Drivers of lung cancer
Lung cancer patients receive significant benefit from targeted therapies aimed at mutationally activated kinases such as EGFR or EML4-ALK, where inhibition of the activated kinase suppresses proliferation and promotes cell death. However, despite the significant advances in targeted therapies for lung cancer patients, only approximately 20% of lung cancer patients can be stratified for treatment with targeted therapies. Therefore, it is necessary to elucidate novel signalling pathways that are essential to maintain lung cancer cell survival. The lab utilises three strategies to identify such pathways in lung cancer. In the first approach we use bioinformatic tools to evaluate the functional impact of somatic mutations in novel or understudied kinases identified in lung cancer genomic screens. We use the following bioinformatics applications to assess the functional impact of somatic mutations: PMUT, polyphen2, mutationtaster, snpeffect, SIFT, and SNPs and go. Kinases where a majority of mutations are predicted to be “likely cancer”, “pathological” or likely to have a “high” functional impact are further evaluated in the lab. Additionally, we model many of the mutations that score highly in our analysis to determine the structural consequences of the putative driver mutations concomitant with experiments in the lab. We have found that this approach works extremely well in identifying LOF mutations and candidate tumour suppressing kinases.
In a second approach, we use genetic dependency screens to identify mutationally activated drivers of lung cancer. Utilising cancer genomic sequencing data from the Sanger Institute we depleted six lung cancer cell lines of all somatically mutated proteins to discover novel genetic dependencies and identify low frequency driver mutations. 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 GOF mutations in lung cancer patients, and these result in hyperactivation of the MEK/ERK pathway (Figure 1). 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. These types of screening approaches have the potential to identify both therapeutic targets and biomarkers.
Figure 1. Targeted genetic dependency screen to identify novel actionable mutations; mutated FGFR4, MAP3K9 and PAK5 are illustrated examples. Patient tumours are exome sequenced and treatment is stratified based upon the mutational profile of each individual. Treatment options include novel pharmacological compounds to specifically inhibit driver oncogenes, and/or targeting downstream the main hyper-activated pro-proliferative signalling pathway, for example inhibition of MEK.
Our final strategy involves an unbiased kinome-wide evaluation of all kinases focusing on amplification and somatic mutations in squamous cell lung cancer. Utilising online data portals such as cBio the lab hones in on kinases that are both amplified and somatically mutated in an effort to identify kinases where increased expression or mutational activation drives lung tumorigenic phenotypes. We start with the kinome wide approach, a candidate kinase is identified that is frequently amplified or mutated in lung cancer, and regions of mutations are highlighted. This kinase is then studied in depth in the laboratory to determine if it is required to maintain lung cancer cell survival and proliferation and what downstream mechanisms are utilised to promote these phenotypes.
Characterisation of mutant kinases
To characterise the mutant kinases, our general strategy is to first assess the functional consequences of somatic mutations on overall kinase activity utilising in vivo and in vitro kinase activity assays. We compare the activity of the kinases harbouring cancer mutations (engineered through site-directed mutagenesis) to WT, kinase dead (KD) and hyperactivated forms of the kinase. Next we determine phenotypic effects of expressing the WT, KD and mutant forms of the target kinase on proliferation, survival and transformed properties of appropriate tumour and normal cell lines. We then verify the function of the kinase using si/shRNA and evaluate the role of the endogenous kinase in regulating cellular phenotypes associated with tumorigenesis. We also investigate the molecular mechanisms utilised by the cancer mutants to promote tumorigenesis. For example, if the mutation is an activating mutation, we will identify cancer relevant substrates that are phosphorylated by the cancer mutants to promote tumorigenesis. Finally we will assess the consequences of somatic mutations utilising cell lines that harbour endogenous mutations in the target kinase. The overall goal of these studies will be to identify common and convergent pathways utilised by cancer cells to promote lung tumorigenesis and identify convergent and essential targets that could be exploited for the development of novel therapeutics for the treatment of lung cancer patients.
The next generation of personalised medicine is becoming a reality in non-small cell lung cancer (NSCLC). Normal cellular growth relies on the interaction of networks of kinases (enzymes) that turn cellular processes ‘on’ and ‘off’. Some NSCLC patients have a specific genetic change that generates an ‘always on’ version of a kinase (EML4-ALK is the name of the genetically activated kinase). Cells with this mutated kinase are unable to turn ‘off’ certain cellular processes and therefore grow out of control to form a tumour. Patients with this defective kinase benefit significantly from treatment with a small molecule inhibitor (Crizotinib) that targets the activated kinase to turn ‘off’ the pathway. The major aim of our research is to identify new druggable targets by screening lung cancer cells to find the next set of hyperactivated genes that are required for lung cancer cell survival.
Kinase mutations in other cancers
In addition, the lab is focused on a novel family of kinases capable of promoting resistance to targeted therapies for melanoma patients. Guided by cancer genomic studies the lab has elucidated a novel family of MEK kinases that can promote resistance to RAF inhibitors in melanoma. Future work will be aimed at understanding the molecular mechanisms that dictate pathway activation downstream of these kinases. Lastly, we have identified a novel tumour suppressing kinase in colon cancer and we aim to elucidate why LOF mutations in this kinase are essential for the development of colon cancer.