AZ304, a novel dual BRAF inhibitor, exerts anti-tumour
effects in colorectal cancer independently of BRAF genetic
, Ling Xu
, Xiujuan Qu
, Xiaofang Che
, Ye Zhang
, Yibo Fan
, Tianshu Guo
, Kezuo Hou
, Xuejun Hu
, Minhui Shen
, Tony Cheung
and Yunpeng Liu
BACKGROUND: BRAF mutation is associated with poor clinical outcome of patients with malignant tumours, and mediates
resistance to chemotherapy and targeted therapy. This study aimed to determine whether V600E mutant and wild type BRAF
colorectal cancers exhibit distinct sensitivities to the dual BRAF inhibitor AZ304.
METHODS: Kinase activity was assessed by the AlphaScreen assay. Then, MTT assay, EdU assay, colony-formation assay and
Western blot were performed to evaluate the anti-tumour effects of AZ304 in vitro. In vivo efﬁcacy was investigated by xenograft
analysis and immunohistochemistry.
RESULTS: AZ304 exerted potent inhibitory effects on both wild type and V600E mutant forms of the serine/threonine-protein
kinase BRAF, with IC
values of 79 nM and 38 nM, respectively. By suppressing ERK phosphorylation, AZ304 effectively inhibited a
panel of human cancer cell lines with different BRAF and RAS genetic statuses. In selected colorectal cancer cell lines,
AZ304 signiﬁcantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. In addition, the EGFR inhibitor
Cetuximab enhanced the potency of AZ304 independently of BRAF mutational status.
CONCLUSIONS: The BRAF inhibitor AZ304 has broad spectrum antitumour activity, which is signiﬁcantly enhanced by combination
with Cetuximab in colorectal cancers in vitro and in vivo.
British Journal of Cancer (2018) 118:1453–1463; https://doi.org/10.1038/s41416-018-0086-x
The oncogenic BRAF mutations are found in approximately 8% of
all human cancers, including 40–70% of melanoma, 36–53% of
thyroid, and 5–22% of colorectal cancer (CRC) cases. In addition,
BRAF mutations are also present in non-small cell lung cancer,
ovarian cancer, gliomas, leukaemia and other malignancies.
serine/threonine-protein kinase BRAF belongs to the RAF family of
kinases, which also include ARAF and CRAF.
As the most common
mutation, BRAF V600E mutation causes constitutive activation of
downstream signalling through the mitogen-activated protein
kinase (MAPK) pathway.
BRAF mutation is currently considered
one of the poor prognostic markers in a series of cancers.
BRAF mutations are also associated with signiﬁcantly low response
rate with Cetuximab used as a single agent or in combination with
chemotherapy for tumours containing wild type RAS.
In the past few years selective BRAF inhibitors, such as
vemurafenib and dabrafenib have been approved by FDA and
EMEA for the treatment of metastatic melanomas harbouring
V600E mutant BRAF.
Although BRAF inhibitors produce
clinical responses, e.g., improvement of progression free survival
and overall survival in patients with mutant BRAF melanoma, the
associated effects are short-lived. Different from V600E
mutant BRAF cells, many existing BRAF inhibitors paradoxically
activate RAF and ERK signalling via a RAS dependent mechanism
in wild type BRAF cells.
Furthermore, malignant tumours
with V600E mutant BRAF do not respond uniformly to BRAF-
The majority of colorectal cancer patients
harbouring V600E mutant BRAF display inherent resistance to
vemurafenib. The overall response rate was only 5% in a
Acquired and intrinsic resistance to BRAF inhibitors
likely due to multiple mechanisms, including MAPK pathway
activation via CRAF, EGFR/MAPK pathway reactivation, BRAFV600E
ampliﬁcation, SRC/STAT3 pathway upregulation, mutation of NRAS
and MEK1, PI3K/AKT pathway activation, and others.
is necessary to identify and develop more potent BRAF inhibitors.
Our results demonstrated that AZ304, a dual BRAF kinase
inhibitor, exerts potent anti-tumour effects on both wild type
and mutant BRAF cancer lines. Moreover, combining AZ304 and
the anti-EGFR monoclonal antibody Cetuximab resulted in
signiﬁcantly improved anti-tumour activity in colorectal cancer
cells both in vitro and in vivo, independently of BRAF mutation
Received: 18 October 2017 Revised: 9 March 2018 Accepted: 23 March 2018
Published online: 14 May 2018
Department of Medical Oncology, The First Hospital of China Medical University, 110001 Shenyang, China;
Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning
Province, The First Hospital of China Medical University, 110001 Shenyang, China;
Department of Respiratory Medicine, The First Hospital of China Medical University, 110001
Shenyang, China and
Oncology iMED, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
Correspondence: Xiujuan Qu (email@example.com) or Yunpeng Liu (firstname.lastname@example.org)
These authors contributed equally: Rui Ma, Ling Xu.
© Cancer Research UK 2018