John Brognard, Ph.D.

The lab utilises a multitude of strategies to identify critical pathways required to promote tumorigeneis. 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 if the mutations likely 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.), 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 identifymutationally 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.

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 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.

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 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.

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.