SCIENTIFIC REPORT 2015

SIGNALLING NETWORKS IN CANCER
www.cruk.manchester.ac.uk/Research/CRUK-MI-Groups/
Signalling-Networks-in-Cancer/Home
Cancer genomic sequencing has significantly impacted our
understanding of the temporal and spatial genetic alterations
that lead to tumorigenesis. This information enables the
development of targeted therapies that result in durable and
less toxic responses in patients.
Figure 1
ABL1 mutations lead to an
intermediate increase in
kinase activity.
A–D (A, C) Root-mean-square
deviation (RMSD) analysis of the
movement of (A) aC-helix and (C)
DFG motif in the active and
inactive conformation of ABL1
kinase with R351W mutation
(orange) compared to the
wild-type (WT) kinase (black). (B,
D) Time-lapse images show
movement within the aC-helix
(B) and DFG motif (D) of the
inactive structure of the WT
(black outline) and R351W
(orange outline) constructs.
A
B
αC helix
inactive
αC helix
active
C
D
DFG
inactive
DFG
active
Group Leader
John Brognard
Postdoctoral Fellows
Anna Marusiak 1
Natalie Stephenson
Pedro Torres-Aryuso
Clinical Fellows
Andrew Hudson
Matthew Howell 2
Senior Scientific Officer
Eleanor Wendy Trotter
Graduate Students
Zoe Edwards
Ewelina Testoni
1
left in 2015
2
joined in 2015
In regard to kinases, the biomedical community
has focused research efforts on approximately
200 kinases among the 538 kinases present in
the human kinome, yet siRNA screens and
cancer genomic studies indicate that the vast
majority of these unexplored kinases
(approximately 300) are implicated in cancer
and harbour putative driver mutations. The
major focus of our research is to elucidate novel
cancer-associated kinases in the unexplored
kinome, guided by bioinformatics and
functional genomic approaches, with an
overarching aim of understanding the
molecular mechanisms utilised by these kinases
to promote tumorigenesis. Through use of in
vivo patient-derived xenograft mouse models,
we will translate these findings to the clinic and
encourage drug development programmes
focused on these novel targets. The overall goal
of our research is to provide a platform for
transformational research to identify novel
druggable drivers so that the vast majority of
cancer patients can begin to benefit from
precision medicine based targeted therapies.
Collectively this research should identify new
genetic drivers, targets for therapeutic
intervention, and novel mechanisms of
tumorigenesis.
Overview
The lab utilises a multitude of strategies to
identify critical pathways required to promote
tumorigenesis. These include high-throughput
bioinformatics and structural modelling, siRNA
screening, and precision genome editing to
establish various functional genomic
approaches to identify novel drivers. Utilising
bioinformatics we identify novel kinases
enriched for functional mutations to hone in
on activated enzymes that can serve as drug
targets. We then assess the structural
consequences of a subset of mutations in the
respective kinases, where crystal structures are
available, to determine whether the mutations
are likely to increase or decrease catalytic
activity. These approaches have been
successful in identifying kinases with activating
mutations in lung cancer (ABL1 – Testoni et al.
EMBO Mol. Med. in press, see also Figure 1), as
well as novel tumour suppressing kinases in
colon and lung cancer that include MLK4 and
DAPK3. In a second approach we use genetic
dependency screens to identify mutationally
activated drivers of lung cancer. Targeted
genetic dependency screens are an effective
way to uncover low frequency oncogenes that
can serve as targets for therapeutic intervention
for tumours of any origin. Specifically we
identified FGFR4, PAK5, and MLK1 as kinases
that harbour novel gain-of-function (GOF)
mutations in lung cancer patients and these
mutations result in hyperactivation of the MEK/
ERK pathway. The mutation frequency for the
genes we identified ranged from 2-10% of lung
cancers; given the frequency of lung cancer in
the population, these targets could be exploited
by pharmaceutical companies for drug
discovery development.
Discrepancies in cancer genomic sequencing
highlight opportunities for driver mutation
discovery
In an additional approach, the lab focuses on
uncovering novel mutations that reside in
unsequenced regions of the exome (cold-spots)
that may have been missed by large cancer
genomic consortiums. To identify these
cold-spots, we performed a comparison of two
of the most prominent cancer genome
sequencing databases from different institutes
(CCLE and COSMIC), which revealed marked
discrepancies in the detection of missense
mutations in identical cell lines. We
demonstrated, using a newly identified PAK4
0 ps 5000 ps
mutation as proof of principle, that specific
sequencing of these GC-rich cold-spot regions
can lead to the identification of novel driver
mutations in known tumour suppressors
and oncogenes.
Cancer-associated protein kinase C
mutations reveal kinase's role as tumour
suppressor
We also focus on studying the PKC family of
kinases, which have been intensely investigated
for over 25 years in the context of cancer.
Historically, this arises from the discovery of
PKC as the receptor for the tumour-promoting
phorbol esters, which suggested that activation
of PKC by phorbol esters promoted
tumorigenesis induced by carcinogens.
However, this interpretation is now open to
question, since long-term treatment with
phorbol esters is known to initiate degradation
of PKC, thus down-regulating its activity. In
collaboration with Dr. Alexandra Newton’s lab at
UCSD, we performed a bioinformatics analysis
to assess the frequency of PKC mutations
present in cancer genomic sequencing studies
and to determine the functional impact of these
mutations. A majority of mutations were
identified to be loss-of-function (LOF) and for
heterozygous mutations they could act in a
dominant negative manner to suppress the
activity of other PKC isoforms. Consistent with
PKCs being tumour suppressors, we identified
0 ps 5000 ps
germline LOF mutations in PKC delta, associated
with increased survival and proliferation of B
cells, and the mutations were causal in juvenile
lupus. In summary, our data provide compelling
evidence that PKCs in general are tumour
suppressors and PKC inhibitors should not be
used to treat cancer patients with mutations in
this family of kinases.
The role of the Mixed Lineage Kinases
in cancer
Lastly, the lab investigates the role of a novel
family of kinases, the Mixed Lineage Kinases
(MLKs), in various forms of cancer including lung
and colon cancer, melanoma, and head and
neck squamous cell carcinoma. We recently
demonstrated that the MLK1-4 promotes
resistance to RAF inhibitors in melanoma by
directly phosphorylating MEK to reactivate the
MEK/ERK pathway. These kinases play a
complex role and can act as both tumour
suppressors and oncogenes depending on the
genetic make-up and origin of the cancer. The
lab will continue to investigate the importance
of this family of kinases and the signalling
pathways they regulate in various forms of
cancer and assess whether inhibition of the
MLKs can be exploited to suppress
tumorigenesis for specific types of cancer,
including lung cancer.
Publications listed on page 58
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