRAF S338 phosphorylation and MAPK activation in ∆BRAF-transfected HEK293 and NIH/3T3 cells.
detect this dimeric species (38). As revealed in Supplementary
Fig. S3A, treatment of HeLa cells by EGF induced a clear PLA
signal for BRAF/CRAF heterodimer. With PLAs for BRAF
homodimers and BRAF/CRAF heterodimers established,
we examined the status of RAF dimers in tumor cells. As
shown in Fig. 3A, H2405, BxPC-3, and OV-90 cells harboring
BRAF in-frame deletions all showed clear evidence for BRAF
homodimers but not for BRAF/CRAF heterodimers, suggesting that the BRAF homodimer is the major RAF dimer in
these cells (Fig. 3A and B).
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RESEARCH ARTICLE
The existence of BRAF homodimers in tumor cells, and
the demonstration that Arg509 is critical for signaling of
these deletion mutants, indicated that the mutations may
activate downstream signaling by promoting the formation of dimers just like many other oncogenic BRAF alterations. To test this idea, we transfected ∆BRAF into HEK293
cells to detect in situ dimerization using PLA. As a positive
control, ectopic expression of BRAFE586K, which has been
shown to promote RAF dimerization (22, 38, 39), exhibited
a strong PLA signal for the BRAF homodimer (Fig. 3C). In
contrast, BRAF homodimers were minimal in cells expressing CRAFE478K, a CRAF dimer–promoting mutation, serving
as a negative control (Supplementary Fig. S3B). Similar to
BRAFE586K, the ectopic expression of the ∆BRAF predominantly promoted BRAF homodimers, while a low level of
BRAF/CRAF heterodimer signal was detected. The homodimer signal was substantially reduced by R509H BRAF dimer–
deficient mutation (Fig. 3C and D). Further, we cotransfected
two constructs encoding the same ∆BRAF with different
tags, FLAG or MYC, into HEK293 cells. As shown in Fig. 3E,
immunoprecipitation (IP) with a FLAG antibody was able to
pull down the MYC-tagged BRAF protein. Similarly, IP with a
MYC antibody was able to pull down the FLAG-tagged BRAF
protein, confirming the formation of BRAF homodimers.
Consistent with the PLA results, BRAF R509H mutation
reduced the BRAF dimer formation in these IP studies (Fig.
3E). All together, the PLA and IP studies show that ∆BRAF
promotes homodimer formation in cells, providing an explanation for oncogenicity of this new class of BRAF mutations.
The low-level PLA signal for BRAF/CRAF heterodimers
may be an artifact of the system used but could, conceivably,
instead reflect a cryptic role of CRAF in the signaling pathway activated by ∆BRAF. To determine whether CRAF may
be active in BRAF deletion–mediated MAPK activation, we
transfected ∆BRAF into HEK293 and NIH/3T3 cells. ∆BRAF
activated phospho-MEK and ERK in both cell lines (Fig. 3F).
However, CRAF protein was not activated in either cell line
based on phosphorylation of serine 338, suggesting that
CRAF is not functionally engaged in ∆BRAF-mediated MAPK
activation. In three tumor cell lines with BRAF deletions
and A375 cells with a BRAFV600E mutation, the endogenous
phospho-CRAF levels are generally low with the exception of
BxPC-3 cells, whereas phospho-CRAF activities are generally
higher in KRAS-mutant tumor cells (Supplementary Fig. S4).
BRAF Deletions Shorten the b3/aC-Helix Loop
and Hinder Its Flexibility by Locking the Helix in
the Active aC-Helix-in Conformation That Favors
BRAF Dimerization
To explain how BRAF in-frame deletions promote protein
dimerization and activation of the MAPK signaling pathway,
we located structural regions corresponding to the identified
deletions in the crystal structure of the BRAF kinase domain
in complex with LY3009120 (PDB 5C9C). Based on the structural information, all in-frame deletions listed in Supplementary Table S1 were mapped to the β3/αC-helix loop, an
essential structural region responsible for a conformational
movement of the αC-helix between the αC-helix-in and αChelix-out conformations (Fig. 4A). Molecular modeling suggests that the in-frame deletions shorten the β3/αC-helix loop
Chen et al.
and hinder the flexibility of αC-helix by locking it in the αChelix-in conformation via the Glu501/Lys483 salt bridge (Fig.
4B). Although this αC-helix-in conformation accommodates
binding of type IIa inhibitors (e.g., LY3009120), it disfavors the
type IIb binders (e.g., vemurafenib) that require the αC-helixout binding conformation. Meanwhile, the αC-helix-in conformation of BRAF promotes protein dimerization through
the network of intermolecular interactions at the dimer surface, including multiple salt bridge/hydrogen bond interactions between Arg506 and Arg509, and Asp449 and Thr508,
respectively (Fig. 4C). To summarize, the active αC-helix-in
conformation stabilized by a shortened β3/αC-helix loop
region in the BRAF in-frame deletion mutants favors BRAF
dimerization, leading to MAPK pathway activation. This
is consistent with previous reports suggesting that the αChelix-in conformation of the RAF proteins promotes dimer
formation (36, 40).
BRAF Deletion–Mediated MAPK Activation Is
Sensitive to LY3009120, a RAF Dimer Inhibitor,
but Resistant to Vemurafenib
Next, we investigated the sensitivity of the BRAF deletion–
mediated MAPK activation to MAPK pathway inhibitors. As
revealed in Fig. 5A, vemurafenib fails to reduce phosphoMEK or ERK, at concentrations below 10 μmol/L in all three
cell lines—H2405, BxPC-3, and OV-90—that harbor BRAF
deletions. Similarly, treatment with another BRAF-selective
inhibitor, dabrafenib, revealed minimal effects on MEK and
ERK phosphorylation in H2405 and BxPC-3 and modest
inhibition in OV-90 (Supplementary Fig. S5A). In contrast,
LY3009120 demonstrated potent and dose-dependent inhibition of phospho-MEK and ERK with significant inhibition
observed at 0.01 μmol/L in all three cell lines (Fig. 5B). Similar
to LY3009120, the MEK inhibitor trametinib is potent and
active at inhibiting the phospho-MEK and ERK activities in
these cells (Fig. 5C). These results further solidify the notion
that the BRAF deletions function as RAF dimers. The sensitivities of BRAF-deleted cells to LY3009120 are similar to
BRAFV600E-mutant A375 cells and are considerably more sensitive than KRAS-mutant HCT116 cells based on phospho-ERK
inhibition (Fig. 5D and E) and cell proliferation (Supplementary Fig. S5B and S5C). To further verify these results,
we compared the inhibitory activities of vemurafenib and
LY3009120 in HEK293 cells transfected with ∆BRAF. Consistent with results obtained in H2405, BxPC-3, and OV-90
cells, LY3009120 exhibited dose-dependent inhibition of
phospho-MEK and ERK in HEK293 cells (Fig. 5F). However,
vemurafenib showed no inhibitory activity (Fig. 5G).
Growth of Tumor Cells Harboring BRAF Deletion
Is Sensitive to LY3009120, but Resistant to
Vemurafenib In Vitro
We then evaluated the in vitro growth inhibitory activities of
MAPK pathway inhibitors to tumor cells harboring BRAF deletions. As shown in Fig. 6A–D, LY3009120 demonstrated a concentration-dependent cell growth inhibition with IC50 values
of 0.04, 0.087, and 0.007 μmol/L against H2405, BxPC-3, and
OV-90 cells, respectively. However, vemurafenib had minimal
activity inhibiting the cell growth of these cells. Again, the MEK
inhibitor trametinib showed potent cell growth inhibition with
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Oncogenic BRAF Deletions Functioning as Homodimers
A
RESEARCH ARTICLE
B
N-termini
β5
β4
β3
Deletion
region
Ala489
Thr488
Pro490
Val487
β2
Asn486
β1
Leu485
LY3009120
∆BRAF
β3
αC-in
Lys483
Glu501
C-termini
C
Loop deletion
D449
αC
R506
T508
R509
αC
T508
R506
∆BRAF #1
D449
∆BRAF #2
Loop deletion
Figure 4. Molecular modeling of the BRAF in-frame deletion using protein coordinates from the BRAF/LY3009120 complex (PDB 5C9C). A, overall
view of the BRAF in-frame deletion in complex with LY3009120. Key structural elements of the kinase domain and BRAF-deleted region are marked.
The ATP-binding regions of ∆BRAF are shown in the following colors: G-loop, green; αC-helix, magenta; hinge, gold; DFG (aspartic acid, phenylalanine and
glycine) motif, turquoise; catalytic loop, red. Pan-RAF type-IIa inhibitor LY3009120 is shown in a space-filled model and colored in the following atom
colors: carbon, green; nitrogen, blue; oxygen, red; fluorine, light green. ∆BRAF is in the DFG-out/αC-helix-in conformation. All identified ∆BRAF in-frame
deletions near L485-A489 (Supplementary Table S1) are located within the β3/αC-helix loop, which provides the essential flexibility to the αC-helix to
toggle between the active (αC-helix-in) and inactive (αC-helix-out) conformations. B, superimposed view of the β3/αC-helix loop region in the ∆BRAFmutant model (colored in gold) and WT BRAF (PDB 5C9C). Residue deletion segment L486-A489 is shown as sticks and colored in the following atom
colors: carbon, green; nitrogen, blue; oxygen, red. Molecular modeling suggests that the in-frame deletions shorten the β3/αC-helix loop and impair its
flexibility by locking the helix in the active αC-helix-in conformation via the Glu501/Lys483 salt bridge. C, surface representation of the dimeric ∆BRAF
mutant viewed approximately down the local 2-fold axis. The αC-helix-in conformation of the mutant protein favors and promotes dimerization through
the network of intermolecular interactions at the dimer interface, including multiple salt bridge/hydrogen bond interactions between R506 and R509 and
D449 and T508, respectively.
IC50 values of 0.079, 0.006, and 0.003 μmol/L against H2405,
BxPC-3, and OV-90 cells, respectively. Further cell-cycle analysis
by flow cytometry illustrated that LY3009120 or trametinib
treatment induced an increase of G1–G0 phase and a decrease
of S phase in these cells (Fig. 6E–G). These cell-cycle effects
were further verified by BrdUrd incorporation (Supplementary
Fig. S6A). LY3009120 at 1 μmol/L or trametinib at 0.2 μmol/L
significantly reduced BrdUrd-positive cells, whereas vemurafenib
at 5 μmol/L had no effect in all three cell lines tested. In addition
to cell-cycle G1–G0 arrest, LY3009120 or trametinib treatment
also induced an increase of sub-G1 population of these cells, suggesting a compound-induced apoptotic effect (Fig. 6E–G). The
apoptotic effects were further verified by LY3009120-induced
increases of cleaved PARP in all three cell lines (Fig. 6H–J). This
PARP cleavage can be inhibited by a pan-caspase inhibitor,
Z-VAD-FMK, in a dose-dependent manner, suggesting that the
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Chen et al.
RESEARCH ARTICLE
A
H2405
BxPC-3
.4
2
0
10
B
.08 .4
OV-90
2
0
10
Vemurafenib
.01
0
.01
.08
.4
2
0
10
.01 .08
.4
2
µmol/L
10
pMEK
Vemurafenib
.01 .08
Vemurafenib
0
.01 .08
.4
2
pERK
β-Actin
0
10
.01 .08
.4
2
10
µmol/L
LY3009120
LY3009120
C
LY3009120
pMEK
0
.01 .08
.4
2
0
10
.01 .08
.4
2
pERK
β-Actin
0
10
.01 .08 .4
2
µmol/L
10
Trametinib
Trametinib
Trametinib
pMEK
pERK
β-Actin
E
3
10
1
3
0.
0.
00
00
0.
pMEK
.03
0.1
0.3
1
β-Actin
3
10
HEK293 ∆BRAF
µmol/L
0
.01
.03
0.1 0.3
1
3
10
µmol/L
pMEK
pMEK
pERK
pERK
FLAG
BRAF
β-Actin
Vemurafenib
.01
pERK
G
HEK293 ∆BRAF
LY3009120
µmol/L
LY3009120
3
0
3
1
0.
03
0.
0.
1
3
01
00
0.
0.
0
LY3009120
F
0
1
00
3
0.
01
0.
03
0.
1
HCT116 (KRASG13D)
03
A375 (BRAFV600E)
0.
D
FLAG
BRAF
β-Actin
Figure 5. BRAF deletion–mediated MAPK activation is sensitive to LY3009120, a RAF dimer inhibitor, and trametinib, but resistant to vemurafenib,
a BRAF monomer inhibitor. A–C, phospho-MEK and ERK levels of H2405, BxPC-3, and OV-90 cells treated with vemurafenib, LY3009120, or trametinib.
Cells were treated at indicated concentrations for 2 hours, and cell lysates were analyzed for MEK and ERK phosphorylation by Western blotting. D and
E, phospho-MEK and phospho-ERK inhibition of BRAFV600E-mutant A375 and KRASG13D-mutant HCT116 cells by LY3009120. F–G, phospho-MEK and
phospho-ERK inhibition by LY3009120 and vemurafenib in ∆BRAF-transfected HEK293 cells. HEK293 cells stably expressing ∆BRAF were treated with
LY3009120 or vemurafenib at indicated concentrations for 2 hours. Cell lysates were analyzed for MEK and ERK phosphorylation by Western blotting.
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Oncogenic BRAF Deletions Functioning as Homodimers
B
0
1
2
3
4
5
Compound: Log (nmol/L)
150
200
100
150
200
Number
0
250
%G1 = 45.10
%S = 43.13
%G2–M = 11.77
%Debris = 11.22
50
100
150
200
250
50
100
150
200
150
200
250
H2405
DMSO LY3009120 Vemurafenib
2 24 48 2 24 48
50
100
150
200
250
150
200
250
80
0
0
50
PI
I
100
%G1 = 57.80
%S = 9.72
%G2–M = 32.48
%Debris = 54.76
320
Number
240
Number
320
%G1 = 43.17
%S = 24.70
%G2–M = 32.12
%Debris = 18.93
80
0
PI
50
400
400
100
250
PI
0
0
50
200
%G1 = 73.67
%S = 14.26
%G2–M = 12.07
%Debris = 26.86
0
250
160
180
60
Number
240
%G1 = 54.80
%S = 9.61
%G2–M = 35.58
%Debris = 61.01
150
PI
PI
120
280
210
Number
140
0
0
100
80
0
PI
%G1 = 43.30
%S = 27.74
%G2–M = 28.97
%Debris = 15.86
50
0
50
0
0
250
PI
%G1 = 71.47
%S = 16.44
%G2–M = 12.09
%Debris = 34.17
0
200
280
210
70
100
50
PI
%G1 = 63.47
%S = 24.47
%G2–M = 12.07
%Debris = 43.94
100 200 300 400 500
600
Number
0
250
0
0
50
70
OV-90
200
140
160
120
80
Number
40
BxPC-3
0
H
150
PI
%G1 = 47.62
%S = 40.60
%G2–M = 11.79
%Debris = 13.77
G
%G1 = 60.88
%S = 26.23
%G2–M = 12.89
%Debris = 9.77
200
100
3.1
Trametinib
0
50
7.1
0
1
2
3
4
5
Compound: Log (nmol/L)
400
0 100 200 300 400 500 600
Number
Number
0
250
6.1
4,033.0
400
200
PI
Tra
79.1
Vemurafenib
%G1 = 70.84
%S = 16.84
%G2–M = 12.32
%Debris = 43.13
LY
H2405 >10,000 40.0
320
150
0
−1
150
100
20
Vem
BxPC-3 >10,000 87.4
OV-90
100
50
40
LY3009120
%G1 = 54.64
%S = 30.90
%G2–M = 14.47
%Debris = 9.98
0
F
60
240
0
−1
DMSO
0 100 200 300 400 500 600
H2405
E
20
IC50
(nmol/L)
Trametinib
Number
1
2
0
3
4
Compound: Log (nmol/L)
40
80
Number
20
−1
60
Vemurafenib
LY3009120
100
160
40
80
OV-90
120
240
80
60
100
D
160
100
BxPC-3
120
Relative luminescence
Relative luminescence
H2405
120
C
Number
Relative luminescence
A
RESEARCH ARTICLE
100
150
200
250
0
PI
BxPC-3
DMSO LY3009120 Vemurafenib
2 24 48 2 24 48
J
50
100
150
200
250
PI
OV-90
DMSO LY3009120 Vemurafenib
2 24 48 2 24 48 h
pERK
pMEK
cPARP
β-Actin
Figure 6. Growth of tumor cells harboring BRAF deletion is sensitive to LY3009120, but resistant to vemurafenib in vitro. A–C, antiproliferation activities of vemurafenib, LY3009120, and trametinib in H2405, BxPC-3, or OV-90 cells assessed by the CellTiter-Glo assay. The cells were treated for 72
hours with different inhibitors at indicated concentrations. D, antiproliferation IC50 of vemurafenib (Vem), LY3009120 (LY), or trametinib (Tra) in H2405,
BxPC-3, and OV-90 cells. IC50 was calculated via sigmoidal dose-response curve using GraphPad Prism 4 software. E–G, cell-cycle analysis by flow cytometry of H2405, BxPC-3, or OV-90 cells treated with vemurafenib (5 μmol/L), LY3009120 (1 μmol/L), or trametinib (0.2 μmol/L). Cells were subjected to PI
staining at 72 hours after treatment. Dead cells are indicated as debris. Representative histograms are shown from three independent experiments. H–J,
apoptosis analysis of H2405, BxPC-3, and OV-90 cells treated with vemurafenib (5 μmol/L) or LY3009120 (1 μmol/L) for 2, 24, and 48 hours,
respectively. Cell lysates were analyzed for MEK and ERK phosphorylation and cleaved PARP (cPARP) induction by Western blotting.
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RESEARCH ARTICLE
apoptosis is caspase dependent (Supplementary Fig. S6B). In
contrast to LY3009120, vemurafenib treatment had minimal
effects on cell-cycle G1–G0 arrest or apoptosis of these tumor
cells (Fig. 6E–J). Similar to vemurafenib, dabrafenib treatment
did not induce apoptosis of H2405 and BxPC-3 cells based on
cPARP (Supplementary Fig. S6C).
To extend the analysis of LY3009120 against tumor cells harboring atypical BRAF mutations where BRAF proteins mostly
function as dimers, we tested 14 additional cell lines, including
2 PDX cell lines, BXF 1218L and RXL 1183L, as shown in Supplementary Table S2. LY3009120 is active against the majority
of these tumor cell lines in vitro. Among them, 12 of 14 cell
lines exhibited absolute IC50 values from 0.045 to 0.58 μmol/L
LY3009120, whereas vemurafenib was generally inactive.
Xenograft Tumors Harboring BRAF Deletions
Are Sensitive to LY3009120, but Resistant to
Vemurafenib In Vivo
We then attempted to develop rat xenograft models with
H2405, BxPC-3, and OV-90 cells. Both H2405 and BxPC-3
cells were able to grow tumors consistently, whereas OV-90
cells failed to grow tumors in nude rats. To assess the in vivo
sensitivity to MAPK pathway inhibitors, we treated the xenograft tumors with LY3009120 or vemurafenib as described.
As demonstrated in Fig. 7A and Supplementary Fig. S7A,
in the H2405 xenograft model, treatment of LY3009120 at
15 or 30 mg/kg achieved almost complete tumor growth
regression, whereas vemurafenib treatment at 20 mg/kg had
no antitumor growth activity despite achieving significant
single-agent activity in melanoma BRAFV600E-mutant models
(36, 41). Similarly, in the BxPC-3 xenograft model, LY3009120
at 15 or 30 mg/kg demonstrated significant tumor growth
inhibition and partial regression, whereas vemurafenib had no
antitumor effect (Fig. 7B; Supplementary Fig. S7B). Western
blot analysis of the tumor lysates from these studies revealed
that LY3009120 significantly inhibited phospho-MEK and
phospho-ERK, whereas vemurafenib did not (Fig. 7C and D).
Further analysis revealed that treatment of LY3009120 at 15
or 30 mg/kg inhibited downstream phospho-MEK and ERK
by approximately 70% and 60%, respectively, in the H2405
model (Fig. 7E), and significant inhibition of phospho-MEK
(61% at 15 mg/kg; 71% at 30 mg/kg) and phospho-ERK
(66% at 15 mg/kg; 75% at 30 mg/kg) by LY3009120 was also
observed in the BxPC-3 model (Fig. 7F). Based on the inhibition of tumor growth and downstream signaling, LY3009120
treatment at 15 mg/kg achieved nearly maximum effect. In
both studies, LY3009120 appeared to be well tolerated at 15
and 30 mg/kg with no significant body weight loss (Supplementary Fig. S7C and S7D). Overall, the results from these
in vivo studies are completely consistent with in vitro observations. Xenograft tumors with a BRAF deletion are sensitive to
RAF dimer inhibitor LY3009120 and resistant to the BRAF
monomer inhibitor vemurafenib. In both studies, the tumor
growth inhibition induced by LY3009120 was correlated with
phospho-MEK and ERK inhibition within the tumors.
DISCUSSION
BRAF inhibitors vemurafenib and dabrafenib are active in
BRAFV600-mutant melanoma. However, these inhibitors are
Chen et al.
less active in cells expressing WT BRAF and paradoxically
activate downstream RAF–MEK–ERK signaling and promote
tumor growth in cells with activating RAS mutations (33, 34).
Consequently, these inhibitors should be used with caution
in patients whose tumors harbor a RAS mutation. Recent
studies have revealed that these BRAF-selective inhibitors
promote BRAF and CRAF dimerization, an essential step
in the paradoxical pathway activation (31–33). It has now
become more evident that vemurafenib and dabrafenib primarily bind one of the two protomers of the asymmetric RAF
dimers and thereby fail to effectively inhibit downstream
signaling (36). LY3009120 is a pan-RAF inhibitor that binds
both protomers of RAF dimers and effectively inhibits downstream signaling (35, 36). Due to their distinct mechanisms
of action, LY3009120, but not vemurafenib, is active against
tumor cells with RAF in-frame deletions identified in this
study and many other atypical BRAF mutations where BRAF
functions as dimers (Table S2). Our data provide additional
evidence that LY3009120 is a RAF dimer inhibitor.
In this study, we have discovered novel oncogenic BRAF
in-frame deletions with a distinct activating mechanism
dependent on BRAF dimer formation in human cancers. In
addition to cell lines, the BRAF deletions were also identified in patients with pancreatic cancer or thyroid carcinoma
with overall prevalence of 0.63% and 0.59%, respectively, and
4.2% frequency occurring in KRAS WT pancreatic cancer. The
prevalence of BRAF deletions is likely underestimated because
the current sequencing technologies and analytic tools are
mainly designed for identification of point mutations and
less favorable for identification of small in-frame deletions.
In addition to BRAF in-frame deletions, other atypical BRAF
mutations and BRAF fusions where BRAF functions as dimers
also occur in many cancer types, including melanoma, lung,
colorectal, and pancreatic cancers. In lung adenocarcinoma,
the overall BRAF mutation frequency is approximately 6.7%,
and 80% of these are atypical (13). In colorectal cancer, BRAF
mutations are present in about 10% of patients, with 50%
atypical (12, 13). Although the frequencies of these BRAF
alterations in lung, colon, and pancreatic cancers are low, the
disease-related mortality of these cancers irrespective of their
mutational subtype remains high: 158,000 and 50,000 deaths
per year in the United States in lung cancer and colon cancer, respectively, which suggests a clear unmet medical need
(42, 43). LY3009120 may have the potential for treatment of
this unique patient population.
We found that BRAF deletions are mutually exclusive
with RAS mutations, suggesting that BRAF deletions are
potential oncogenes. Indeed, we have confirmed that they
are activating and oncogenic alterations. In three cancer cell
lines, BxPC-3, H2405, and OV-90, harboring BRAF deletions,
siRNA knockdown of BRAF, but not CRAF or ARAF, significantly reduced phospho-MEK and ERK levels, and ectopic
expression of ∆BRAF enhanced phospho-MEK and ERK activation in HEK293 and NIH/3T3 cells. Knockdown of BRAF
by siRNA or inhibition by LY3009120 inhibited proliferation
of tumor cells harboring BRAF deletions. Ectopic expression
of ∆BRAF is able to transform NIH/3T3 cells and promotes
anchorage-independent growth that is comparable with that
caused by the BRAFV600E mutation. Finally, xenograft models
developed with tumor cells harboring a BRAF deletion are
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Oncogenic BRAF Deletions Functioning as Homodimers
A
RESEARCH ARTICLE
B
H2405
BxPC-3
Vehicle
1,200
Vem (20 mg/kg, BID)
LY (15 mg/kg, BID)
4,000
LY (30 mg/kg, BID)
3,000
2,000
1,000
1,000
LY (15 mg/kg, BID)
800
LY (30 mg/kg, BID)
600
400
200
0
0
18
21
24 27 30 33 36
Day after tumor implant
39
18 21 24 27 30 33 36 39 42 45 48 51
Day after tumor implant
42
C
D
LY
15 mg/kg
Vehicle
LY
30 mg/kg
LY
15 mg/kg
Vehicle
Vem
E
LY
30 mg/kg
Vem
pMEK
pMEK
pERK
pERK
MEK
MEK
ERK
ERK
β-Actin
β-Actin
F
H2405
BxPC-3
120
100
100
100
80
60
40
****
****
20
0
Relative densitometry
(pMEK/MEK)
120
Relative densitometry
(pERK/ERK)
120
80
60
***
***
40
20
0
e
kg
cl
hi
Ve
LY
15
m
LY
kg
kg
g/
g/
g/
30
m
m
Ve
20
m
BxPC-3
120
Relative densitometry
(pERK/ERK)
H2405
Relative densitometry
(pMEK/MEK)
Vehicle
Vem (20 mg/kg, BID)
5,000
Tumor volume (mm3)
Tumor volume (mm3)
6,000
80
60
****
40
****
20
0
e
LY
m
LY
g/
g/
g/
15
kg
kg
kg
cl
hi
Ve
30
m
m
Ve
20
m
100
80
60
40
****
****
20
0
kg
e
cl
hi
Ve
LY
15
m
LY
kg
kg
g/
g/
g/
30
m
m
Ve
20
m
LY
kg
kg
le
ic
h
Ve
m
LY
kg
g/
g/
15
30
m
m
Ve
g/
20
m
Figure 7. Xenograft tumors harboring BRAF deletion are sensitive to LY3009120, but resistant to vemurafenib. A and B, antitumor activities of LY3009120
(LY) and vemurafenib (Vem) in H2405 (A) and BxPC-3 (B) models. Xenografts were treated with vemurafenib (20 mg/kg), LY3009120 (15 or 30 mg/kg), or
vehicle twice daily for 3 to 4 weeks (8 animals per treatment group) and tumor volumes (mean ± SEM) were measured every 3 to 5 days. C and D, inhibition of
phospho-MEK and phospho-ERK in tumor lysates. H2405 and BxPC-3 tumors were lysed following the completion of the treatment and analyzed with Western
blotting for MEK and ERK phosphorylation. E and F, densitometric analysis (mean ± SEM, relative to vehicle groups) of the levels of phospho-MEK and phosphoERK in H2405 and BxPC-3 tumors after normalized to total MEK and ERK using ImageJ software (***, P < 0.001; ****, P < 0.0001, one-tailed t test).
highly sensitive to inhibition by the RAF dimer inhibitor
LY3009120, and the tumor growth inhibition is associated
with downregulation of phospho-MEK and ERK. Overall,
these data support the conclusion that the novel BRAF deletions are activating and oncogenic alterations.
We found that MAPK activation by BRAF deletions is dependent on homodimerization. Ectopic expression of ∆BRAF activated phospho-MEK and ERK in HEK293 and NIH/3T3 cells,
and BRAF dimer-deficient mutation R509H significantly
reduced MEK and ERK activation, suggesting that BRAFengaged dimer is important for pathway activation. Similarly
in soft-agar culture, BRAF deletion with a dimer-deficient
R509H substitution failed to transform NIH/3T3 cells. In situ
PLA demonstrated that the BRAF homodimer is the major
RAF dimer formed in tumor cells or transfected HEK293 cells
harboring these BRAF in-frame deletions, and co-IP analysis
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Published OnlineFirst January 5, 2016; DOI: 10.1158/2159-8290.CD-15-0896
Chen et al.
RESEARCH ARTICLE
revealed that the BRAF deletion is able to form BRAF homodimers. Structural analysis revealed that these BRAF deletions
shorten the β3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors
BRAF dimerization. Finally, tumor cells with these BRAF deletions are sensitive to the RAF dimer inhibitor LY3009120 but
resistant to the BRAF monomer inhibitor vemurafenib in vitro
and in vivo. As indirect evidence, transfection of ∆BRAF into
HEK293 or NIH/3T3 cells activated the MAPK activity in a
CRAF-independent manner. All together, these data suggest
that these BRAF deletions function as BRAF homodimers.
In the original description of BRAF mutations in cancer,
BRAFV600E was only 1 of the 14 BRAF alterations identified in
cell lines and primary tumor samples (1). Since then, nearly
300 distinct missense mutations have been found in tumor
samples and cancer cell lines (44). These missense mutations
encompass over 100 of the 766 BRAF amino acids, but most of
the mutations occur in the activation loop (A-loop) near V600,
or in the phosphate-binding loop (P-loop) at residues 464–469
(10). According to a model based on crystal structures of the
BRAF kinase domain, both loops interact with each other via
hydrophobic interactions (2). Disruption of this interaction
by V600 mutations results in a conformational change within
the kinase domain and full activation of BRAF. Many nonV600 oncogenic variants of BRAF have been shown to activate
signaling by promoting the formation of active dimers, and
our data suggest that BRAF deletions also promote dimer
formation. Structural analysis suggests an explanation for
this ability and points to a novel mechanism of dimer promotion. The BRAF deletions described here all have a 5-amino
acid deletion just outside the P-loop, within the β3/αC-helix
loop, a region at the interface critical for RAF dimerization.
The β3/αC-helix loop provides flexibility to the αC-helix,
allowing movement between the active (αC-helix-in) and inactive (αC-helix-out) conformation. The 5-amino acid deletion
shortens the β3/αC-helix loop, impairing its flexibility and
fixing the helix in the active αC-helix-in conformation. The
αC-helix-in conformation of the BRAF proteins favors and
promotes BRAF dimerization as described (36, 40). Consistent
with this model, tumor cells with BRAF deletions are sensitive
to the RAF dimer inhibitor LY3009120, but resistant to the
BRAF monomer inhibitor vemurafenib in vitro and in vivo. In
principle, the structural studies do not preclude the possible
stabilization of BRAF heterodimers with ARAF or CRAF.
However, we have not found evidence for a substantial contribution of heterodimers. For example, transfection of ∆BRAF
into HEK293 or NIH/3T3 cells activated the MAPK activity
in a CRAF-independent manner. The activation mechanism
proposed here is consistent with recent findings showing that
N-terminally truncated BRAF proteins, and many other atypical BRAF mutations, function as BRAF homodimers (5, 23).
The activating effect of the BRAF deletion described here
highlights a region of the kinase domain that could play an
important role in the normal control of RAF activity. In addition to BRAF, similar 5-amino acid deletions near the αC-helix
domain of a protein kinase were also identified in other targets, such as EGFR and HER2. In EGFR, the exon 19 deletions
including E746-A750 were characterized in non–small cell
lung cancer (45, 46). For HER2, another EGFR family member, the L755-T799 deletion was recently identified in breast
cancer (47). All these deletions have been found to be activating mutations. Therefore, the activating mechanism of BRAF
deletions identified in this study might represent a common
mechanism for activating other protein kinases.
METHODS
Cell Culture, Antibodies, and Reagents
BxPC-3, H2405, OV-90, A375, HCT116, NIH/3T3, and HEK-293
cells were obtained from the ATCC from 2010 to 2013 and stored
within a central cell bank that performs cell line characterizations. All
these cells were passaged for less than 2 months, after which new cultures were initiated from vials of frozen cells. Characterization of the
cell lines was done by a third-party vendor (RADIL), which included
profiling by PCR for contamination by various microorganisms of
bacterial and viral origin. As a result, no contamination was detected.
The samples were also verified to be of human origin without mammalian interspecies contamination. The alleles for 9 different genetic
markers were used to determine that the banked cells matched the
genetic profile that has been previously reported. H2405 cells were
grown in ACL-4 medium (ATCC), whereas NIH/3T3, HEK-293, and
A375 cells were maintained in DMEM supplemented with 10% FBS
(Invitrogen). HCT116 cells were cultured in McCoy’s 5A with 10%
FBS (Invitrogen), and BxPC-3 cells were cultured in RPMI with 10%
FBS. OV-90 cells were grown in a 1:1 mixture of MCDB 105 medium
containing a final concentration of 1.5 g/L sodium bicarbonate and
Medium 199 containing a final concentration of 2.2 g/L sodium
bicarbonate with 15% FBS (Thermo Scientific). The BRAF-selective
inhibitor vemurafenib, the pan-RAF inhibitor LY3009120, and the
MEK inhibitor trametinib were synthesized by Eli Lilly and Company. All siRNAs were obtained from Dharmacon (ON-TARGETplus
siRNA). siRNA transfections were performed using Lipofectamine
RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions. All plasmids were created using standard
cloning methods with pcDNA3.1 (Invitrogen) as a vector. All plasmid
transfections were carried out using FuGENEHD transfection reagents (Promega) as per the manufacturer’s instructions.
Deletion Detection from Sequencing Analysis
BRAF deletion calls on cancer cell lines were aggregated by searching through the repositories COSMIC v71 and Sanger Institute’s
Cancer Cell Line Project, and exome sequencing variant calls from
Broad Institute’s Cancer Cell Line Encyclopedia (CCLE), as well as
internally generated exome sequencing data. Internal exome data
were prepared using Agilent Sure-Select 38 Mbp all-exon capture
library sequenced on the Illumina HiSeq 2000 platform, generating approximately 80× paired-end reads. Variant calling on internal
exome and CCLE exome data were performed using the BWA-mem
aligner v0.7.4 (mapped to GRCh37) and called with GATK lite v2.3,
Freebayes v0.9.10, and Samtools mpileup v0.1.19. TCGA mutation
data (MAF files) were downloaded from the Broad Institute’s GDAC
firehose (2014_10_17 release). RNA-sequencing data (fastq files)
from TCGA were downloaded from http://cghub.ucsc.edu under
controlled access in accordance with the data-user agreement, and
mapped to human genome GRCh37 using the GSNAP (2013-11–27)
aligner. BRAF deletions based on RNA-sequencing data from TCGA
and CCLE were identified by searching for reads with at least 5
base deletions in the BRAF genomic region, and analysis was done
to determine the consequence of the change if it resulted in an inframe deletion. For TCGA pancreatic and thyroid cancer samples, we
further confirmed the deletion identified by searching through the
mapped reads from the whole exome sequencing data where there
were cases in which the deletion was not reported from the mutation
data downloaded from Broad’s firehose. The ICGC data (release 17)
was accessible from http://dcc.icgc.org.
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Published OnlineFirst January 5, 2016; DOI: 10.1158/2159-8290.CD-15-0896
Oncogenic BRAF Deletions Functioning as Homodimers
Preparation of Cell Lysates, Western Blot Analysis, and
Cell Proliferation Assay
Cell lysate preparation, Western blot analysis, and cell proliferation
assay were performed as described previously (41, 48).
Transfections and Coimmunoprecipitation
A375 cells transfected with BRAFV600E with deleted amino acids
169–380 (A375 p61V600E) and HEK293 transfected with BRAF
E586K, CRAF E478K, ∆BRAF, and ∆BRAF R509H were generated
using pcDNA3.1 vectors under G418-containing medium selection
and evaluated for BRAF and FLAG or MYC-tagged protein expression
as described previously (36). For coimmunoprecipitation, HEK293
cells stably expressing FLAG-tagged ∆BRAF or ∆BRAF R509H proteins were transfected with pcDNA3.1 vector encoding MYC-tagged
∆BRAF followed by immunoprecipitation using anti-FLAG (Sigma)
or anti-MYC magnetic beads (Cell Signaling Technologies). The IPprepared proteins were next subjected to Western blot analysis as
previously described (36).
Colony Transformation Assay in Soft-Agar Culture
For ectopic expression, NIH/3T3 cells transfected with ∆BRAF,
∆BRAF with R509H, BRAFV600E, or BRAF WT constructs or parental
vector (pcDNA3.1) were selected in G418-containing medium for
2 weeks. The transfected cells (3 × 104) in growth media with 0.3%
agar were plated on top of 0.5% agar medium in 6-well tissue culture
plates. Formation of spherical colonies was evaluated after 3 weeks
under a microscope. For target protein knockdown with siRNAs, the
transformation assay for H2405 and OV-90 cells was performed with
the same procedure at 48 hours after transfection.
In Situ Proximity Ligation Assay
PLA was conducted and validated according to the manufacturer’s instructions (Olink Bioscience) as previously described (37, 38).
Briefly, cells grown on glass slides or 96-well plates were fixed with
4% formaldehyde and permeabilized with 0.2% Triton X-100 before
being incubated with 1% BSA-blocking solution overnight at 4°C.
For detection of BRAF homodimers, monoclonal BRAF antibodies
were first conjugated to PLUS and MINUS PLA oligonucleotides
using the Duolink II Probemaker system (37). For detection of BRAF
and CRAF heterodimers, the primary antibodies were directly used
and followed by incubation with PLUS and MINUS oligonucleotide-conjugated PLA probes (38). The bound proximity probes were
then visualized as red spots with Duolink In Situ Detection Reagents Orange (Olink Bioscience) and detected under a confocal fluorescent microscope. The nuclear staining with Hoechst 33342 was
used to delineate the cells, and the quantification of the number of
in situ PLA signals per cell was further analyzed by Cellomics ArrayScan
VTI Reader and HCS software (Thermo Scientific). At least 1,000 cells
were analyzed in each 96-well plate for all reactions in triplicate.
Cell-Cycle Analysis
Cell-cycle analysis was performed as described previously (36, 41).
Cells treated with DMSO or inhibitors for 72 hours were collected
and fixed in 70% ethanol for 30 minutes at −20°C. After being washed
with PBS, fixed cells were stained with propidium iodide/Triton
X-100 staining solution and incubated for 30 minutes at room temperature. Fixed cells were then subjected to flow cytometric analysis
on a Beckman Coulter FC 500 Cytomics flow cytometer. Data were
analyzed with ModFit LT 3.0 (Verity House Software).
BrdUrd Incorporation Assay
Tumor cell lines H2405, BxPC-3, and OV-90 were grown in 6-well
plates, and the growth medium was changed and the cells were
treated with DMSO or inhibitors the following day. One hour prior
RESEARCH ARTICLE
to the end of the 72 hours of treatment time, cell medium was
spiked with 10 μmol/L BrdUrd. After 1 hour of incubation with
BrdUrd, cells were harvested and fixed in 70% EtOH at −20°C. Cells
were then washed with PBS/BSA, incubated with 2N HCl/FBS for
20 minutes, and treated with sodium borate. To determine the
amount of BrdUrd incorporation, cells were stained with isotype
control or FITC-conjugated anti-BrdUrd antibody (BD Pharmingen)
for 30 minutes, washed with PBS/BSA, and incubated in propidium
iodide (Life Technologies) for 30 minutes before reading on a flow
cytometer. Data were analyzed with FlowJo software.
In Vivo Xenograft Studies
In vivo studies were performed in accordance with the American
Association for Laboratory Animal Care institutional guidelines.
All the experimental protocols were approved by The Eli Lilly and
Company Animal Care and Use Committee. Briefly, 5 × 106 to 10
× 106 tumor cells in a 1:1 Matrigel mix (0.2 mL total volume) were
injected subcutaneously into the right hind flank of female NIH
nude rats (Taconic Biosciences). After tumors reached a desired size
of approximately 300 mm3, animals were randomized into groups
of 8 for efficacy studies. Drugs (LY3009120 or vemurafenib) were
administered orally (gavage) in 0.6-mL volume of vehicle with the
dose schedules described in each study. Tumor growth and body
weight were monitored over time to evaluate efficacy and signs of
toxicity as described (41).
Disclosure of Potential Conflicts of Interest
Y.G. Yue is Director, Computational Biology, at Boehringer Ingelheim. No potential conflicts of interest were disclosed by the other
authors.
Authors’ Contributions
Conception and design: S.-H. Chen, S. Buchanan, V. Yadav, J.R.
Henry, J.J. Starling, G.D. Plowman, S.-B. Peng
Development of methodology: S.-H. Chen, Y. Zhang, R.D. Van
Horn, T. Yin, S.-B. Peng
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): S.-H. Chen, Y. Zhang, R.D. Van
Horn, T. Yin, L. Huber
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): S.-H. Chen, Y. Zhang,
S. Buchanan, I. Mochalkin, S.S. Wong, Y.G. Yue, S.-B. Peng
Writing, review, and/or revision of the manuscript: S.-H. Chen,
V. Yadav, I. Mochalkin, S.S. Wong, I. Conti, J.R. Henry, G.D. Plowman,
S.-B. Peng
Administrative, technical, or material support (i.e., reporting or
organizing data, constructing databases): S.-H. Chen, R.D. Van
Horn, T. Yin, S.S. Wong, L. Huber, G.D. Plowman
Study supervision: S.-B. Peng
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received July 27, 2015; revised December 28, 2015; accepted
December 30, 2015; published OnlineFirst January 5, 2016.
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Oncogenic BRAF Deletions That Function as Homodimers and
Are Sensitive to Inhibition by RAF Dimer Inhibitor LY3009120
Shih-Hsun Chen, Youyan Zhang, Robert D. Van Horn, et al.
Cancer Discov Published OnlineFirst January 5, 2016.
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