Prof Andrew Fry E: email@example.com
Dr Sam Khan E: firstname.lastname@example.org and Professor Don Jones E: email@example.com
Lung cancer is the largest cause of cancer-related deaths in the UK and worldwide. However, while improving the long-term survival rates of lung cancer patients remains a major challenge, we now have good understanding of the genetic drivers and this is leading to successful application of targeted treatments in the clinic. 5% of lung cancers are driven by EML4-ALK oncogenic fusions and many of these initially respond well to targeted ALK inhibitors. Unfortunately though, patients with some EML4-ALK variants respond poorly and all patients eventually relapse. Hence, there is a pressing need for better understanding of resistance mechanisms and new therapeutic approaches. We have discovered a novel pathway involving NEK protein kinases and stabilised microtubules that potentially drives the metastatic dissemination of EML4-ALK tumours. Our goal is to better understand this pathway, examine whether it sensitises these tumours to alternative drugs, and develop a novel mass spectrometry diagnostic approach to EML4-ALK identification that will enable patient stratification to the most effective treatments.
Aims & Objectives
The aims of this project are to examine a novel pathway that drives metastasis of lung cancer cells expressing EML4-ALK oncogenic fusion variants, determine whether this sensitizes cells to alternative chemotherapeutic agents and establish a novel mass spectrometry based approach to the diagnosis of EML4-ALK lung cancers.
The specific objectives are to
1. Examine how this novel EML4-ALK variant 3 (V3)/NEK9/NEK7 pathway modifies the cytoskeleton to promote migration and invasion of cells, with a focus on proteins involved in focal adhesion assembly and signaling (e.g. paxillin, FAK, src).
2. Test whether microtubule poisons (vinorelbine, paclitaxel) or inhibitors of focal adhesion signaling (FAK, SRC inhibitors) inhibit the growth and survival of EML4-ALK expressing lung cancer cells, alone or in combination with ALK inhibitors (crizotinib, alectinib, ceritinib).
3. Develop a novel diagnostic approach to determination of specific EML4-ALK variant expression using mass spectrometry.
Experimental plan and techniques to be used
The experimental plan (and key techniques) in which the student will be trained are:
- expression analysis, drug treatment and imaging (fixed and live) of human cultured cells, including Beas-2B (bronchial epithelial) cells that stably express YFP-tagged EML4-ALK V1 and V3 fusion proteins, and H3122 and H2228 NSCLC patient-derived cells that express EML4-ALK V1 and V3; cell lines and techniques established in the Fry lab (e.g. reference 5).
- 2D (wound healing and individual cell tracking) and 3D (spheroid) migration and invasion assays that represent single cell and collective migration and invasion; established in the Fry lab.
- flow cytometry and clonogenic assays for cell death and survival in response to drug treatments; routinely undertaken by Khan.
- isotopic protein labelling, cell lysis, tryptic digestion, and mass spectrometry for detection and quantitation of EML4-ALK variant using SKLYINE and MSI software; expertise in the Jones lab. Serum from patients who have confirmed EML4-ALK lung cancers will be used to assess this methodology in a clinical setting. Paired histology from these patients will be used to confirm EML4-ALK variants.
What the student will gain from the project
The student will gain a detailed understanding of the fundamental and translational research that underlies the development of novel anti-cancer precision medicine drugs and diagnostic approaches. They will learn cutting-edge molecular cell biology techniques and the importance of obtaining quantified data that meets statistical significance. They will also gain an appreciation of the challenges associated with applying laboratory-based experimental data to patient treatment and the legal and ethical frameworks in place when using patient samples e.g. HTA requirements.
The translational goals of this project are to (i) identify clinically relevant drug combinations that might be effective in treatment of EML4-ALK V3 driven lung cancers, and (ii) develop a novel approach to EML4-ALK variant diagnosis using mass spectrometry. A clinical study designed to recruit patients during the diagnostic pathway or when a patient progresses through their cancer treatment could be used to validate the assay in real-time.
1. Yuan M, Huang L-L, Chen J-H, Wu J and Xu Q (2019) The emerging treatment landscape of targeted therapy in non-small-cell lung cancer. Signal Transduction and Targeted Therapy 4:61. doi: 10.1038/s41392-019-0099-9.
2. Rotow & Bivona (2018) Understanding and targeting resistance mechanisms in NSCLC. Nature Reviews Cancer 17:637-658. doi: 10-1038/nrc.2017.84.
3. Bayliss R, Choi J, Fennell DA, Fry AM and Richards M (2016) Molecular mechanisms that underpin EML4-ALK driven cancers and their response to targeted drugs. Cell and Molecular Life Sciences 73, 1209-1224. doi: 10.1007/s00018-015-2117-6.
4. O’Regan L, Barone G, Adib R, Woo CG, Jeong HJ, Richardson E, Richards MW, Muller PAJ, Collis SJ, Fennell DA, Choi J, Bayliss R and Fry AM (2020) EML4-ALK V3 drives cell migration through NEK9 and NEK7 kinases in non-small-cell lung cancer. Journal of Cell Science 133. doi: 10.1242/jcs.241505.