Understanding activating PIK3CA mutations in human disease

Each one of us is the result of an extraordinary developmental process during which a single fertilised egg turns into more than a trillion cells. This is critically dependent on exquisite coordination of fundamental cell behaviours including growth, migration and “decisions” to differentiate into cell types with specialised functions. When co-ordination is lost, either during development or in adulthood, disease results. Cancer is one severe consequence of dysregulation, but in some people, loosening of growth controls may arise during development and lead to non-malignant disorders featuring patchy, asymmetric excess growth. The genetic mutations underlying these defects occur at different times and in different tissues during the development of affected individuals; as a result, these so called “mosaic” disorders may range from isolated overgrowth of a single digit to lethal overgrowth of the brain and/or major blood vessels (Keppler-Noreuil et al., 2015). Seminal discoveries in the past decade have revealed that both cancer and early-onset non-malignant overgrowth are critically dependent on mutations in the human PIK3CA gene (Thorpe, Yuzugullu and Zhao, 2015), which codes for a protein that controls when cells grow, move, and die in response to external cues and nutrient availability. Many biologists know the product of the PIK3CA gene as p110α – the catalytic subunit of class IA phosphatidylinositol 3-kinase which catalyses the formation of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) downstream of multiple receptor tyrosine kinases, including the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R). Disease-linked mutations in the PIK3CA gene leave the encoded protein stuck in a partially active state, leading to enhanced cell survival and growth. Consequently, the p110α protein is a major target for cancer drug development.

Studies of PIK3CA mutations in cancer are complicated by the presence of many other mutant genes. In contrast, their isolated occurrence in rare overgrowth individuals has opened exciting opportunities to test targeted, specific therapies and to make observations about the fundamental mechanisms underlying human development. As one of the first groups to discover PIK3CA mutations as the cause of congenital asymmetric overgrowth (Lindhurst et al., 2012), grouped under the clinical term PIK3CA-related overgrowth spectrum (PROS), my supervisor and his team have assembled and continue to follow up a large cohort of 160+ patients. Based on observations in these patients, the aim of my PhD is to characterise PIK3CA mutant-specific effects in a developmental context, and to explore if and how different levels of protein activation lead to distinct cellular characteristics. A hallmark of my project is the generation of the first developmental human cellular models of PROS by capitalising on two scientific revolutions from the past decade – the first already recognised by a Nobel Prize in 2012 and the second imminently expected to receive one:

• The ability to turn adult skin cells into induced pluripotent stem cells, i.e. cells that can generate every cell type found in the adult body (reviewed in (Takahashi and Yamanaka, 2016)); importantly, this bypasses the ethical considerations associated with the use of human embryonic stem cells.
• The development of the most advanced DNA editing technology to date, namely CRISPR/Cas9, that enables rapid and precise gene engineering. Consequently, disease-relevant mutations can be introduced into normal cells with minimal off-target effects (reviewed in (Hockemeyer and Jaenisch, 2016))

As most cell biologists will know, pluripotent stem cells (PSCs) are notoriously difficult to work with. Moreover, when I started my PhD in 2013, CRISPR was still in its infancy and was not yet thoroughly optimised for use in PSCs in our lab. Nonetheless, I embraced the challenges with great motivation and have successfully achieved the following in the past two years:

• The optimisation of a highly efficient CRIPSR-based gene editing protocol that works well in multiple cell types including human PSCs. This prompted me to make my workflow freely accessible to other scientists via the research platform Benchling.
• The optimisation of a reproducible PSC workflow of critical importance for the quality of my experimental findings as well as future projects in the lab.
• The development of the first stem cell-based models of PIK3CA-related overgrowth, encompassing a series of mutations that allow us to study mutant-specific effects in a developmental context and in more specialised cell types derived from the parental stem cells.

Overall, the versatile nature of our models enable us to address how different levels of endogenous chronic p110α activation in PSCs affect their differentiation behaviour. As we are working with models free from additional cancer-linked mutations, we are also able to study the isolated signalling consequences that underlie the aforementioned phenotypic changes. Thus, we hope that this work will enable us to better understand diseases caused by activating PIK3CA mutations and ultimately promote the development of novel treatments for affected individuals.

Hockemeyer, D. and Jaenisch, R. (2016) ‘Induced pluripotent stem cells meet genome editing’, Cell Stem Cell, pp. 573–586. doi: 10.1016/j.stem.2016.04.013.

Keppler-Noreuil, K. M., Rios, J. J., Parker, V. E. R., Semple, R. K., Lindhurst, M. J., Sapp, J. C., Alomari, A., Ezaki, M., Dobyns, W. and Biesecker, L. G. (2015) ‘PIK3CA -related overgrowth spectrum (PROS): Diagnostic and testing eligibility criteria, differential diagnosis, and evaluation’, American Journal of Medical Genetics Part A, 167(2), pp. 287–295. doi: 10.1002/ajmg.a.36836.

Lindhurst, M. J., Parker, V. E. R., Payne, F., Sapp, J. C., Rudge, S., Harris, J., Witkowski, A., Zhang, Q., Groeneveld, M. P., Scott, C. E., Daly, A., Huson, S. M., Tosi, L. L., Cunningham, M. L., Darling, T. N., Geer, J., Gucev, Z., Sutton, V. R., Tziotzios, C., Dixon, A. K., Helliwell, T., O’Rahilly, S., Savage, D. B., Wakelam, M. J. O., Barroso, I., Biesecker, L. G. and Semple, R. K. (2012) ‘Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA’, Nature Genetics. Nature Publishing Group, 44(8), pp. 928–933. doi: 10.1038/ng.2332.

Takahashi, K. and Yamanaka, S. (2016) ‘A decade of transcription factor-mediated reprogramming to pluripotency.’, Nature reviews. Molecular cell biology, 17(3), pp. 183–93. doi: 10.1038/nrm.2016.8.

Thorpe, L. M., Yuzugullu, H. and Zhao, J. J. (2015) ‘PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting’, Nature Reviews Cancer. Nature Publishing Group, 15(1), pp. 7–24. doi: 10.1038/nrc3860.