Recent Progress 2015

Approximately 70% of all lung cancer patients present with late stage disease and there is a pressing need to develop better therapies for these patients. This remains a major challenge as the underlying genetic causes of greater than half of all lung cancers remain unknown. Compounding this problem, it is becoming clear that initiating events are dispensable for maintenance of tumorigenic phenotypes, for example mutationally activated KRAS (one of the more prevalent driver mutations in NSCLC) is not required to maintain tumor cell survival in a subset of cancer cell lines. Given the dismal success rates of current treatments, it is imperative that genetic mechanisms driving maintenance of tumorigenic phenotypes are discovered, new drug targets identified, and targeted therapeutics developed to improve treatment for lung cancer patients. Recent success with therapies targeting mutationally activated EGFR and constitutively active ALK serve as proof of principle that drugs targeting activated drivers of lung cancer result in better, more durable therapeutic responses in patients. Recent clinical trials, such as the TRACERx trial, are allowing unprecedented flexibility in tailoring therapeutic treatments to the underlying genetic aberrations that drive an individual’s specific lung cancer. This is based on patient mutational profiles and other biomarkers from real time biopsies. This type of personalised clinical trial is revolutionising the way we treat patients with lung cancer. Unfortunately, as we do not understand the mechanisms that drive greater than 50% of lung cancers, we lack personalised therapies for a large proportion of the patient population. A major aim of our research is to systematically fill in these knowledge gaps by identifying novel mutationally activated oncogenes, focusing mainly on kinases, as they are readily druggable targets.

Genetic Drivers of Lung Cancer

The lab utilises a multitude of strategies to identify critical pathways required to promote lung tumorigeneis. These include high-throughput bioinformatics and structural modelling, siRNA screening, and precision genome editing to establish a functional genomic approach to identify novel drivers. Utilising bioinformatics we identify novel kinases enriched for functional mutations or amplifications in lung cancer 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 if the mutations likely increase or decrease catalytic activity. These approaches have been successful in identifying kinases with activating mutations in lung cancer, 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 GOF mutations in lung cancer patients and this results 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.

Exploiting ‘cold-spots’ to elucidate novel drivers of cancer
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. The main reason for these discrepancies is inadequate sequencing of GC-rich areas of the exome, or cold-spots (Figure 1). We mapped over 400 regions of consistent inadequate sequencing in known cancer-causing genes and kinases in which neither institute found mutations. We demonstrated, using a newly identified PAK4 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. Going forward we will continue to mine these cold-spot regions of the exome to elucidate novel drivers of lung cancer, missed by cancer genome consortium studies. 

 

Figure 1: The 20 largest cold-spots detected in cancer census or kinase gene transcripts (of those that were sequenced by both COSMIC and CCLE hybrid capture) using CCLE whole-exome sequencing data. All but one of these cold-spots was located in a high GC-content area and resulted in no mutations being detected by either institute. The TET2 cold spot was not located in high-GC content areas and contained mutations detected by COSMIC, indicating that this cold spot was not present in the COSMIC data. The outer shaded grey plot shows the GC content at each base (calculated as 50 bp either side) with GC content over 70% shaded in red. The middle light green plot shows sequencing read coverage with white troughs representing poor read coverage. The inner three rings record the position of mutations found by both institutes (orange), COSMIC-only (violet), and CCLE (green). Light blue shards show cold-spots over 100 bp in length with the top 20 shaded darker. Data were plotted using a combination of Circos and custom scripts.   

PKCs in cancer
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. Crispr/Cas genome editing was used to verify the importance of PKC LOF mutations, and correcting these mutations with genome editing suppressed tumour growth in vivo consistent with a tumour suppressive role for PKCs in cancer.  Lastly, germ line loss-of-function mutations in PKC delta are associated with development of genetic disorders associated with hallmarks of cancer including increases in proliferation and suppression of cell death in immune cells (mainly B cells).  Specifically, LOF mutations have been identified in lymphoproliferative syndromes and we elucidated a casual LOF mutation in PKC delta in Juvenile Systemic Lupus Erythematous patients that promoted proliferation and suppressed cell death in B cells acquired from the patients.  In summary these 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.

MLKs 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 if inhibition of the MLKs can be exploited to suppress tumorigenesis for specific types of cancer, including lung cancer.