2019 phd projects and supervisory teams doctoral fellowships … · 2018-10-03 · 2019 phd...

2019 PhD projects and supervisory teams Doctoral Fellowships for Clinicians

The role of hepatic cell metabolism in liver regeneration and liver cancer. Supervisory team: Dimitrios Anastasiou (primary supervisor, Crick) and Massimo Pinzani (UCL) Using iPSC to investigate a novel disorder of insulin dysregulation caused by a promoter mutation in PMM2. Supervisory team: Paola Bonfanti (primary supervisor, Crick) and Detlef Bockenhauer (UCL/Great Ormond Street Hospital for Children NHS Foundation Trust) Understanding human thymopoiesis within an engineered thymus for treatment of primary immune deficiencies. Supervisory team: Paola Bonfanti (primary supervisor, Crick) and Adrian Thrasher (UCL Great Ormond Street Institute of Child Health) Mechanisms and impact of plasma membrane polar head recycling in M. tuberculosis. Supervisory team: Luiz Pedro Carvalho (primary supervisor, Crick), Edward Tate (Imperial College London) and Robert Wilkinson (Crick) Resistance to infection and Parkinson’s disease: an exploration of LRRK2, PD risk alleles and macrophage function. Supervisory team: Maximiliano Gutierrez (primary supervisor, Crick) and Huw Morris (UCL) The molecular mechanism underlying Loeys-Dietz syndrome. Supervisory team: Caroline Hill (primary supervisor, Crick), David Abraham (UCL) and Nitha Naqvi (Royal Brompton Hospital) Developing small bowel intestine for the treatment of short bowel syndrome. Supervisory team: Vivian Li (primary supervisor, Crick) and Paolo De Coppi (UCL Great Ormond Street Institute of Child Health) Identification of novel molecular pathways in congenital hypopituitarism. Supervisory team: Robin Lovell-Badge (primary supervisor, Crick) and Mehul Dattani (UCL Great Ormond Street Institute of Child Health) Aberrant intron retention and RBP mislocalization: a new mechanism for ALS. Supervisory team: Nicholas Luscombe (primary supervisor, Crick), Rickie Patani (Crick/UCL) and Jernej Ule (Crick/UCL) An evolutionary approach to understand regulation of cellular energy metabolism in normal physiology and disease. Supervisory team: Snezhana Oliferenko (primary supervisor, Crick) and Paul Gissen (UCL Great Ormond Street Institute of Child Health) Identifying regulators of immune surveillance and cancer cachexia in lung cancer. Supervisory team: Charles Swanton (primary supervisor, Crick) and Nicholas McGranahan (UCL) Exploring CDKL5 substrates in humans. Supervisory team: Sila Ultanir (primary supervisor, Crick) and Helen Cross (UCL Great Ormond Street Institute of Child Health) Elucidating the evolutionary history of rare atypical cancer cells by single-cell laser capture microdissection and genome and transcriptome sequencing. Supervisory team: Peter Van Loo (primary supervisor, Crick) and Nischalan Pillay (UCL)

Tracing the origin of disseminated tumour cells in bone marrow. Supervisory team: Peter Van Loo (primary supervisor, Crick) and Samra Turajlic (The Institute of Cancer Research, Royal Marsden) Towards deciphering the role of long noncoding RNA mediated gene regulation in mammalian and cancer cells. Supervisory team: Folkert van Werven (primary supervisor, Crick) and Jessica Downs (The Institute of Cancer Research). Clinical supervisor tba Structural determinants of the T cell contribution to tuberculosis and HIV-tuberculosis pathogenesis. Supervisory team: Robert Wilkinson (primary supervisor, Crick) and Xiao-Ning Xu (Imperial College London) Metabolic stratification of heterogenous breast tumours. Supervisory team: Mariia Yuneva (primary supervisor, Crick), Robert Stein (UCL) and Gyorgy Szabadkai (UCL)

2019 Doctoral fellowships for clinicians

The role of hepatic cell metabolism in liver regeneration and liver cancer A PhD project for the 2019 doctoral clinical fellows programme with Dimitrios Anastasiou (primary supervisor, Crick) and Massimo Pinzani (UCL)

In the last two decades, there has been an alarming increase in the number of patients with liver disease linked to obesity, alcohol consumption, environmental toxins and pathogens [1]. If current epidemiological trends continue, about a fifth of these patients will develop hepatocellular carcinoma (HCC), which has a high mortality rate due to lack of effective therapy options. It is unclear what causes progression from liver disease to HCC, but stopping this transition would halt the imminent increase in HCC incidence. This project will use mouse models to understand how metabolic changes in specific hepatic cell populations contribute to liver disease and HCC development.

The liver has a remarkable capacity to regenerate after injury [2], but failure in this regeneration process is thought to lead to cancer [3]. To preserve its important physiological functions in the mammalian body, the liver undergoes significant metabolic adaptations after damage and during tumorigenesis. The Anastasiou Lab are studying, among other topics, how whole-body and liver metabolism adapt during the development of HCC and after chemical or mechanical damage, as models of the regenerative processes that occur during liver disease. In recent unpublished work, we have found changes in carbohydrate and lipid metabolism occurring during mouse liver regeneration that are similar, at the level of the whole tissue, to those seen in livers with HCC. We have also shown that these changes are driven, in part, by metabolic reprogramming in hepatic non-parenchymal (NP) cells, which have many essential signalling functions for both regeneration and cancer. However, how metabolic adaption of NP cell metabolism influences liver homeostasis is unclear.

In this project, we will address this question using mouse genetics to interfere with metabolic pathways that we found altered in NP cells during liver regeneration and cancer. We will then use in vivo and ex vivo metabolic measurements to understand the consequences of these genetic manipulations in normal liver function as well as during regeneration and HCC development. To this end, the student will combine stable isotope tracers in cultured cells and in mice, with nuclear magnetic resonance (NMR) and mass spectrometry (MS) metabolomics. The student will also use flow cytometry to isolate and characterise hepatic NP cell populations. In addition to uncovering fundamental physiological functions of hepatic NP cell metabolism in mammals, this project will also provide important insights towards targeting NP cell metabolism for cancer therapy [4].

The partner institution for this project is UCL.

References: 1. http://www.thelancet.com/pb/assets/raw/Lancet/stories/commissions/lancet-liver-disease-

infographic.pdf 2. Karin M, Clevers H., Reparative inflammation takes charge of tissue regeneration, Nature (2016),

529(7586):307-15 [PMID: 26791721] 3. Sun B, Karin M., Obesity, inflammation, and liver cancer, Journal of Hepatology (2012),

56(3):704-13 [PMID: 22120206] 4. Anastasiou D., Tumour microenvironment factors shaping the cancer metabolism landscape.

British Journal of Cancer (2017), 116(3):277-286 [PMID: 28006817]


Using iPSC to investigate a novel disorder of insulin dysregulation caused by a promoter mutation in PMM2 A PhD project for the 2019 doctoral clinical fellows programme with Paola Bonfanti (primary supervisor, Crick) and Detlef Bockenhauer (UCL/Great Ormond Street Hospital for Children NHS Foundation Trust)

We recently reported a novel disorder, which we named HIPKD, an acronym for the key manifestations of HyperInsulinism and Polycystic Kidney Disease.[1] In addition, patients can have liver cysts. As underlying cause, we identified a promoter mutation in a gene called PMM2. The encoded enzyme Phosphomannomutase2 is a key enzyme in protein glycosylation and ubiquitously expressed. Bi-allelic coding mutations in PMM2 cause a severe and typically lethal multiorgan disorder with obligate neurological involvement called congenital disorder of glycosylation type 1a (CDG1A). In contrast, the promoter mutation, either homozygous or in trans with a PMM2 coding mutation causes the organ-specific HIPKD. Such organ-specificity conferred by a promoter mutation has not been described previously and suggests a critical role for this part of the promoter for expression of PMM2 in the key organs affected by HIPKD: pancreatic beta-cells, kidney and liver.[2] This provides a unique opportunity to better understand the time and place-specific regulation of gene expression. Our preliminary data suggest that the promoter mutation affects chromatin-looping and we hypothesise that it interferes with transactivation by HNF4A, a transcription factor specifically expressed in pancreatic beta cells, kidney and liver.[3] Indeed, several HNF4 binding sites are present in the predicted chromatin loop. We have obtained peripheral blood monocytes from patients and heterozygous promoter mutation carriers (parents), which are currently used to derive patients’ and parents’ induced pluripotent stem cells (iPSC). iPS cells can be first induced to a stage corresponding to a common pancreatic progenitor that we can expand in vitro thus allowing a detailed molecular analysis of a well-defined population [4,5].

The aims of the project are: 1) to differentiate these iPSC into pancreatic beta cells 2) to investigate the effects of the promoter mutation on glycosylation of key proteins involved in insulin secretion (e.g. ABCC8 and KCNJ11) 3) to assess chromatin conformation and transcription factor binding (especially HNF4A) of wildtype versus mutant promoter sequence

In summary, this project involves several exciting and highly topical aspects: • a novel disorder with a novel mechanism of organ-specific manifestations due to a promoter mutation • differentiation of iPSC into pancreatic beta-cells • Understanding the complex regulation of time- and tissue specific gene expression through chromatin

looping The partner institution for this project is UCL.

References: 1. Cabezas, O.R., et al., Polycystic Kidney Disease with Hyperinsulinemic Hypoglycemia Caused by a

Promoter Mutation in Phosphomannomutase 2. J Am Soc Nephrol, 2017. 28(8): p. 2529-2539. 2. Carney, E.F., Polycystic kidney disease: PMM2 mutation causes PKD and hyperinsulinism. Nat Rev

Nephrol, 2017. 13(6): p. 321. 3. Ihara, A., et al., Functional characterization of the HNF4alpha isoform (HNF4alpha8) expressed in

pancreatic beta-cells. Biochem Biophys Res Commun, 2005. 329(3): p. 984-90. 4. Huch M., Bonfanti P., et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors

through the Lgr5/Rspondin axis. EMBO J. 2013 16;32(20):2708-21 5. Cai Q., Bonfanti P., et al. Prospectively isolated NGN3-expressing progenitors from human

embryonic stem cells give rise to endocrine hormone-expressing cells. Stem Cells Transl Med. 2014;3(4):489-99


Understanding human thymopoiesis within an engineered thymus for treatment of primary immune deficiencies A PhD project for the 2019 doctoral clinical fellows programme with Paola Bonfanti (primary supervisor, Crick) and Adrian Thrasher (UCL Great Ormond Street Institute of Child Health)

Complete DiGeorge Syndrome (cDGS) and Foxn1-Deficiency (Nude) are congenital disorders characterised by severe T-cell deficiency, due to complete absence of the thymus gland. They are caused by genetic mutations that impair the development of the stromal component of the thymus, while the haematopoietic lineage is not affected. cDGS and Nude patients suffer opportunistic infections and die early in life unless treated. Current treatment is Allogeneic Thymus Transplantation (ATT) [1] which restores some immune function, though T cell counts remain low. ATT has limited safety and efficacy because of donor organ availability and the variable quality of thymic slices cultivated in vitro that can impact on the success of treatment; moreover, incomplete removal of donor lymphocytes may result in Graft versus Host Disease.

We are developing an in vitro system that allows culture and manipulation of human thymic stromal cells for the generation of a bioengineered thymus, suitable for transplantation in cDGS and Nude patients.

This project focuses on reconstructing the cellular and molecular interactions between human thymic stromal cells, cultured inside a decellularised thymus scaffold and lymphocyte progenitors from different sources (i.e. cord blood, bone marrow, peripheral blood). We aim to identify which signals are provided by specific subpopulations of the thymus stroma - both as extra cellular matrix (ECM) molecules and cytokines production - that support progenitor differentiation into different types of mature T cells and that could validate these engineered constructs as substitute of current ATT.

Therefore, the candidate will seek to reconstruct key thymic functions ex vivo by (i) re-assembling different human stromal cells (in various combinations) with T cell precursors within a thymus scaffold where molecular cell-cell interactions can be extensively studied; (ii) defining and phenotyping the thymic stromal cell subtypes capable to induce T cell maturation; (iii) validating T cell function by stimulation and proliferation assays and estimating T cell repertoire diversity.

These aims represent a novel and multidisciplinary approach that combines stem cell biology, basic and clinical immunology and the newest bioengineering technologies in scaffold preparation.

The UCL Great Ormond Street Hospital is the only centre in Europe to report transplant cases for cDGS [2]. The Crick supervisor unique expertise in isolation and long-term expansion of thymic stromal cells will offer a rigorous training in cell biology and tissue culture [3]. In addition, the candidate will learn tissue engineering protocols for the preparation of scaffolds that retain the tissue architecture and ECM proteins of the native microenvironment [4,5] and will be trained in protein biochemistry, FACS analysis, confocal microscopy and advanced imaging techniques. Moreover, world class immunologists at Crick will provide a rich and stimulating environment for the student. The partner institution for this project is UCL.

References: 1. Markert M.L.,et al. (1999) Transplantation of thymus tissue in complete DiGeorge syndrome N Engl

J Med 341(16):1180–9. 2. Davies G.E., et al. (2017) Thymus Transplantation for Complete DiGeorge Syndrome: European

Experience. J Allergy Clinical Immunology. S0091-6749(17)30576-6 3. Bonfanti P., et al. (2010) Microenvironmental Reprogramming of Thymic Epithelial Cells to Skin

Multipotent Stem Cells. Nature. 466(7309):978-82 4. Elliott M.J., et al., (2012) Stem-cell-based, tissue engineered tracheal replacement in a child: a

two-year follow-up study. Lancet 380:994-1000 5. Urbani L., et al. (2018) Multi-stage bioengineering of a layered oesophagus with in vitro expanded

muscle and epithelial adult progenitors. Nature Communications in press


Mechanisms and impact of plasma membrane polar head recycling in M. tuberculosis A PhD project for the 2019 doctoral clinical fellows programme with Luiz Pedro Carvalho (primary supervisor, Crick), Edward Tate (Imperial College London) and Robert Wilkinson (Crick)

Tuberculosis continues to be one of the most important infectious diseases affecting mankind. In spite of its impact in global health we still lack fundamental understanding of processes that allow Mycobacterium tuberculosis to survive, persist and evade sterilization by the immune system and by antibiotics. Several of these mechanisms are highly specific to this bacterium and are not present even in closely related organisms, such as M. smegmatis, M. leprae and M. ulcerans.

This project aims at addressing the role of the bacterium plasma membrane remodelling in its ability to survive, cause disease, resist and persist. The lipid composition of plasma membranes is directly responsible for its overall structure and function, as well as the activity of a number of membrane anchored or integral membrane proteins such as the ones involved in signal transduction, nutrient acquisition and antibiotic uptake/efflux. Using metabolomics methods that we have pioneered in M. tuberculosis (de Carvalho LP. et al. 2010a,b), we have discovered that M. tuberculosis degrades its phospholipid polar heads, using a pathway that was not thought to exist in mycobacteria before (Larrouy-Maumus G. et al. 2013). The goal of this project is to define which changes are observed in plasma membrane lipid composition, membrane proteome and what physiological effects they trigger in the bacterium. In addition to the fundamental knowledge that this project will generate, it might lead to the validation of novel targets or approaches to develop improved antibiotics to treat human tuberculosis.

The work will involve significant volume of microbiology with virulent M. tuberculosis and mutants, in containment level 3 laboratory. In addition, the successful candidate will also investigate how parent and mutant strains respond to antibiotics, infect macrophages, and we will carry out metabolomics, lipidomics and (chemo)proteomics analysis employing liquid chromatography mass spectrometry. Mouse infection experiments will also be carried out in parallel to estimate the impact of the pathway on murine infection. A number of additional alternative experiments could be employed in the future to gain further insight on plasma membrane remodelling, depending on the initial results.

The partner institution for this project is Imperial College London.

References: 1. Tate EW, Kalesh KA, Lanyon-Hogg T, Storck EM, Thinon E. Global profiling of protein lipidation using

chemical proteomic technologies. (2015) Curr Opin Chem Biol. 24:48-57. 2. Larrouy-Maumus G, Biswas T, Hunt DM, Kelly G, Tsodikov OV and de Carvalho LP. (2013) Discovery

of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 110(28): 11320- 11325

3. de Carvalho LP, Fisher SM, Marrero J, Ehrt S, Nathan C and Rhee KY. (2010b) Metabolomics of Mycobacterium tuberculosis Reveals Compartimentalized Co-Catabolism of Carbon Substrates. Chem & Biol 17(10): 1122-1131

4. de Carvalho LP, Zhao H, Dickinson CE, Arango NM, Lima CD, Ouerfelli O, Fisher SM, Nathan C and Rhee KY. (2010a) Activity-based metabolomic profiling of enzyme function. Chem & Biol 17(4): 323-332


Resistance to infection and Parkinson’s disease: an exploration of LRRK2, PD risk alleles and macrophage function A PhD project for the 2019 doctoral clinical fellows programme with Maximiliano Gutierrez (primary supervisor, Crick) and Huw Morris (UCL)

LRRK2 is the most important Mendelian gene causing Parkinson’s disease (PD) and the G2019S mutation is particularly common in Ashkenazi Jewish and North African populations, with the G2385R mutation common in Asian populations [1]. The LRRK2 gene has also been implicated in the risk of developing mycobacterial infections and inflammatory bowel disease, raising the possibility that PD risk alleles may be protective in early life and in populations with high risk of infectious disease (ID), and deleterious in later life leading to neurodegeneration [2]. Understanding these relationships will have important implications for understanding global variation in neurodegenerative disease risk, and in predicting beneficial and harmful effects of LRRK2 directed therapy. The Gutierrez lab has recently shown that LRRK2 regulates phagosome maturation [3] and Prof. Morris leads a community based study of familial PD and over 100 UK based LRRK2 PD families have been identified. This project will involve using clinical and genetic data from large scale datasets to identify the relationship between infectious disease and PD (cross disorders analysis systematically exploring the overlap, at a genetic and clinical level, between PD and ID), including patients carrying rare and common LRRK2 risk alleles and using population genetic data related to LRRK2 haplotypes and rare variants to explore the possibility of balancing positive selection for LRRK2-PD risk alleles in specific global populations.

The major part of the project will involve studying biological samples from PD patients in the Gutierrez lab to determine the effects of variation at the LRRK2 locus on macrophage function. For that, blood samples from patients with different types of Parkinson’s disease will be collected and blood monocyte-derived macrophages isolated and differentiated under different conditions. Macrophage function such as phagocytosis, lysosomal function, ROS and RNS production as well as the response to infection with several pathogens (e.g. M. tuberculosis) will be analysed to establish possible correlations with clinical manifestations in the LRRK2 PD families.

This project will offer training in cell biology and infection as well as bioinformatics/data analysis/genetics, developing evolutionary approaches to studying neurodegeneration and in cellular approaches to studying inflammatory and immune function. The partner institution for this project is UCL.

References: 1. Cookson MR. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson’s disease. Nat Rev

Neurosci. 2010 Dec;11(12):791–7. 2. Wang D, Xu L, Lv L, Su L-Y, Fan Y, Zhang D-F, et al. Association of the LRRK2 genetic

polymorphisms with leprosy in Han Chinese from Southwest China. Genes Immun. 2015 Mar;16(2):112–9.

3. Härtlova A, Herbst S, Peltier J, Rodgers A, Bilkei-Gorzo O, Fearns A, et al. LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages. EMBO J [Internet]. 2018 Jun 15;37(12). Available from: http://dx.doi.org/10.15252/embj.201798694


The molecular mechanism underlying Loeys-Dietz syndrome A PhD project for the 2019 doctoral clinical fellows programme with Caroline Hill (primary supervisor, Crick), David Abraham (UCL) and Nitha Naqvi (Royal Brompton Hospital)

Background Loeys-Dietz syndrome (LDS) is an autosomal dominant connective tissue disorder, related to Marfan syndrome and characterised by vascular tortuosity and aneurysm in association with craniofacial and skeletal manifestations (1). Marfan syndrome itself is caused by mutations in the extracellular matrix protein, Fibrillin 1 (FBN1) (2), which are thought to increase the bioavailablity of TGF-b. Interestingly, six components of the TGF-b signalling pathway have been shown to be mutated in LDS. Loss-of-function mutations have been found in the type I and type II TGF-b receptors and in two TGF-b ligands – TGFB2 and TGFB3, and mutations have also been found in two downstream signal transducers of the TGF-b signalling pathway – SMAD2 and SMAD3 (1). In SMAD3, 61% are missense mutations and 23% are frameshift mutations; in SMAD2 all the mutations are missense mutations. Another Marfan-related syndrome, Sprintzen-Goldberg syndrome (SGS), is caused by mutations in a transcriptional repressor, SKI that also functions in the TGF-b pathway (3).

TGF-b signalling is initiated by ligand binding to the type I and type II serine/threonine kinase receptors. The type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates receptor-regulated SMADs, SMAD2 and SMAD3. The phosphorylated SMADs form complexes with the common mediator SMAD4, which accumulate in the nucleus. There, they are recruited to DNA in conjunction with other transcription factors and both positively and negatively regulate target gene transcription (4). Prior to signalling, some target genes are repressed by SKI and/or a related protein, SKIL. In response to signal, SKI and SKIL are rapidly degraded, allowing the target genes to be activated by phosphorylated SMAD2/3–SMAD4 complexes. Recent work on SGS in the Hill lab at the Francis Crick Institute has shown that the reported SGS mutations in SKI result in a protein that is resistant to degradation by TGF-b, and thus has a dominant negative effect on the TGF-b signalling pathway. The project Given the role of excess TGF-b signalling in Marfan syndrome, it has been difficult to understand how what appear to be loss-of-function mutations in components of the signalling pathway, can result in a disease with remarkable similarities. Paradoxically, mouse knockins of selected TGFBR1 and TGFBR2 mutations actually show elevated TGF-b signalling in the aortic wall and upregulation of TGF-b1. How this result relates to the diminished activity of the mutated receptors has not been resolved (5). Furthermore, little is known about the functional effects of the LDS SMAD2/3 mutations.

The project aims to use CRISPR/Cas9-mediated genome editing to determine the effects of the LDS SMAD2/3 mutations on TGF-b pathway activity and also on the activity of other TGF-b superfamily pathways. Given that aortic aneurysms are a major feature of LDS, we will focus mainly on generating the mutations in human aortic endothelial cells and vascular smooth muscle cells, cultured separately and together. RNA-seq will be used to investigate how the gene expression programmes are altered by these mutations in response to TGF-b superfamily signals. The project will also involve strong links with patients at the Royal Brompton Hospital through the Dr Nitha Naqvi.


References: 1. Schepers, D., Tortora, G., Morisaki, H., MacCarrick, G., Lindsay, M., Liang, D., Mehta, S. G.,

Hague, J., Verhagen, J., van de Laar, I., Wessels, M., Detisch, Y., van Haelst, M., Baas, A., Lichtenbelt, K., Braun, K., van der Linde, D., Roos-Hesselink, J., McGillivray, G., Meester, J., Maystadt, I., Coucke, P., El-Khoury, E., Parkash, S., Diness, B., Risom, L., Scurr, I., Hilhorst-Hofstee, Y., Morisaki, T., Richer, J., Desir, J., Kempers, M., Rideout, A. L., Horne, G., Bennett, C., Rahikkala, E., Vandeweyer, G., Alaerts, M., Verstraeten, A., Dietz, H., Van Laer, L., and Loeys, B. (2018) A mutation update on the LDS-associated genes TGFB2/3 and SMAD2/3. Hum Mutat 39, 621-634.

2. Verstraeten, A., Alaerts, M., Van Laer, L., and Loeys, B. (2016) Marfan Syndrome and Related Disorders: 25 Years of Gene Discovery. Hum Mutat 37, 524-531.


3. Doyle, A. J., Doyle, J. J., Bessling, S. L., Maragh, S., Lindsay, M. E., Schepers, D., Gillis, E., Mortier, G., Homfray, T., Sauls, K., Norris, R. A., Huso, N. D., Leahy, D., Mohr, D. W., Caulfield, M. J., Scott, A. F., Destree, A., Hennekam, R. C., Arn, P. H., Curry, C. J., Van Laer, L., McCallion, A. S., Loeys, B. L., and Dietz, H. C. (2012) Mutations in the TGF-b repressor SKI cause Shprintzen-Goldberg syndrome with aortic aneurysm. Nat Genet 44, 1249-1254.

4. Wu, M. Y., and Hill, C. S. (2009) TGF-b superfamily signaling in embryonic development and homeostasis. Dev Cell 16, 329-343.

5. Gallo, E. M., Loch, D. C., Habashi, J. P., Calderon, J. F., Chen, Y., Bedja, D., van Erp, C., Gerber, E. E., Parker, S. J., Sauls, K., Judge, D. P., Cooke, S. K., Lindsay, M. E., Rouf, R., Myers, L., ap Rhys, C. M., Kent, K. C., Norris, R. A., Huso, D. L., and Dietz, H. C. (2014) Angiotensin II-dependent TGF-b signaling contributes to Loeys-Dietz syndrome vascular pathogenesis. J Clin Invest 124, 448-460.


Developing small bowel intestine for the treatment of short bowel syndrome A PhD project for the 2019 doctoral clinical fellows programme with Vivian Li (primary supervisor, Crick) and Paolo De Coppi (UCL Great Ormond Street Institute of Child Health)

Small Bowel Syndrome (SBS) is a condition where segments of small bowel are lost, resulting in intestinal failure. While the majority of patients preserve their colon, the outcome is no better because the colon does not absorb nutrients. This project aims to generate an absorptive colon replacing the colonic mucosa with a small bowel organoid (SBO)-derived mucosa. Our group is working on a 3-dimensional “mini-gut” culture protocol with unlimited expansion potential of intestinal epithelial cells in vitro. Intestinal crypts can be expanded in culture creating crypt-villus organoid structures in vitro while maintaining chromosomal stability. These organoids are capable to functionally engraft in vivo providing an ideal source of cells for this project. Human derived intestinal stem cells and organoids will be characterised prior to transplantation in the mucosected colon.

The project is based on the principle that intestinal stem cells possess intrinsic programme to remodel the surrounding extracellular matrix and microenvironment for tissue regeneration. To test the capacity of small bowel intestinal stem cells to repopulate a mucosected colon, a humanized model will be established. Colonic surgical mucosectomy is a routine procedure for some of surgical techniques used in paediatric surgery. In order to test the engraftment of the intestinal organoids and to describe the relevant mechanisms for their functional integration, a model will be established by using colonic samples from patients undergoing colonic resections. Specifically, a colonic sample will be undergoing in vitro mucosectomy, whilst maintaining its vascular supply intact. Mucosectomy of the isolated colonic segment will be optimized using different techniques such as brushing, manual or chemical detachment. To reconstruct an “absorptive” colon, the extremities of the isolated colonic loop will be sutured after mucosectomy in order to create a ‘sausage-like’ structure, followed by intraluminal injection of human-derived small bowel organoids. The efficiency of organoid engraftment will be studied through tracking green fluorescent protein (GFP)-labelled organoids. To test the successful remodeling of colonic mucosa into small intestine, the mucosal microstructure and cell type differentiation will be examined via histology analysis and immunostaining of small bowel-specific mucosa markers. The absorptive function of the engineered colon will further be tested using bioreactors established in the lab to maintain the seeded bowel loops with injected labelled nutrient. This in vitro-based humanized intestine will provide information for in vivo translation in pre-clinical models where the constructs will be validated.

Candidate with experience on tissue culture and surgical procedures will be advantageous. The partner institution for this project is UCL.

References: 1. Meran L, Baulies A, Li VSW. Intestinal Stem Cell Niche: The Extracellular Matrix and Cellular

Components. Stem Cells Int. 2017;2017:7970385. 2. Li VS, Clevers H. In vitro expansion and transplantation of intestinal crypt stem cells.

Gastroenterology. 2012 Jul;143(1):30-4. 3. Totonelli G, Maghsoudlou P, Garriboli M et al. A rat decellularized small bowel scaffold that

preserves villus-crypt architecture for intestinal regeneration. Biomaterials. 2012 Apr;33(12):3401-10.

4. Elliott MJ, De Coppi P, Speggiorin S et al. Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet. 2012 Sep 15;380(9846):994-1000.

5. Sato T, Stange DE, Ferrante M et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology. 2011 Nov;141(5):1762-72.


Identification of novel molecular pathways in congenital hypopituitarism A PhD project for the 2019 doctoral clinical fellows programme with Robin Lovell-Badge (primary supervisor, Crick) and Mehul Dattani (UCL Great Ormond Street Institute of Child Health)

The pituitary gland, in the midline of the brain, consists of the anterior, intermediate and posterior lobes, which secrete hormones that regulate growth, puberty and reproduction, metabolism, stress responses, fluid balance and lactation. Normal hypothalamo-pituitary (HP) development depends upon interactions between the oral and overlying neural ectoderm, and is regulated by a complex cascade of transcription factors and signalling molecules. These may be intrinsic or extrinsic to the developing Rathke's pouch, the primordium of the anterior pituitary (AP). A series of tightly regulated steps resulting in cell proliferation and differentiation give rise to the five specialized AP cell types that secrete 6 different hormones: somatotrophs [growth hormone (GH)], thyrotrophs [thyroid-stimulating hormone (TSH)], gonadotrophs [luteinizing hormone (LH) and follicle-stimulating hormone (FSH)], lactotrophs [prolactin (PRL)] and corticotrophs [adrenocorticotropic hormone (ACTH)]. Congenital hypopituitarism (CH) is a highly variable disorder including a range of syndromes comprising septo-optic dysplasia (SOD), holoprosencephaly (HPE) and Hypogonadotrophic hypogonadism (HH)/Kallmann syndrome (KS), arising as a result of disordered development of the hypothalamus and/or pituitary. Patients may present in the neonatal period with severe hypoglycaemia due to ACTH deficiency, central hypothyroidism and diabetes insipidus, or later with growth and/or pubertal failure due to GH and gonadotrophin deficiencies with/without other pituitary hormone deficiencies. The lifelong condition is associated with significant morbidity and mortality if untreated, and is evolving, emphasizing the need for careful follow up. Additionally, severe visual impairment with blindness, obesity with Type 2 Diabetes, autism and learning difficulties may affect these children. Collaborations between Professors Lovell-Badge and Dattani have resulted in several high impact publications identifying novel molecular mechanisms underlying CH. However, mutations have only been identified in ~10-15% of patients. Professor Dattani’s group has therefore generated a large cohort (n=~500) of patients with CH and related disorders, with >1900 DNA samples collected worldwide from CH patients. Novel approaches such as whole exome/genome sequencing have been used to identify the molecular basis of these conditions. To date, we have submitted 81 samples from 52 pedigrees with familial and/or unique CH phenotypes to GOSgene, at UCL GOS ICH, for NGS. Professor Dattani has also recruited a large cohort of patients to the 100000 Genomes project. Preliminary data have revealed novel pathways, such as Ras, that may be implicated in the aetiology of these disorders. Additionally, we found variants in members of the Sonic Hedgehog pathway, SLC20A1 (homozygous, sodium-dependent phosphate transporter 1) and CCDC149 (homozygous, ciliary gene of unknown function) in pedigrees with CH, representing good candidates segregating with the disease phenotype. We aim to investigate these genetic variants using a combination of approaches, including gene expression studies in human embryonic tissues, and functional studies, such as genome editing, in human and murine cells. Induced pluripotent stem cells (IPSC), which we will derive from patient fibroblasts, will provide important material for this and further analysis, such as in vitro disease modelling. These studies will enable the identification of novel molecular pathways associated with CH, and the characterisation of the aetiology and pathogenesis of these syndromes, with the aim to improve therapies. The partner institution for this project is UCL.

References: 1. Gregory LC, Alatzoglou KS, McCabe MJ, Hindmarsh PC, Saldanha JW, Romano N, Le Tissier P,

Dattani MT (2016) Partial loss of function of the GHRH Receptor leads to mild Growth Hormone Deficiency. J Clin Endocrinol Metab 101(10):3608-3615.

2. Gaston-Massuet C, McCabe MJ, Scagliotti V, Young RM, Carreno G, Gregory LC, Jayakody SA, Pozzi S, Gualtieri A, Basu B, Koniordou M, Wu CI, Bancalari RE, Rahikkala E, Veijola R, Lopponen T, Graziola F, Turton J, Signore M, Mousavy Gharavy SN, Charolidi N, Sokol SY, Andoniadou CL, Wilson SW, Merrill BJ, *Dattani MT, *Martinez-Barbera JP (2016) (*Co-Senior Authors) Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans. Proc Natl Acad Sci USA 13(5):E548-57.

3. Sun Y, Bak B, Schoenmakers N, van Trotsenburg ASP, Oostdijk W, Voshol P, Cambridge E, White JK, le Tissier P, Mousavy Gharavy SN, Martinez-Barbera JP, Stokvis-Brantsma WH, Vulsma T, Kempers MJ, Persani L, Campi I, Bonomi M, Beck-Peccoz P, Zhu H, Davis TME, Hokken-Koelega ACS, Del Blanco DG, Rangasami JJ , Ruivenkamp CAL, Laros JFL, Kriek M, Kant SG, Bosch CAJ,


Biermasz NR, Appelman-Dijkstra NM, Corssmit EP, Hovens GCJ, Pereira AM, den Dunnen JT, Breuning MH, Hennekam RC, Chatterjee KK*, Dattani MT*, Wit JM*, Bernard DJ* (*Co-Senior Authors) (2012) Loss-of-function mutations in IGSF1 cause a novel X-linked syndrome of central hypothyroidism and testicular enlargement Nature Genetics 44(12): 1375-1381.

4. Kelberman D, Rizzoti K, Lovell-Badge R, Robinson ICAF, Dattani MT (2009) Genetic Regulation of pituitary gland development in human and mouse. Endocrine Reviews 30(7): 790-829.

5. Kelberman D, Rizzoti K, Avilion A, Bitner-Glindzicz M, Cianfarani S, Collins J, Chong WK, Kirk JM, Achermann JC, Ross R, Carmignac D, Lovell-Badge R, Robinson IC, Dattani MT (2006) Mutations within Sox2/SOX2 are associated with abnormalities in the hypothalamo-pituitary-gonadal axis in mice and humans. J Clin Invest 116 (9):2442-2455.


Aberrant intron retention and RBP mislocalization: a new mechanism for ALS A PhD project for the 2019 doctoral clinical fellows programme with Nicholas Luscombe (primary supervisor, Crick), Rickie Patani (Crick/UCL) and Jernej Ule (Crick/UCL)

Amyotrophic lateral sclerosis (ALS) is progressive and fatal, with a lifetime risk of ~1 in 400. ALS is characterized by motor neuron (MN) degeneration, but its etiology remains unknown. For therapeutic development, we need a unified understanding of common primary events leading to ALS pathogenesis. This project explores the earliest molecular and cellular events in ALS.

90% of ALS is sporadic and least understood. Past studies have tended to focus on familial cases and have implicated RNA deregulation (Bakkar et al. 2018). RNA deregulation has turned out to be a potential common mechanism across all ALS, whether sporadic or familial (Taylor et al. 2016). Our recent breakthrough discovery of Aberrant Intron Retention (AIRiALS) is the earliest detectable molecular phenotype in ALS (Luisier et al. 2017). Combining cellular models of MN differentiation (Patani), transcriptomic measurements of protein-RNA interactions (Ule Lab) and detailed statistical analysis (Luscombe), we identified reproducible AIRiALS events during early motor neurogenesis. These defects are universal to ALS mutations in multiple genes.

Functional analysis of AIRiALS transcripts converges on RNA regulation. The introns with strongest retention are in transcripts encoding for SFPQ and FUS, RBPs already linked to ALS (Thomas-Jinu et al. 2017; Vance et al. 2009). In turn, SFPQ binds strongly to the retained intron in its own transcript. We revealed universal nuclear-to-cytoplasmic mislocalization of the SFPQ protein in human stem cell cultures, transgenic mouse models and human post-mortem tissues from sporadic cases, thus encompassing the full spectrum of ALS. This suggests that AIRiALS and the nuclear loss of SFPQ are unifying molecular hallmarks (Luisier et al. 2018).

Our hypothesis is that AIRiALS are linked to mislocalization of RPBs in ALS. By examining these defects during MN development, we will chronicle the earliest molecular events leading to ALS. We will complement these with study of our previously reported cellular phenotypes, including endoplasmic reticulum stress, mitochondrial depolarization, oxidative stress, synaptic perturbation and electrophysiological hypoactivity to understand how these events conspire to cause ALS.

We model ALS with human iPSC libraries from familial and sporadic ALS patients, including isogenic controls. Our protocols achieve efficient iPSC differentiation into fully functional MNs and ACs, with mutant lines presenting robust disease phenotypes (TDP43 mislocalization and accelerated cell death; Hall et al. 2017). Findings are validated in transgenic mouse models and post-mortem spinal cord tissues from ALS patients.

We will computationally and experimentally analyze AIRiALS introns to highlight sequence properties that distinguish them from other introns; this will also allow shortlisting of the top introns for detailed investigation. Using CRISPR/Cas9, we shall progressively delete intronic segments to identify minimal regions that are sensitive to promoting or inhibiting AIRiALS in control and ALS lines. These AIRiALS sensitive regions will be reinserted into transcripts, and the molecular and cellular phenotypes of ALS motor neurons will be assessed in order to establish whether AIRiALS is a possible causal event or an early hallmark of processes leading to disease phenotypes.


References: 1. Luisier R, Tyzack GE, Hall CE, Mitchell JS, Devine H, Taha DM, Malik B, Meyer I, Greensmith L,

Newcombe J, Ule J, Luscombe NM, Patani R. Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS. Nat Commun. 2018 May 22;9(1):2010.

2. Hall CE, Yao Z, Choi M, Tyzack GE, Serio A, Luisier R, Harley J, Preza E, Arber C, Crisp SJ, Watson PMD, Kullmann DM, Abramov AY, Wray S, Burley R, Loh SHY, Martins LM, Stevens MM, Luscombe NM, Sibley CR, Lakatos A, Ule J, Gandhi S, Patani R. Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS. Cell Rep. 2017 May 30;19(9):1739-1749.

3. Sugimoto Y, Vigilante A, Darbo E, Zirra A, Militti C, D'Ambrogio A, Luscombe NM, Ule J. hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature. 2015 Mar 26;519(7544):491-4.


4. Zarnack K, König J, Tajnik M, Martincorena I, Eustermann S, Stévant I, Reyes A, Anders S, Luscombe NM, Ule J. Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell. 2013 Jan 31;152(3):453-66.

5. Tollervey JR, Curk T, Rogelj B, Briese M, Cereda M, Kayikci M, König J, Hortobágyi T, Nishimura AL, Zupunski V, Patani R, Chandran S, Rot G, Zupan B, Shaw CE, Ule J. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nat Neurosci. 2011 Apr;14(4):452-8.


An evolutionary approach to understand regulation of cellular energy metabolism in normal physiology and disease A PhD project for the 2019 doctoral clinical fellows programme with Snezhana Oliferenko (primary supervisor, Crick) and Paul Gissen (UCL Great Ormond Street Institute of Child Health)

Altered energy metabolism is implicated in common diseases such as Alzheimer’s dementia and cancer. Furthermore, numerous single gene defects cause metabolic failure, leading to such rare disorders as mitochondrial respiratory chain abnormalities or fatty acid oxidation defects. Energy metabolism occurs through oxidative phosphorylation in mitochondria and glycolysis in the cytoplasm. Most cell types can combine both energy generation pathways. In animals, differentiated cells tend to rely on oxidative phosphorylation but dividing cells or abnormally proliferating cancers may adopt aerobic glycolysis that favours increased macromolecular synthesis (Warburg effect). How is energy production regulated? How do cells choose a particular energy generation route, and how do they switch between alternative strategies? How do these metabolic strategies influence the rest of cellular physiology? We want to answer these fundamental, yet surprisingly poorly understood questions by exploiting the naturally occurring divergence in metabolism between related species.

We will use two related eukaryotic model organisms, the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, as ‘living test tubes’ for conserved cell-autonomous processes relevant for human disease. In spite of similar genome content and genome structure, these species show remarkable differences in fundamental biological processes such as cell proliferation, with S. japonicus utilizing mechanisms previously perceived to be common in animal cells rather than in simple yeasts. Importantly, unlike S. pombe, S. japonicus appears to be an obligate ‘Warburg’ organism, relying exclusively on aerobic glycolysis to generate energy. S. japonicus grows and divides considerably faster than S. pombe, suggesting that it greatly optimized its glycolytic program.

We propose to combine our complementary expertise in yeast cell biology and metabolism (Oliferenko lab) and human metabolic diseases (Gissen lab) to tackle the question how regulatory networks controlling glycolysis and other parts of central carbon metabolism remodel. We will identify critical flux-controlling elements in both species and investigate how they can be modified to produce novel behaviours. This framework will hopefully allow us to translate our fundamental findings to the clinic. In particular, we would like to tackle the problem of unexplained hypoglycaemia in paediatric patients. The 100,000 Genome Project identified numerous potentially pathogenic genetic variants responsible for this phenotype. Many of these genes have not been previously implicated in human disease. Interestingly, a number of these variants occur in evolutionarily conserved genes encoding the components of central carbon metabolism. We will use S. pombe and S. japonicus as a composite system to probe the potential importance of these variants in cellular metabolism and physiology.

Using an evolutionary cell biology approach in related species will provide us with a wider view of biology without compromising on the depth of insight, and hopefully make unanticipated connections between central metabolism and other cellular features.

The details of the project can be adjusted to the interests and expertise of the student. The partner institution for this project is UCL.

References: 1. Ward PS, Thompson CB. 2012. Metabolic reprogramming: a cancer hallmark even Warburg did not

anticipate. Cancer Cell. 21:297-308. 2. Makarova, M., Gu, Y., Chen, J-S., Beckley, J., Gould, K. and S. Oliferenko. 2016. Temporal

regulation of Lipin activity diverged to account for differences in mitotic programs. Current Biology. 26: 237-243.

3. Banushi, B., et al and P. Gissen. 2016. Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis. Nature Communications. 7:12111. doi: 10.1038/ncomms12111

4. Oliferenko. 2018. Understanding eukaryotic chromosome segregation from a comparative biology perspective. J. Cell Sci. 131: doi: 10.1242/jcs.203653.

5. Peplow, M. 2016. The 100,000 Genomes Project. BMJ. 353: doi: 10.1136/bmj.i1757


Identifying regulators of immune surveillance and cancer cachexia in lung cancer A PhD project for the 2019 doctoral clinical fellows programme with Charles Swanton (primary supervisor, Crick) and Nicholas McGranahan (UCL)

Cancer associated cachexia (CAC) is a catabolic state resulting in the loss of skeletal muscle, driven by anorexia and metabolic changes, including increased energy expenditure and inflammation. A clinical diagnosis of CAC is made by demonstrating a loss of >5% of body weight over a period of 6 months. A molecular basis for the clinical syndrome remains poorly described.

TRACERx is a longitudinal cancer evolutionary program that aims to track cancer genetic adaptations through space and time from diagnosis to cure or recurrence and death through the PEACE autopsy study.

Through analysis of serial radiological imaging (axial lumbar CT scans) we have classified the cancer cachexia syndrome in the first 450 patients. Through preliminary computational analysis of multi-region TRACERx DNA and RNA sequencing data, we have found evidence for a number of candidate regulators of cancer cachexia, enriched for immune-regulatory and metabolic functions.

We aim to validate and refine such regulators through an in-depth computational analysis of the full 842 patient TRACERx cohort and integration with mass spectrometry analysis of blood derived proteins and metabolites. In parallel, the candidate will develop a comprehensive analysis of immune-therapy response and toxicity in the TRACERx cohort to explore the relationships between clonal neo-antigen burden, HLA loss of heterozygosity and immune-paresis with response to checkpoint inhibitor blockade and clinical outcome.

The successful candidate will have an interest in clinical bioinformatics, metabolism and/or the immune-cancer interface and enjoy working in a multi-disciplinary team and have aspirations to become a fully competent cancer bioinformatician within a 3-year time frame. The partner institution for this project is UCL.

References: 1. Jamal-Hanjani et al NEJM 2017 Jun 1;376(22):2109-2121 2. McGranahan et al Cell. 2017 Nov 30;171(6):1259-1271 3. Abbosh et al Nature. 2017 Apr 26;545(7655):446-451 4. Turajlic, et al Cell. 2018 Apr 19;173(3):581-594 5. Turajlic et al. Cell. 2018 Apr 19;173(3):595-610


Exploring CDKL5 substrates in humans A PhD project for the 2019 doctoral clinical fellows programme with Sila Ultanir (primary supervisor, Crick) and Helen Cross (UCL Great Ormond Street Institute of Child Health)

Cyclin Dependent Kinase Like-5 (CDKL5) is a serine/threonine kinase in CDKL family, most similar to cyclin dependent kinases and MAP kinases. Cdkl5 gene is on the X-chromosome. Loss of function mutations in CDKL5 causes a severe neurodevelopmental disorder now termed the CDKL5 Deficiency Disorder, CDD 1,2. Patients with CDKL5 mutations have early-onset hard-to-control seizures and widespread neurodevelopmental delays. CDD mostly affects girls but is also observed in boys in line with a classical X-chromosome linked dominant genetic disorder. Genetic data strongly suggest the disease-causing mutations in CDKL5 are loss of function mutations. Due to stochastic nature of X-chromosome inactivation, girls are mosaics in defective CDKL5 allele.

CDKL5 is highly brain enriched and it is widely expressed in the brain during development and in adult 3,4. In two separate CDKL5 knockout (KO) mouse lines, impaired learning and memory and autistic-like social behaviour was observed. However, no evidence for spontaneous seizures or increased seizure susceptibility was found 2. Albeit multiple studies on dendrite and spine deficits and electrophysiological characterization in CDKL5 knockout mice, consistent and robust cellular differences in CDKL5 knockout mice remain to be determined.

Recently, we have used chemical genetic methods to identify direct substrates of CDKL5. We find that CDKL5 phosphorylates three microtubule binding proteins EB2, MAP1S and ARHGEF2 (Baltussen et al, under review). Using rabbit polyclonal phosphospecific antibodies we find that pEB2 and pMAP1S are greatly reduced in CDKL5 KO mouse brain. In collaboration with Dr Alysson Muotri’s lab we showed that pEB2 is greatly reduced in patient neurons derived from induced pluripotent stem cells when compared to healthy controls. Discovery of CDKL5’s physiological substrates is a major landmark for CDKL5 field.

Functional and molecular biomarkers are important read-outs of cellular activities and can aid translational and/ or clinical research greatly. Peripheral blood in particular, is an accessible resource for evaluating signalling pathway activities in humans. Blood is a source for potential molecular biomarkers and due to difficulty in accessing the brain. Two systemic therapeutic approaches are being tested/ developed for restoring lost CDKL5 activity in humans. First Ataluren 5 is a drug that allows read-through of stop codons allowing CDKL5 to be translated in patients with nonsense mutations. Second, X-chromosome reactivation strategy is being developed to transcribe good copy of CDKL5 from inactive X chromosome. In order to test the effectiveness of these treatments in altering CDKL5 activity EB2 phosposerine 222 (pSer222) antibody and antibodies targeting other CDKL5 substrates can be used.

We find that microtubule dependent trafficking and microtubule plus-tip dynamics are altered in the dendrites of CDKL5 knockout mice. We would like to this purpose we would like to use human Induced Pluripotent Stem Cell (hIPSC) lines to examine microtubule dependent functions in human neurons. Establishing human derived neurons will enable translating finding from mouse models to human neurons. We will use CDD patient derived hIPSCs and shRNA mediated transduction of human neurons or Crispr mediated gene editing to alter the signalling pathways. We will use imaging and biochemistry methods to then determine microtubule dependent functions in human neurons.

1- Testing if EB2 pSer222 is altered in blood derived PMBCs or other blood cell types in humans with CDD 2- Testing if pEB2 can be detected in skin biopsies or cerebrospinal fluid in humans 3- Determining if microtubule dependent functions are altered in human neurons using differentiated

hIPSCs.


References: 1. Bahi-Buisson, N. & Bienvenu, T. CDKL5-Related Disorders: From Clinical Description to Molecular

Genetics. Mol Syndromol 2, 137-152, doi:000331333 (2012). 2. Zhou, A., Han, S. & Zhou, Z. J. Molecular and genetic insights into an infantile epileptic

encephalopathy - CDKL5 disorder. Front Biol (Beijing) 12, 1-6, doi:10.1007/s11515-016-1438-7 (2017).


3. Chen, Q. et al. CDKL5, a protein associated with rett syndrome, regulates neuronal morphogenesis via Rac1 signaling. The Journal of neuroscience : the official journal of the Society for Neuroscience 30, 12777-12786, doi:10.1523/JNEUROSCI.1102-10.2010 (2010).

4. Rusconi, L. et al. CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail. The Journal of biological chemistry 283, 30101-30111, doi:10.1074/jbc.M804613200 (2008).

5. McDonald, C. M. et al. Ataluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT DMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet (London, England) 390, 1489-1498, doi:10.1016/S0140-6736(17)31611-2 (2017).


Elucidating the evolutionary history of rare atypical cancer cells by single-cell laser capture microdissection and genome and transcriptome sequencing A PhD project for the 2019 doctoral clinical fellows programme with Peter Van Loo (primary supervisor, Crick) and Nischalan Pillay (UCL)

Cancers acquire somatic mutations as they evolve, including single-nucleotide variants, insertions and deletions, structural variants and copy number alterations. A tumour’s genome therefore carries an archaeological record of its evolutionary past, and much of this history can be reconstructed from the tumour’s genome sequence (1, 2). In addition, single-cell DNA and RNA sequencing approaches allow tracing the origin of single cancer cells to cancer clones or subclones (3) and inferring their cell states, respectively.

Interestingly, some mutational processes, such us chromothripsis (4), can generate large numbers of changes to the genome in a single event. Such punctuated events can substantially reconfigure the cancer genome, leading to large leaps in tumour evolution. Chromothripsis is observed in multiple cancer types, and is particularly common in several sarcomas, including osteosarcoma and liposarcoma. We hypothesise that due to the extensive damage chromothripsis causes to the genome, most of these events are detrimental to their host cells, and therefore, most chromothripsis events remain unobserved through genome sequencing of bulk tumour tissue. Single-cell sequencing approaches hold the potential to observe and study these unselected chromothripsis events.

Some cancers, particularly connective tissue tumours, harbour scattered morphologically highly atypical cells. Such cells are observed in a number of sarcomas, specifically undifferentiated pleomorphic sarcomas, pleomorphic rhabdomyosarcoma, pleomorphic liposarcoma and pleomorphic leiomyosarcoma, and also in glioblastomas. It is noteworthy that scattered atypical cells with enlarged polymorphic nuclei are also seen in some benign connective tissue tumours, in particular schwannomas, osteoblastomas, and chondromyoid fibroma. The genotypes of these cells and their role in tumour evolution have not been characterised.


Fig. 1: atypical cells across cancer types. A and B: monster cells (200 µm and 150 µm respectively) in undifferentiated sarcomas. C: Mitosis in this same tumour as B, demonstrating multipolarity as well as micronuclei. D: giant cell glioblastoma (source: Oh et al., Brain Pathology). In this project, we plan to study these rare scattered atypical cells across sarcoma and benign connective tissue tumours, using single-cell laser capture microdissection (5) and single-cell DNA and RNA sequencing. Our aims are three-fold:

(i) Study the evolutionary history of these rare atypical cells and their relationship to their neighbouring tumour cells, across multiple sarcomas, as well as a few benign connective tissue tumours with scattered highy atypical cells such as those seen in ‘degenerate schwannona’, using single-cell DNA sequencing.

(ii) Study their phenotype and cell state using single-cell RNA sequencing. (iii) Evaluate whether these rare atypical cells (or other cancer cells studied through single-

nucleus sequencing), are chromothriptic, and if so, leverage them to study the landscape of chromothripsis outside of the context of positive selection.

This project is suitable for a pathologist in training, with an interest in genomics.


References: 1. Nik-Zainal, S.#, Van Loo, P.#, Wedge, D.C.#, et al. (2012). The life history of 21 breast cancers.

Cell, 149:994-1007. 2. Gerstung, M.#, Jolly, C.#, Leshchiner, I.#, Dentro, S.C.#, … [38 authors], Spellman, P.T.#, Wedge,

D.C.#, Van Loo, P.#, on behalf of the PCAWG Evolution and Heterogeneity Working Group and the PCAWG network (2017). The evolutionary history of 2,658 cancers. bioRxiv preprint, doi: https://doi.org/10.1101/161562.

3. Demeulemeester, J.#, Kumar, P.#, Møller, E.K.#, Nord, S., Wedge, D.C., Peterson, A., Mathiesen, R.R., Fjelldal, R., Zamani Esteki, M., Theunis, K., Fernandez Gallardo, E., Grundstad, A.J., Borgen, E., Baumbusch, L.O., Børresen-Dale, A.L., White, K.P.#, Kristensen, V.N.#, Van Loo, P.#, Voet, T.#, Naume, B.# (2016). Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing. Genome Biology, 17:250.

4. Stephens, P.J., Greenman, C.D., Fu, B., Yang, F., Bignell, G.R., Mudie, L.J., Pleasance, E.D., Lau, K.W., Beare, D., Stebbings, L.A., McLaren, S., Lin, M.L., McBride, D.J., Varela, I., Nik-Zainal, S., Leroy, C., Jia, M., Menzies, A., Butler, A.P., Teague, J.W., Quail, M.A., Burton, J., Swerdlow, H., Carter, N.P., Morsberger, L.A., Iacobuzio-Donahue, C., Follows, G.A., Green, A.R., Flanagan, A.M., Stratton, M.R., Futreal, P.A., Campbell, P.J. (2011). Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell, 144:27-40.

5. Casasent, A.K., Schalck, A., Gao, R., Sei, E., Long, A., Pangburn, W., Casasent, T., Meric-Bernstam, F., Edgerton, M.E., Navin, N.E. (2018). Multiclonal Invasion in Breast Tumors Identified by Topographic Single Cell Sequencing. Cell, 172:205-217.e12.


Tracing the origin of disseminated tumour cells in bone marrow A PhD project for the 2019 doctoral clinical fellows programme with Peter Van Loo (primary supervisor, Crick) and Samra Turajlic (The Institute of Cancer Research, Royal Marsden)

Note additional eligibility criterion: candidates with a background in veterinary medicine are not eligible for the funding of this position.

Cancer is a disease of the genome, arising through the accumulation of somatic driver mutations in normal cells, leading to successive clonal expansions. Cancer cells of solid tumors may intravasate, travel through the blood stream as circulating tumor cells and subsequently extravasate in distant organs like bone marrow. These disseminated tumor cells (DTCs) are often refractory to therapy and can lay dormant for years. Alternatively, they may progress to cause overt distant metastases, often resulting in the death of the patient.

Patients diagnosed with non-metastatic breast cancer for instance still have a significant risk of relapse, even after complete surgical removal of the tumor, most likely due to the existence of DTCs, reported in up to 40% of cases. In bone marrow, these DTCs can be sampled through aspirates and can be identified using epithelial or tissue-specific markers. Their presence has been shown to be prognostic for poor survival in breast cancer. The concentration of DTCs in bone marrow is typically estimated at one cell per 107–108 blood cells in patients with advanced disease.

Unfortunately, the molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. We recently applied single-cell sequencing to identify and trace the origin of DTCs, isolated by micromanipulation from the bone marrow of six primary breast cancer patients using the established markers for epithelial tumor cells (1). Our genomic analyses revealed that a quarter of the cells are DTCs disseminating relatively late from the tumour. The remaining cells represented non-aberrant ‘normal’ cells and ‘aberrant cells of unknown origin’ (AUs) with mutational landscapes evidencing an origin outside of the lineage of the observed tumour. AU-like cells have been interpreted as DTCs in previous studies, supporting an early dissemination and parallel evolution model. Instead, AUs (and perhaps also the normal cells) may represent either a likely epithelial cell type of breast or non-breast origin homing to the bone marrow or derive from a haematopoietic cell lineage. Alternatively, these cells may originate from another neoplasm in the patient, an undetected synchronous primary breast tumor, or an undetectable tumor cell clone residing in the primary tumor.

In this project we aim to characterise the three classes of epithelial-like cells (normal, AU and DTC) in healthy individuals and cancer patients. Our goals are threefold:

1) Characterise and trace epithelial-like cells (‘normal’/AU) in healthy volunteers and cancer patients.

2) Characterise and trace the origins of DTCs across different cancer types (renal cancer, breast cancer, prostate cancer, melanoma, lung cancer) in relation to the subclonal composition and evolutionary history of the primary tumour.

3) Trace the evolutionary history of metastases in relation to DTCs. The results from this project will contribute to our understanding of how somatic mutations accumulate under normal and pathological conditions. Our findings will provide both broad and in-depth insights into dissemination of both pre-malignant and cancer cell and the cellular states involved. They are also expected to yield optimized markers for clinical and potentially subclinical neoplasms.

The partner institution for this project is The Institute of Cancer Research.

References: 1. Demeulemeester, J.#, Kumar, P.#, Møller, E.K.#, Nord, S., Wedge, D.C., Peterson, A., Mathiesen,

R.R., Fjelldal, R., Zamani Esteki, M., Theunis, K., Fernandez Gallardo, E., Grundstad, A.J., Borgen, E., Baumbusch, L.O., Børresen-Dale, A.L., White, K.P.#, Kristensen, V.N.#, Van Loo, P.#, Voet, T.#, Naume, B.# (2016). Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing. Genome Biology, 17:250.

2. Gundem, G., Van Loo, P., Kremeyer, B., Alexandrov, L.B., Tubio, J.M.C., Papaemmanuil, E., Brewer, D.S., Kallio, H.M.L., Hӧgnäs, G., Annala, M., Kivinummi, K., Goody, V., Latimer, C., O’Meara, S., Dawson, K.J., Isaacs, W., Emmert-Buck, M.R., Nykter, M., Foster, C., Kote-Jarai, Z., Easton, D., Whitaker, H.C., ICGC Prostate UK Group, Neal, D.E., Cooper, C.S., Eeles, R.A.,


Visakorpi, T., Campbell, P.J., McDermott, U.#, Wedge, D.C.#, Bova, G.S.# (2015). The evolutionary history of lethal metastatic prostate cancer. Nature, 520:353-357.

3. Nik-Zainal, S.#, Van Loo, P.#, Wedge, D.C.#, Alexandrov, L.B., Greenman, C.D., Lau, K.W., Raine, K., Jones, D., Marshall, J., Ramakrishna, M., Shlien, A., Cooke, S.L., Hinton, J., Menzies, A., Stebbings, L.A., Leroy, C., Jia, M., Rance, R., Mudie, L.J., Gamble, S.J., Stephens, P.J., McLaren, S., Tarpey, P.S., Papaemmanuil, E., Davies, H.R., Varela, I., McBride, D.J., Bignell, G.R., Leung, K., Butler, A.P., Teague, J.W., Martin, S., Jönsson, G., Mariani, O., Boyault, S., Miron, P., Fatima, A., Langerød, A., Aparicio, S.A.J.R., Tutt, A., Sieuwerts, A.M., Borg, Å., Thomas, G., Salomon, A.V., Richardson, A.L., Børresen-Dale, A.-L., Futreal, P.A., Stratton, M.R., Campbell, P.J.; for the Breast Cancer Working Group of the International Cancer Genome Consortium (2012). The life history of 21 breast cancers. Cell, 149:994-1007.

4. Jamal-Hanjani, M.#, Wilson, G.A.#, McGranahan, N.#, Birkbak, N.J.#, Watkins, T.B.K.#, Veeriah, S.#, Shafi, S., Johnson, D.H., Mitter, R., Rosenthal, R., Salm, M., Horswell, S., Escudero, M., Matthews, N., Rowan, A., Chambers, T., Moore, D.A., Turajlic, S., Xu, H., Lee, S.M., Forster, M.D., Ahmad, T., Hiley, C.T., Abbosh, C., Falzon, M., Borg, E., Marafioti, T., Lawrence, D., Hayward, M., Kolvekar, S., Panagiotopoulos, N., Janes, S.M., Thakrar, R., Ahmed, A., Blackhall, F., Summers, Y., Shah, R., Joseph, L., Quinn, A.M., Crosbie, P.A., Naidu, B., Middleton, G., Langman, G., Trotter, S., Nicolson, M., Remmen, H., Kerr, K., Chetty, M., Gomersall, L., Fennell, D.A., Nakas, A., Rathinam, S., Anand, G., Khan, S., Russell, P., Ezhil, V., Ismail, B., Irvin-Sellers, M., Prakash, V., Lester, J.F., Kornaszewska, M., Attanoos, R., Adams, H., Davies, H., Dentro, S., Taniere, P., O'Sullivan, B., Lowe, H.L., Hartley, J.A., Iles, N., Bell, H., Ngai, Y., Shaw, J.A., Herrero, J., Szallasi, Z., Schwarz, R.F., Stewart, A., Quezada, S.A., Le Quesne, J., Van Loo, P., Dive, C., Hackshaw, A., Swanton, C.; TRACERx Consortium (2017). Tracking the Evolution of Non-Small-Cell Lung Cancer. New England Journal of Medicine, 376:2109-2121.

5. Turajlic, S.#, Xu, H.#, Litchfield, K.#, Rowan, A.#, Horswell, S.#, Chambers, T.#, O'Brien, T.#, Lopez, J.I.#, Watkins, T.B.K., Nicol, D., Stares, M., Challacombe, B., Hazell, S., Chandra, A., Mitchell, T.J., Au, L., Eichler-Jonsson, C., Jabbar, F., Soultati, A., Chowdhury, S., Rudman, S., Lynch, J., Fernando, A., Stamp, G., Nye, E., Stewart, A., Xing, W., Smith, J.C., Escudero, M., Huffman, A., Matthews, N., Elgar, G., Phillimore, B., Costa, M., Begum, S., Ward, S., Salm, M., Boeing, S., Fisher, R., Spain, L., Navas, C., Grönroos, E., Hobor, S., Sharma, S., Aurangzeb, I., Lall, S., Polson, A., Varia, M., Horsfield, C., Fotiadis, N., Pickering, L., Schwarz, R.F., Silva, B., Herrero, J., Luscombe, N.M., Jamal-Hanjani, M., Rosenthal, R., Birkbak, N.J., Wilson, G.A., Pipek, O., Ribli, D., Krzystanek, M., Csabai, I., Szallasi, Z., Gore, M., McGranahan, N., Van Loo, P., Campbell, P., Larkin, J., Swanton, C.; TRACERx Renal Consortium (2018). Deterministic Evolutionary Trajectories Influence Primary Tumor Growth: TRACERx Renal. Cell, 173:595-610.e11.


Towards deciphering the role of long noncoding RNA mediated gene regulation in mammalian and cancer cells A PhD project for the 2019 doctoral clinical fellows programme with Folkert van Werven (primary supervisor, Crick) and Jessica Downs (The Institute of Cancer Research). Clinical supervisor tba

Note additional eligibility criterion: candidates with a background in veterinary medicine are not eligible for the funding of this position.

Eukaryotic genomes are transcribed into protein coding, noncoding and long noncoding RNAs (lncRNAs). Despite increasing efforts, the function of the majority of lncRNA transcripts remains unknown. Some of lncRNAs act in cis and thereby regulate gene expression locally. There is growing evidence that lncRNAs play critical role in regulating various cellular processes including cell differentiation and development. Mis-regulation of lncRNAs has also been implicated in cancer. A long term goal of the Cell Fate and Gene Regulation laboratory is to understand how transcription of lncRNAs regulates gene expression locally, and how this is important for cellular processes globally such as cell differentiation, development and cancer.

Transcription of lncRNAs through promoter sequences represses local gene expression in yeast 1,2. Transcription coupled chromatin changes are critical for this process. In particular, transcription of lncRNAs deposits Set2-dependent histone H3 lysine 36 methylation to generate a repressive chromatin state 3. In cancer cells, loss of function mutations of SETD2, the human Set2 orthologue, have been implicated in cancer progression 3,4. Like in yeast, SETD2 can act as a repressor of the transcription 3,5. Although the lncRNA mediated gene regulation facilitated by Set2 has been well studied in yeast, the presence of related mechanisms in mammalian cells has not been well characterized.

The goal of this project is to (1) identify genes in mammalian cells that are regulated locally by transcription of long noncoding RNAs and (2), dissect the role of SETD2 in lncRNA mediated gene regulation during cell differentiation programs and disease (2). The work is a direct extension from our findings in yeast 1,2. First, we will study lncRNA mediated gene regulation in a model for cell differentiation: e.g. the mouse embryonic stem cells to neurons cell fate program. To identify genes that are regulated by lncRNAs, a combination of established techniques from our laboratory will be used, such as transcription-start-site sequencing (TSS-seq) together with nascent RNA-seq. We will generate null mutants of SETD2 and other chromatin factors using gene editing technology and examine their involvement in lncRNA mediated gene regulation. Finally, we will investigate how lncRNA mediated gene regulation is affected in cancer cell lines harbouring loss of function mutations in SETD2. So far the role of lncRNAs in reprogramming cancer stem cell fate through transcriptome regulation has been less focussed. The project will be a first step towards deciphering how transcription of long noncoding RNAs regulates gene expression during mammalian development and how mis-regulation of this process affects gene expression in cancer.

The partner institution for this project is The Institute of Cancer Research.

References: 1. Chia, M. et al. Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes

and interference of a downstream promoter. Elife 6, doi:10.7554/eLife.27420 (2017). 2. van Werven, F. J. et al. Transcription of two long noncoding RNAs mediates mating-type control of

gametogenesis in budding yeast. Cell 150, 1170-1181, doi:10.1016/j.cell.2012.06.049 (2012). 3. McDaniel, S. L. & Strahl, B. D. Shaping the cellular landscape with Set2/SETD2 methylation. Cell

Mol Life Sci 74, 3317-3334, doi:10.1007/s00018-017-2517-x (2017). 4. Li, J. et al. SETD2: an epigenetic modifier with tumor suppressor functionality. Oncotarget 7,

50719-50734, doi:10.18632/oncotarget.9368 (2016). 5. Neri, F. et al. Intragenic DNA methylation prevents spurious transcription initiation. Nature 543,

72-77, doi:10.1038/nature21373 (2017).


Structural determinants of the T cell contribution to tuberculosis and HIV-tuberculosis pathogenesis A PhD project for the 2019 doctoral clinical fellows programme with Robert Wilkinson (primary supervisor, Crick) and Xiao-Ning Xu (Imperial College London)

Tuberculosis remains an important problem whose global control would be enhanced by a novel vaccine that protects adults and thereby reduces transmission. Understanding however of the antigenic specificity and phenotype of protective or potentially pathogenic T cell responses is incomplete. Most clinical trials therefore rely on vaccines antigens whose selection has been narrowly based on their ability to restimulate, from peripheral T cells in sensitised or vaccinated donors, the secretion of cytokines (e.g. TNF, interferon (IFN)-g, L-2) known to contribute, but which are insufficient alone, to impart protection. In dysregulation, these same cytokines may also contribute to pathology.

We will test the hypothesis that specific protein determinants of M. tuberculosis differentially induce the TNF superfamily molecule CD153 (TNFSF8) that we have recently demonstrated necessary for IFNg-independent control of pulmonary Mtb infection by CD4 T cells (Sallin et al. Nature Microbiology 2018). Background data, collaboration, expertise and existing funding supporting the planning project are as follows

1. Whilst initial experience of large-scale novel tuberculosis vaccine trials was disappointing Wilkinson has co-authored, via his role as Director of a Wellcome Centre in Cape Town, a recent Phase IIB trial that suggests two specific antigens are of interest (Van der Meeren et al. New Engl J Med 2018). Another recent phase IIB clinical trials evidence also points to two further antigens of interest (Nemes, N Engl J Med, 2018). These hints of specificity are important when considering our experimental approach

2. In Cape Town, Wilkinson is also Co-PI (with Alan Sher, NIAID) of a NIH U01 collaborative program entitled ‘Inflammatory and cellular determinants of disease severity and treatment outcome in South African TB patients’. This is carefully recruiting (April 2015-March 2020), and biobanking T cells from, 500 tuberculosis patients and controls, equal numbers HIV infected and uninfected and includes sampling of lung, pericardial and pleural fluids that will allow rarely-performed analysis of responses at disease sites as well as blood. This program has also already identified CD153 as above.

3. Using peptide pools (Crick peptide STP) from the selected four antigens and other standardised stimuli we will expand T cells in vitro from both blood and disease sites in the variously sensitised groups of persons as described above. Activated cells will be single-cell sorted using a BD influx sorter available in the BSLIII in Cape Town and lysed for RNA analysis. Single cell RNAseq and analysis of TcR variants will be undertaken in the laboratory of the Co-supervisor Xiao-Ning Xu at Imperial. Frequency analysis will focus on a) compartment (disease site versus blood); b) HIV status; c) Disease status

4. TcR sequences over-represented either in blood or at disease sites in patients with distinct ‘protected’, ‘susceptible’ or pathological phenotypes will be transfected via a lentiviral vector into a TcR -/- T cell line and undergo functional analyses at Crick. This will include a) Fine specificity mapping using individual peptides b) Flow cytometric analysis of surface molecule expression and cytokine production c) Ability to restrict the growth of M. tuberculosis in macrophages The partner institution for this project is Imperial College London.

References: 1. Esmail, H., Riou, C.R., du Bruyn, E., Lai, R.P-J., Harley, Y.X.R., Meintjes, G., Wilkinson, K.A.,

Wilkinson, R.J. The Immune Response in Tuberculosis in HIV-1-Coinfected Persons Annual Review of Immunology (2018) 36: 603-638 PMID 29490165

2. Riou, C., Berkowitz, N., Goliath, R.T., Burgers, W.A., and Wilkinson, R.J. Analysis of the phenotype of Mtb-specific CD4+ T cells to discriminate latent from active tuberculosis in HIV-uninfected and HIV-infected individuals Frontiers in Immunology (2017) 8:968 PMID 28848561 PMCID PMC5554366

3. Van Der Meeren, O., Hatherill, M., Nduba, V., Wilkinson, R.J., Muyoyeta, M., Van Brakel, E., Ayles, H.M., Henostroza, G., Thienemann, F., Scriba, T.J., Diacon, A., Blatner, G.L., Demoitié, M-A., Tameris, M., Malahleha, M., Innes, J.C., Hellstrom, E., Martinson, N., Singh, T., Akite, E.J., Azam, A.K., Bollaerts, A., Ginsberg, A.M., Evans, T.G., Gillard, P., Tait, D.R. Phase 2b placebo-controlled


trial of M72/AS01E candidate vaccine to prevent active tuberculosis in adults New England Journal of Medicine (2018) DOI: 10.1056/NEJMoa1803484

4. Wilkinson T, Li CKF, Chui CSC, Huang AKY, Perkins M, Julia C Liebner JC, Williams RL, Gilbert A, Oxford J, Dong T, Douek DC, McMichael AJ & Xu XN (2012). Pre-existing influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans. Nature Medicine 18(2):274-80 PMID 22286307

5. Sallin, M.A., Kauffman, K.D., Riou, C.R., Du Bruyn, E., Foreman, T.W., Sakai, S., Hoft, S.G., Myers, T.G., Gardina, P.J, Sher, A., Moore, R., Wilder-Kofie, T., Moore, I.N., Sette, A., Lindestam Arlehamn, C.S., Wilkinson, R.J., Barber, D.L. CD153 expression by CD4 T cells is required for control of pulmonary Mycobacterium tuberculosis infection Nature Microbiology (2018) https://doi.org/10.1038/s41564-018-0231-6 PMID 30202016


Metabolic stratification of heterogenous breast tumours A PhD project for the 2019 doctoral clinical fellows programme with Mariia Yuneva (primary supervisor, Crick), Robert Stein (UCL) and Gyorgy Szabadkai (UCL)

The overarching aim of our research programme is to understand the correlation between transcriptional regulation of nuclear encoded mitochondrial genes, cancer cell bioenergetics and genome scale metabolic networks and tumour biology, progression, architecture and chemosensitivity. We use (i) unbiased approaches, including bioinformatic analysis of genome sequencing, modifications and gene expression data as well as (ii) high content functional imaging of metabolic function on cellular and patient derived organotypic cultures and xenograft mouse models, (iii) quantify metabolic fluxes using isotope labelled metabolites and mass spectrometry. Overall, we aim (i) to establish gene expression based biomarkers for metabolic stratification of tumours to predict tumour progression and ultimately chemosensitivity, and (ii) to identify therapeutic targets in metabolic pathways to develop novel treatment strategies. The PhD studentship will address the following project: Identification and functional characterisation of metabolic subtypes of breast cancer. Here we will validate a novel, predictive biomarker tool based on mitochondrial gene expression (mGEP), which reveals fundamental biology of tumours and thus can more effectively predict pathology and clinical outcome. The algorithm effectively predicts the metabolic phenotype of breast tumours, which in turn informs on their chemosensitivity. The identified clusters of metabolic pathways are characteristic of specific tumour types, which can lead to the identification of novel metabolic pharmacological targets, occurring in specific subgroups of tumours. We have tested and functionally verified the prediction tool in cellular models. Accordingly, the principal objective of the project is to perform validation on human tumours. In addition to the aim to stratify breast cancers using mGEPs as biomarkers, we also plan to identify molecular mechanisms underlying the relationship between mitochondrial activity and nutrient requirements of tumours and develop novel mitochondria/metabolism targeting therapeutic protocols for the treatment of these tumours. We will: a. Classify human breast cancer samples according to their mitochondrial biogenesis patterns driven by specific transcription factors/nuclear receptors and co-regulators. b. Verify the functional mitochondrial and metabolic phenotype in in vivo human tumour xenograft models and ex vivo organotypic cultures using the biochemical and functional imaging platform (UCL) and metabolomics (Crick) facilities to identify possible metabolic targets. c. Identify the functional relationship between mitochondrial and metabolic phenotypes using cellular and in vivo models. d. Identify the correlation between mGEP patterns and chemosensitivity using cellular and in vivo models. The partner institution for this project is UCL.

References: 1. Jones AWE, Yao Z, Vicencio JM, Karkucinska-Wieckowska A, Szabadkai G: PGC-1 family

coactivators and cell fate: roles in cancer, neurodegeneration, cardiovascular disease and retrograde mitochondria-nucleus signalling. Mitochondrion 2012, 12:86–99

2. Yuneva MO, Fan TWM, Allen TD, Higashi RM, Ferraris D V, Tsukamoto T, Matés JM, Alonso FJ, Wang C, Seo Y, Chen X, Bishop JM: The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type. Cell Metab 2012, 15:157–170.

3. Yao Z, Jones AWE, Fassone E, Sweeney MG, Lebiedzinska M, Suski JM, Wieckowski MR, Tajeddine N, Hargreaves IP, Yasukawa T, Tufo G, Brenner C, Kroemer G, Rahman S, Szabadkai G: PGC-1β mediates adaptive chemoresistance associated with mitochondrial DNA mutations. Oncogene 2013, 32:2592–600

4. Eirew P, Steif A, Khattra J, Ha G, Yap D, Farahani H, Gelmon K, Chia S, Mar C, Wan A, Laks E, Biele J, Shumansky K, Rosner J, Mcpherson A, Nielsen C, Roth AJL, Lefebvre C, Bashashati A, Souza C De, Siu C, Aniba R, Brimhall J, Oloumi A, Osako T, Bruna A, Sandoval JL, Algara T, Greenwood W, Leung K, et al.: Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution. Nature 2014, 518:422–426


5. Blacker TS, Mann ZF, Gale JE, Ziegler M, Bain AJ, Szabadkai G, Duchen MR: Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat Commun 2014, 5(May):3936.

2019 phd projects and supervisory teams doctoral fellowships … · 2018-10-03 · 2019 phd...

Documents