Intrahepatic cholangiocarcinoma (iCCA) is an aggressive and lethal malignancy arising
from the bile ducts. Patients with iCCA typically present with genetically
heterogeneous tumours. There is an exception of a small minority of patients where
actionable mutations are present, but the heterogeneity of this cancer has hampered
the development of targeted therapeutics as the number of recurrent mutations
between patients is limited. The majority of iCCA patients are not amenable to surgery,
and standard of care chemotherapies act to extend life and are not considered as a
curative treatment. As such, there is a significant requirement to explore the
functional genetics of iCCA with the goal of developing more effective treatments and
therapies. Deep sequencing studies have identified oncogenic mutations in KRAS in
iCCA and these represent one of the most recurrent alterations in this cancer.
Therefore I focussed on the role of KRAS mutations and their interactions with loss of
function mutations.
During this work, I identified and validated in vivo which loss of function mutations
were capable of interacting with oncogenic KRASG12D mutations. To do this, I
developed and carried out a CRISPR-spCas9 library screen informed by mutations that
have been previously identified as pathogenic in patient iCCA. Using this approach, I
identified the loss of cytoskeletal signalling protein Neurofibromin 2 (NF2) as a novel
driver event in iCCA and that loss of this gene interacts with mutant KRAS to initiate
cancer.
To investigate the role of NF2 further, I explored the relationship between Nf2-loss,
Trp53-loss and mutant KRAS, and I found that Nf2 loss cooperated with Trp53 loss to
lead to cancer with a severely accelerated phenotype and increased lethality.
Additionally, these tumours were representative of a rare and aggressive sarcomatoid
subtype of iCCA.
To understand how loss of both Trp53 and Nf2 leads to this aggressive phenotype, I
used proteomic analysis of a number of signalling pathways, and RNA sequencing to
define which mechanisms are altered and contribute to the aggressive cancer
phenotype. These analyses identified the co-activation of Wnt/ β-catenin and Pi3K/AKT
signalling as recurrently activated in iCCA. To test whether the aggressive nature of
dual Trp53 and Nf2 loss was a result of changes in these signalling pathways, I inhibited
them pharmacologically with inhibitors targeting Wnt/β-catenin and Pi3K/AKT
signalling. Treatment with both of these inhibitors significantly improved survival of
tumour bearing mice. Finally, I established a genetically engineered mouse model of
iCCA not reliant on Nf2-loss for its progression to address whether inhibition of Wnt/β-catenin and Pi3K/AKT could represent a wide-spanning therapeutic and determine
whether the treatment of a different pathological subtype of iCCA would be with these
inhibitors would reduce disease progression. I showed this form of well-differentiated
iCCA is also amenable to inhibition with this combination of therapies.
These data demonstrate that Nf2 is a rare driver gene of iCCA that acts in a
cooperative manner with oncogenic KRAS to accelerate tumourigenesis in vivo.
Examination of these tumours highlighted a concurrent Wnt and PI3K signalling
signature, and I demonstrate that pharmacological co-inhibition substantially impedes
iCCA growth, highlighting the use of Wnt/β-catenin and Pi3K/AKT signalling inhibition
as a broad treatment for iCCA patients
