John Brognard, Ph.D.
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. 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 my research will be 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 programs 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.
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.
Selected Recent Publications
- EMBO Mol Med. 8(2): 105-16, 2016. [ Journal Article ]
- Cancer Research . [Epub ahead of print], 2017. [ Journal Article ]
- Cell. 160(3): 489-502, 2015. [ Journal Article ]
- Nature Commun. 5: 3901, 2014. [ Journal Article ]
Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer.Proc Natl Acad Sci U S A. 110(30): 12426-31, 2013. [ Journal Article ]
John received a BSc degree in chemistry from James Madison University (Harrisonburg, Virginia), followed by a MSc degree in biotechnology from Johns Hopkins University (Baltimore, Maryland). He then joined Dr. Phillip Dennis’s laboratory at the National Cancer Institute in Bethesda, Maryland, where they discovered signaling pathways that promote resistance to chemotherapy, elucidated mechanisms implicated in the initial stages of lung tumorigenesis, and developed small molecule inhibitors targeting constitutively active Akt in lung cancer. John obtained his PhD from University of California, San Diego in Dr. Alexandra Newton’s laboratory, where he discovered a novel class of phosphatases (PHLPP1 and PHLPP2) that act directly on Akt and PKC to decrease signalling through these pathways. Lastly, John trained as a postdoctoral fellow in Dr. Tony Hunter’s laboratory at the Salk Institute. Utilizing bioinformatic tools they screened cancer genomes to identify novel kinases implicated in cancer by the presence of functional somatic mutations. He joined the CRUK Manchester Institute as a group leader in September of 2010 and the lab was focused on identifying mechanisms to promote lung tumorigenesis. John then moved to the Laboratory of Cell and Developmental Signaling at the NCI as a Earl Stadtman Investigator in the summer of 2016. His research will be focused on defining novel enzymes that act to suppress or promote tumorigenesis and in some cases can serve as novel targets for therapeutic intervention. The lab has several ongoing collaborations with pharmaceutical companies including Genentech and AstraZeneca to investigate novel inhibitors targeting newly identified kinases implicated in cancer.
|Elvira An BSc.||Postbaccalaureate Fellow (CRTA)|
|Van Barnes Ph.D.||Postdoctoral Fellow (CRTA)|
|Amy Funk Ph.D.||Postdoctoral Fellow (CRTA)|
|Katherine Nyswaner MSc.||Research Biologist|
|Pedro Torres-Ayuso Ph.D.||Postdoctoral Fellow (Visiting)|
|Christina Young Ph.D.||Postdoctoral Fellow (CRTA)|