ESR01 – Aravind Sankar – based in Cambridge

Project Title: The prioritisation and functional validation of genetic variants that contribute to melanomagenesis.
PhD Award Expected: Spring 2020

As a computer science student in school, I was interested in studying the applications of computers in addressing real-world problems, especially biological ones. This led me to pursue my Bachelor’s degree in Bioinformatics at SASTRA University, India. During this period, I got the opportunity to complete my final year project at the Karlsruhe Institute of Technology, Germany where I worked on the development of web-based platforms used for storing and visualizing experimental microscopy assays. To further refine my understanding of the field , I did my Master’s degree in Bioinformatics at the University of Helsinki, Finland. Concurrently, I worked for a year and a half at the Helsinki Institute for Information Technology as a research assistant. My work involved the design, development and evaluation of a bioinformatics method used to identify bacterial strains in a mixed sample and estimate their individual abundances. The experience I got during this project, which eventually became my thesis, reaffirmed my interest in pursuing a career in bioinformatics research, with a graduate degree being the natural next step in the process.

When I am not working, I like to relax by reading books, playing football and quizzing. I also like to travel extensively whenever possible.


Research Summary

Since the discovery of CDKN2A as the primary driver gene in familial melanoma, several other driver genes have been identified including BAP1, TERT and POT1. However, the germline mutations responsible for more than half of the individuals affected by familial melanoma globally are still unknown. This project aimed at identifying novel germline variants that predispose the individuals and the families carrying these variants to develop familial melanoma. A total of 308 individuals belonging to 133 families of European descent were identified from 9 different institutions across the world. These individuals were sequenced through a combination of exome and whole genome sequencing. Several different procedures were implemented for the analysis of these families, with a brief summary of each approach given below.

The samples that were selected to be studied as part of this project were split into 4 different datasets. The criteria for the sample selection were different for each of these datasets, with different sequencing methodologies applied for each set as well. Once the samples had been sequenced, sequence alignment and joint variant calling were performed uniformly across all 4 datasets.

Once the samples and been sequenced and variant calling had been performed, the next step was to identify rare variants within the dataset. Several quality control filters were applied to eliminate low quality variants. In order to effectively define candidate driver genes, a strategy was developed to determine genes with an increased burden of mutations. Individuals from gnomAD were chosen as a control dataset. Variants in the cases and the controls were filtered using the same workflow. An association analysis was performed on the filtered variants to identify genes with an increased burden of mutations in the cases. In addition to BAP1, candidate genes including MUC4, UBR5, ITGAV and EPHA7 were discovered. To account for the family structure of the samples in the dataset, a joint association-linkage analysis using pVAAST was also implemented. LOD scores were combined with the association scores to generate CLRT scores for every gene which was used to rank the genes, resulting in more novel candidate genes.

Variants in previously identified melanoma driver genes were checked to ensure that the families in the dataset did not carry a nonsense variant in such genes. A mixture of known and novel variants was identified in 8 families in genes including BAP1, BRCA2, CDKN2A, POT1 and MITF. In a second method, the proportion of samples in a pedigree carrying a specific variant were estimated for all variants. Loss-of-function mutations in ATR, TP53AIP1 and EXO5 were found in 10 pedigrees. All of these genes have previously been associated with cancer development and in the case of TP53AIP1 and EXO5, specifically to melanoma development. The third and final secondary analysis of the exonic region variants focussed on determining the presence of known pathogenic variants within the cases. ClinVar, a curated database of variants, their estimated pathogenicity and the associated disorders, was utilised for this purpose. Pathogenic variants in genes associated with oculocutaneous albinism and hereditary cancer syndrome were identified.

A subset of the individuals selected for the project were whole genome sequenced. The availability of variant information across the entire genome allowed for the analysis of both small and large non-coding changes and their potential impact on melanoma oncogenesis; this is an aspect of familial melanoma research that has been relatively unexplored. Two complementary analyses were identified for this purpose. The first approach focussed on variation in the regions of the genome that contained transcription factor binding motifs. Known transcription factor binding motif sites were identified from Ensembl. An association analysis was performed to identify genes with an increased burden of transcription factor binding motif variants. VAV1, SKI and SRC were identified as potential candidates. The second approach centred on the impact of large-scale structural variation on melanoma onset. Insertions, deletions, translocations and duplications were identified on the 123 whole-genome individuals belonging to the pilot whole-genome dataset. Novel structural variants were determined by estimating large overlapping variations that were present in all sequenced members of pedigree. This led to the discovery of a 233,780 base pair deletion upstream of the transcription start site of CDKN2A. This deletion was observed in 10/11 members of a pedigree from Sydney.

In summary, a multipronged approach was utilized to determine novel germline variants in familial melanoma patients affecting both the coding and the non-coding regions of the genome to identify candidate melanoma driver genes. Several candidate genes affecting smaller number of families were discovered across all applied methods.

Publications

Artomov M, Stratigos AJ, Kim I, Kumar R, Lauss M, Reddy BY, Miao B, Robles-Espinoza CD, Sankar A, Njauw C-N, Shannon K, Gragoudas ES, Lane AM, Iyer V, Newton-Bishop JA, Bishop DT, Holland EA, Mann GJ, Singh T, Daly MJ, Tsao H. Rare Variant, Gene-Based Association Study of Hereditary Melanoma Using Whole-Exome Sequencing. J Natl Cancer Inst. 2017 Dec 1;109(12). Available at: https://academic.oup.com/jnci/article/109/12/djx083/3861235

Find out about ESR01 – Aravind Sankar – based in Cambridge's research

ESR02 – Eirini Christodoulou – based in Leiden

Project Title: Using functional studies to explore potential high penetrance melanoma susceptibility genes identified using whole exome and genome sequencing.
PhD Award Expected: Autumn 2020

A journey in scientific research is my dream career pathway! My passion to study Biology developed when my mother was diagnosed with Breast Cancer. During my Undergraduate course at Swansea University I was fascinated by the multi-factorial and complex mode of Cancer Development and undertook my research project in Cancer Chemo-prevention. I then pursued my Master’s degree at The University of Manchester in Cancer Research where I completed two research placements enhancing my laboratory techniques and scientific critique. I also organized my own vacation work experience at Larnaca General Hospital and Karaiskakio Foundation where I became familiar with diagnosis and processing of clinical samples. A highlight of my University life is being the president of Swansea University Dance Society with more than 400 members. Our contribution to charity events was a success with our donations to different organizations and our end-of-year show being dedicated to the Cyprus Anticancer Society, a charity organization dear to me and my mother. I am a young, passionate researcher ready to undertake my PhD project in Melanoma Genetics and I am highly excited to join MELGEN training network and collaborate with fellow scientists! My future goals are to complete my PhD successfully, contribute to the success of MELGEN and discover my opportunities as a Post-Doc!


Research Summary

Cutaneous Melanoma (CM) develops from malignant transformation of melanocytes, the pigment producing cells colonizing our skin. CM is one of the deadliest types of skin cancer due to its high metastatic propensity. There is an estimated 232 100 CM cases diagnosed and about 55 500 (24%) reported deaths annually worldwide [1].

A family history of melanoma has a significant role in determining an individual’s risk of developing the disease. About 10-12% of reported CM cases occur in familial kindreds (Figure 1) therefore, familial (or hereditary) melanoma is arbitrarily defined by the clustering of at least two or more melanomas in first degree relatives [2]. Back in the 90s, scientists have revealed CDKN2A as the first gene responsible for familial clustering of melanoma [3]. A year later, a specific founder mutation has been identified in Dutch melanoma families, known as the p16-Leiden mutation [4]. CDKN2A is the most common melanoma susceptibility gene known today (Figure 1) [5]. Carriers with germline CDKN2A mutations have 70% risk of developing melanoma [6] and an additional 15-20% risk of developing pancreatic carcinoma (PC) [7]. Interestingly, clinical studies suggest variability in occurrence of melanoma and PC within families, indicating that modifying factors may contribute to the risk of developing these two tumor types in p16-Leiden carriers [7]. Determination of genetic risk factors that modulate the risk of PC in p16-Leiden families would therefore allow for a better identification of patients at increased risk that might benefit from personalized clinical management. One of the aims of this project, was to identify genetic factors, responsible for PC risk in p16-Leiden families. We investigated a variable genomic region (SNP) within a high-cancer risk locus as a modifying genetic risk factor for PC and melanoma in p16-Leiden families [8]. We did not find a significant association of PC and melanoma risk with SNP variant presence as determined by statistical analysis [8]. Therefore, genetic modifiers for the prediction of PC and melanoma in p16-Leiden carriers remain to be determined.

Figure 1: Summary of currently identified melanoma susceptibility genes. The first pie chart on the left represents two settings of CM where 90% of cases are found in the general population (sporadic) and 10% report a family history of melanoma (familial). Zooming into the familial setting, several melanoma susceptibility genes have been identified thus far with CDKN2A accounting for most families (40%) and several other genes, each responsible for about <1% of families. There is still a proportion of unknown genetic variants in occurrence of hereditary melanoma. Furthermore, there is probably also a proportion of families (20%) explained by polygenic risk factors; an effect of multiple genes but also environmental risk factors.

Moreover, even though scientific studies provide evidence for CDKN2A bi-allelic loss to be an important event in the transition to invasive melanoma in sporadic cases, there is no evidence about functional inactivation of this tumor suppressor gene in the progression stages of hereditary melanoma [9]. An additional project aim, was to investigate CDKN2A inactivation through loss-of-heterozygosity (LOH) in patients carrying a germline CDKN2A mutation. Application of sensitive digital PCR (dPCR) technology allowed for precise quantification for CDKN2A LOH in primary melanoma and common melanocytic nevi from p16-Leiden mutation carriers. Remarkably, we found CDKN2A LOH to be an early event in the development of familial melanoma.

Since the identification of CDKN2A, other genes have been reported as melanoma susceptibility genes including CDK4 [10], BAP1 [11], MITF [12], TERT [13], POT1 [14], ACD, TERF2IP [15], GOLM1 [16], EBF3 [17] and POLE [18] .These genes account for about 10% of germline variation, however, still about 50% requires investigation (Figure 1). Herein, we aimed to elucidate the genetic basis of familial melanoma and discover novel melanoma predisposition genes by application of whole exome sequencing (WES) analysis. We have identified a novel co-segregating variant, in the checkpoint regulatory gene, NEK11 (Figure 2). We showed somatic loss of the wildtype allele in the melanoma tumor of a NEK11 mutation carrier and demonstrated loss-of-function through protein instability. Therefore, our findings support NEK11 as a candidate melanoma susceptibility gene [19]. This work is absolutely essential to improve genetic testing and counselling of familial melanoma kindreds.

Figure 2: Co-segregation of NEK11 alteration in a Dutch melanoma family. Co-segregation of NEK11 alteration was confirmed by analyzing germline DNA from all family members.

In addition to susceptibility genes, a second class of genes is relevant for the biology and treatment of cancer [20]. These are fitness genes that could serve as therapeutic targets and their identification seems mandatory since relapse is a common event upon initial treatment of CM. In a collaboration with the Welcome Trust Sanger Institute (UK), we aimed to apply CRISPR-Cas9 screening technology and data to identify genetic vulnerabilities in melanoma (Figure 3). Previously reported melanoma fitness genes such as SOX10, BRAF and MITF were among the most significant hits, suggesting that results from this screen analysis are specific for melanoma phenotypes. Remarkably, genetic inhibition of novel fitness genes that are negative regulators of cell growth had a pronounced effect on decreased proliferation in cell-lines, pointing towards possible targets for melanoma therapy.

Figure 3: Schematic overview of a CRISPR-CAS9 knockout screen. The cell-lines of interest (different cancer lineage) are infected by the human library that knocks out all known genes. The endpoint of depleted and enriched genes is read using Next Generation Sequencing technology at days 0 and 14 (or 21) after infection. Those genes that are depleted compared to the initial abundance depict genes essential for cell growth whereas genes that are enriched indicate genes that may serve as possible tumor suppressors.

Collectively, through application of novel genomic techniques in this PhD project, we hope to have explored in detail the genetic mechanisms in familial and sporadic melanoma.

  1. Schadendorf, D., A.C.J. van Akkooi, C. Berking, K.G. Griewank, R. Gutzmer, A. Hauschild, et al., Melanoma. Lancet, 2018. 392(10151): p. 971-984.
  2. Leachman, S.A., J. Carucci, W. Kohlmann, K.C. Banks, M.M. Asgari, W. Bergman, et al., Selection criteria for genetic assessment of patients with familial melanoma. J Am Acad Dermatol, 2009. 61(4): p. 677.e1-14.
  3. Kamb, A., D. Shattuck-Eidens, R. Eeles, Q. Liu, N.A. Gruis, W. Ding, et al., Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nature Genetics, 1994. 8(1): p. 22-26.
  4. Gruis, N.A., P.A. van der Velden, L.A. Sandkuijl, D.E. Prins, J. Weaver-Feldhaus, A. Kamb, et al., Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds. Nature Genetics, 1995. 10: p. 351.
  5. Goldstein, A.M., M. Chan, M. Harland, N.K. Hayward, F. Demenais, D.T. Bishop, et al., Features associated with germline CDKN2A mutations: a GenoMEL study of melanoma-prone families from three continents. J Med Genet, 2007. 44(2): p. 99-106.
  6. Bishop, D.T., F. Demenais, A.M. Goldstein, W. Bergman, J.N. Bishop, B. Bressac-de Paillerets, et al., Geographical variation in the penetrance of CDKN2A mutations for melanoma. J Natl Cancer Inst, 2002. 94(12): p. 894-903.
  7. Vasen, H.F., N.A. Gruis, R.R. Frants, P.A. van Der Velden, E.T. Hille, and W. Bergman, Risk of developing pancreatic cancer in families with familial atypical multiple mole melanoma associated with a specific 19 deletion of p16 (p16-Leiden). Int J Cancer, 2000. 87(6): p. 809-11.
  8. Christodoulou, E., M. Visser, T.P. Potjer, N. van der Stoep, M. Rodriguez-Girondo, R. van Doorn, et al., Assessing a single SNP located at TERT/CLPTM1L multi-cancer risk region as a genetic modifier for risk of pancreatic cancer and melanoma in Dutch CDKN2A mutation carriers. Fam Cancer, 2019.
  9. Shain, A.H., I. Yeh, I. Kovalyshyn, A. Sriharan, E. Talevich, A. Gagnon, et al., The Genetic Evolution of Melanoma from Precursor Lesions. New England Journal of Medicine, 2015. 373(20): p. 1926-1936.
  10. Zuo, L., J. Weger, Q. Yang, A.M. Goldstein, M.A. Tucker, G.J. Walker, et al., Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma. Nature Genetics, 1996. 12(1): p. 97-99.
  11. Wiesner, T., A.C. Obenauf, R. Murali, I. Fried, K.G. Griewank, P. Ulz, et al., Germline mutations in BAP1 predispose to melanocytic tumors. Nature Genetics, 2011. 43(10): p. 1018-1021.
  12. Yokoyama, S., S.L. Woods, G.M. Boyle, L.G. Aoude, S. MacGregor, V. Zismann, et al., A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma. Nature, 2011. 480(7375): p. 99-103.
  13. Horn, S., A. Figl, P.S. Rachakonda, C. Fischer, A. Sucker, A. Gast, et al., TERT promoter mutations in familial and sporadic melanoma. Science, 2013. 339(6122): p. 959-61.
  14. Robles-Espinoza, C.D., M. Harland, A.J. Ramsay, L.G. Aoude, V. Quesada, Z. Ding, et al., POT1 loss-of-function variants predispose to familial melanoma. Nature Genetics, 2014. 46: p. 478.
  15. Aoude, L.G., A.L. Pritchard, C.D. Robles-Espinoza, K. Wadt, M. Harland, J. Choi, et al., Nonsense Mutations in the Shelterin Complex Genes ACD and TERF2IP in Familial Melanoma. JNCI: Journal of the National Cancer Institute, 2015. 107(2): p. dju408-dju408.
  16. Teerlink, C.C., C. Huff, J. Stevens, Y. Yu, S.L. Holmen, M.R. Silvis, et al., A Nonsynonymous Variant in the GOLM1 Gene in Cutaneous Malignant Melanoma. JNCI: Journal of the National Cancer Institute, 2018. 110(12): p. 1380-1385.
  17. Artomov, M., A.J. Stratigos, I. Kim, R. Kumar, M. Lauss, B.Y. Reddy, et al., Rare Variant, Gene-Based Association Study of Hereditary Melanoma Using Whole-Exome Sequencing. J Natl Cancer Inst, 2017. 109(12).
  18. Aoude, L.G., E. Heitzer, P. Johansson, M. Gartside, K. Wadt, A.L. Pritchard, et al., POLE mutations in families predisposed to cutaneous melanoma. Fam Cancer, 2015. 14(4): p. 621-8.
  19. Christodoulou, E., R. van Doorn, M. Visser, A. Teunisse, M. Versluis, P. van der Velden, et al., NEK11 as a candidate high-penetrance melanoma susceptibility gene. J Med Genet, 2019.
  20. Dempster, J.M., C. Pacini, S. Pantel, F.M. Behan, T. Green, J. Krill-Burger, et al., Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets. Nature Communications, 2019. 10(1): p. 5817.

Publications

Christodoulou E, Visser M, Potjer TP, van der Stoep N, Rodriguez-Girondo M, van Doorn R, Gruis N. Assessing a single SNP located at TERT/CLPTM1L multi-cancer risk region as a genetic modifier for risk of pancreatic cancer and melanoma in Dutch CDKN2A mutation carriers. Familial Cancer 2019; 18: 439-444.  Available at: https://link.springer.com/article/10.1007%2Fs10689-019-00137-5

Christodoulou E, van Doorn R, Visser M, Teunisse A, Versluis M, van der Velden P, Hayward NK, Jochemsen A, Gruis N. NEK11 as a candidate high-penetrance melanoma susceptibility gene. J Med Genet. 2020 Mar;57(3):203-210. Available at: https://jmg.bmj.com/content/57/3/203.long

Find out about ESR02 – Eirini Christodoulou – based in Leiden's research

ESR03 – Marina Juraleviciute – based in Oslo

Project Title: iPSC derived melanocytes as in vitro melanoma models
PhD Award Expected: Summer/Autumn 2020

Hi everyone! I am Marina Juraleviciute and I was born and raised in a wonderful city of Vilnius. My interest in natural sciences arose in the high school and after finishing it I entered bachelor studies of Modern Technologies Physics at Vilnius University. As my enthusiasm only grew I finished my master’s in Biophysics at the same alma mater. During the study years I participated in Erasmus student exchange program and went for one semester to study to Ljubljana (Slovenia). I took the chance and participated at Erasmus+ graduate programme. It was a six months placement at The Norwegian Radium Hospital, Institute for Cancer Research. Those were an extremely beneficial and inspiring experiences. Quite recently I became a part of MELGEN network which I am very excited about.

Science is fun and interesting, but there are other things that I enjoy as well. Whenever it is possible I travel and explore new destinations. I am passionate about the photography so these two activities often complement each other. I like reading, there is no particular literature genre I prefer, but I’m hung up on Haruki Murakami’s novels now.


Research Summary

Melanoma is the most aggressive form of skin cancer that arises from dark pigmented cells called melanocytes. Mutation of the BRAF gene is the most common mutation detected in as many as 50% melanomas. Up to 10% of melanoma cases are of familial origin (arise in melanoma-prone families) where alterations in CDKN2A and CDK4 genes account for about 25% of melanomas occurring in such families. However, current knowledge only allows us to explain less than 30% of the genetic susceptibility in melanoma-prone families, which suggests that other genetic and environmental factors might be contributing to the remaining risk. Recent studies have identified new melanoma susceptibility genes by comparing genetic mutations between healthy individuals and melanoma patients. Molecular mechanisms by which these genes alone or together with others influence melanoma susceptibility are still poorly understood. Therefore, my project aimed to investigate how these newly identified susceptibility genes and environmental factors (e.g., UV) are involved in melanoma initiation.

To implement the project, we applied a novel model based on innovative and powerful cell reprogramming technology. Melanoma patients carrying genetic mutations predisposing them to melanoma donated skin biopsies, from which we extracted fibroblasts. We reprogrammed these into stem cells (induced pluripotent stem cells – iPSC) and subsequently differentiated these into melanocytes.  Since iPSC can be cultivated for an infinite time, this approach allows us to obtain an unlimited supply of melanocytes predisposed to melanoma for further extensive studies (Figure 1).

First, we investigated and confirmed that iPSC-derived melanocytes in laboratory settings retain patients’ traits. A cohort of melanocytes was exposed to ultraviolet radiation, a known environmental risk factor for melanoma, and sensitivity was evaluated. We observed a good correlation between melanocyte sensitivity to UV and patients’ pigment factors. Pigmentation factors were calculated by evaluating and scoring a number of self-reported parameters: skin reaction to intense sun exposure, usual skin reaction to sunlight, hair and eye color, the total number of nevi on the body, skin type. Into account was also taken the status of the MC1R gene, which regulates pigmentation pathway. Mutations in this gene are associated with red hair and fair skin and increase UV sensitivity. Furthermore, by investigating UV sensitive and resistant melanocytes, we observed apparent differences in accumulation and repair of UV caused DNA damage. We demonstrated that this model is a novel tool to investigate melanoma initiation as it is more accurate than existing models since it takes into account individual variability in genes.

Next, we focused on one specific risk gene, namely MX2. Previous studies have reported that it is associated with reduced risk to develop melanoma; however, its function in tumorigenesis was utterly unclear. We discovered that the presence of MX2 protein in primary melanoma tumors is associated with better patient survival and that it is often absent in metastatic tumors. To determine the function of the MX2 gene (Figure 2), we engineered melanoma cell lines that contained a high level of MX2 protein and compared their behavior to the corresponding cell lines that had low levels of MX2 protein. Interestingly, cells with a high amount of MX2 protein proliferated much slower than control cells. We injected these melanoma cells into mice to investigate if the MX2 gene is associated with tumor formation. Fifty days after injection, mice that received cells with a low amount of MX2 protein had much bigger tumors compared to the ones that were injected with cells containing high levels of MX2 protein. In this study, we were the first to provide evidence of the MX2 gene function in melanoma by demonstrating that the MX2 gene slows down the growth of cancer cells and has tumor-suppressive properties. These studies improve our understanding of how particular genes and mutations contribute to or prevent melanoma development and might aid in discovering novel therapeutic approaches.

Publications

Juraleviciute M, Pozniak J, Nsengimana J, Harland M,  Randerson-Moor J, Wernhoff P, Bassarova A, Øy G.F, Trøen G, Flørenes VA, Bishop DT, Herlyn M, Newton-Bishop J, Slipicevic A. MX 2 is a novel regulator of cell cycle in melanoma cells. Pigment Cell Melanoma Res. 2019; 00: 1– 12. Available at: https://onlinelibrary.wiley.com/doi/full/10.1111/pcmr.12837

Find out about ESR03 – Marina Juraleviciute – based in Oslo's research

ESR05 – Joanna Pozniak – based in Leeds

Project Title: Gene expression variation in primary melanoma in immunological pathways related to survival.
PhD awarded: March 2019

I have obtained a BSc degree in Molecular Biology, and an International Master degree in Medical Biotechnology, co-funded by the European Union within the European Social Fund, at the Adam Mickiewicz University (AMU) in Poznan, Poland.

I have been interested in cancer since I started my studies. Therefore, I took part in many activities to gather and share knowledge about cancer. I have taken part in conferences in Poland and abroad. I founded a Student Association Section of Molecular Cancer Research aiming at informing students and teenagers via organizing lectures/quizzes/laboratory performances in various science festivals.

There is not only science, which amazes me in this world. I love to explore nature gifts such as wind and snow by windsurfing and snowboarding. I enjoy spending time at the sea side and in the mountains with my friends and family, especially with my one-year-old son Hugo. Additionally, from time to time I try to play drums, which raises many protests from my family members.


Project Summary

I completed my PhD at the University of Leeds (UK) in the framework of a Marie Skłodowska-Curie Early Training Network (MELGEN). My PhD project focused on analyses of gene expression, DNA copy number, and clinical data from primary (very early stage) cutaneous melanoma samples from one of the largest primary melanoma cohorts – Leeds Melanoma Cohort (LMC) (Pozniak et al, 2019). The aim of my project was to improve our understanding of immune responses to primary, treatment naïve melanoma using various bioinformatic, statistical, and computational methods, and some laboratory techniques. Using gene expression data, I created immune cell scores and classified patients based on their expression intensity. I have identified three immune subgroups of the patients, which were associated with prognosis and various clinico-pathological factors. I have also interrogated these patients’ subgroups using environmental exposures, such as smoking and showed that immune responses within tumors are not protective for melanoma patients if one ever smoked cigarettes.

For my PhD project, I have been utilizing software packages such as STATA and R to perform statistical and bioinformatics analysis of gene expression, copy number and clinical data. I did not receive training in bioinformatics prior my PhD, hence I have been learning all the in-silico approaches during the PhD period. In order to learn more about data handling and processing, I undertook an internship with the Eagle Genomics company on the Welcome Trust Genome Campus, in Cambridge UK. During my doctoral studies, I have learnt the basics of high-performance computing and the Unix operating system. Additionally, to my main PhD goals I have also performed some gene wide association study (GWAS) analysis to find germline single nucleotide polymorphisms (SNPs) associated with immune cell score imputed using gene expression data from melanoma tumors.

Regarding laboratory work, I was involved in the project focusing on analysis of copy number data from formalin paraffin embedded (FFPE) melanoma samples, for which I have performed Multiplex Ligation-independent Probe Amplification (MLPA) and analyzed the results (Filia et al, 2019). Moreover, I performed immunohistochemical staining for several proteins of interest that have been chosen (based on gene expression data) as candidates associated with immune responses to melanoma. I also evaluated the staining intensity and location using light microscopy.

To meet the aims of my PhD project, I collaborated with scientists from the host institution as well as with researchers from the University of Zurich. Additionally, I collaborated with researchers from Indian Institute of Science in India, for whom I have used the LMC gene expression and survival data to validate the results obtained from their in-vitro experiments (Metri et al, 2017). I have also performed analyses of gene expression and copy number data of the LMC for Prof. David Fisher (Harvard Medical School, USA). This collaborative work is currently under revision in Cancer Discovery. Lastly, I have performed some statistical analysis using gene expression and single nucleotide pleomorphism data for a study with scientists from Oslo University Hospital in Norway, which was recently published in Pigment Cell & Melanoma Research (Juraleviciute et al, 2019).

Publications

Metri R, Mohan A, Nsengimana J, Pozniak J, Molina-Paris C, Newton-Bishop J, Bishop D, Chandra N. Identification of a gene signature for discriminating metastatic from primary melanoma using a molecular interaction network approach. Sci Rep. 2017 Dec 11;7(1):17314. Available at: https://www.nature.com/articles/s41598-017-17330-0

Nsengimana J, Laye J, Filia A, O’Shea S, Muralidhar S, Poźniak J, Droop A, Chan M, Walker C, Parkinson L, Gascoyne J, Mell T, Polso M, Jewell R, Randerson-Moor J, Cook GP, Bishop DT, Newton-Bishop J. β-Catenin-mediated immune evasion pathway frequently operates in primary cutaneous melanomas. J Clin Invest. 2018 May 1;128(5):2048-2063. Available at: https://www.jci.org/articles/view/95351

Poźniak J, Nsengimana J, Laye JP, O’Shea SJ, Diaz JMS, Droop AP, Filia A, Harland M, Davies JR, Mell T, Randerson-Moor JA, Muralidhar S, Hogan SA, Freiberger SN, Levesque MP, Cook GP, Bishop DT, Newton-Bishop J. Genetic and Environmental Determinants of Immune Response to Cutaneous Melanoma. Cancer Res. 2019 May 15;79(10):2684-2696 Available at: https://cancerres.aacrjournals.org/content/79/10/2684

Filia A, Droop A, Harland M, Thygesen H, Randerson-Moor J, Snowden H, Taylor C, Diaz JMS, Pozniak J, Nsengimana J, Laye J, Newton-Bishop JA, Bishop DT. High-Resolution Copy Number Patterns From Clinically Relevant FFPE Material. Sci Rep. 2019 Jun 20;9(1):8908. Available at: https://www.nature.com/articles/s41598-019-45210-2

Juraleviciute M, Pozniak J, Nsengimana J, Harland M,  Randerson-Moor J, Wernhoff P, Bassarova A, Øy G.F, Trøen G, Flørenes VA, Bishop DT, Herlyn M, Newton-Bishop J, Slipicevic A. MX 2 is a novel regulator of cell cycle in melanoma cells. Pigment Cell Melanoma Res. 2019; 00: 1– 12. Available at: https://onlinelibrary.wiley.com/doi/full/10.1111/pcmr.12837

Thakur R, Laye JP, Lauss M, Diaz JMS, O’Shea SJ, Poźniak J, Filia A, Harland M, Gascoyne J, Randerson-Moor JA, Chan M, Mell T, Jönsson G, Bishop DT, Newton-Bishop J, Barrett JH. Transcriptomic Analysis Reveals Prognostic Molecular Signatures of Stage I Melanoma. Clin Cancer Res. 2019 Dec 15;25(24):7424-7435. Available at: https://clincancerres.aacrjournals.org/content/25/24/7424

Find out about ESR05 – Joanna Pozniak – based in Leeds's research

ESR06 – Sathya Muraldihar – based in Leeds

Project Title: Establishing the role of smouldering inflammation in suppression of adaptive immunity and therefore survival from melanoma.
PhD awarded: December 2019

I have been fascinated by cancer ever since I realised that it is caused not by foreign germs, but by rogue cells within our own selves. This fascination led me to major in Cancer Biology at the German Cancer Research Centre in Heidelberg, thus helping me understand the disease better. Fascination eventually became an obsession, motivating me to conduct cancer research as part of my PhD. Being part of MELGEN, my research aims to understand how melanoma is influenced by genetic factors as well as our own immune system, thus leading to the possibility of more effective detection and treatment choices for melanoma patients.

When I’m not dabbling with science I enjoy cooking, painting, travelling and practicing yoga. I am also a movie buff and an ardent science fiction fan, which often help me realise that facts and fiction are not very far apart!


Research Summary

My PhD project was to understand the role of vitamin D in melanoma. Previous research suggested that melanoma patients with higher vitamin D levels in their blood were more likely to have less aggressive melanomas and improved survival. However, the mechanism behind this effect was not fully understood. As part of my PhD project, I studied the relationship between patients’ vitamin D levels and their melanomas, by focusing on the genetic material from the melanomas. My research revealed that the effect of vitamin D levels on patient prognosis was strongly linked to the expression of a specific gene: the vitamin D Receptor, also known as VDR. This could be owing to the fact that vitamin D is dependent on VDR in order to be effective. Patients whose tumours expressed high levels of VDR were also likely to have reduced expression of genes that accelerated cancer growth and increased expression of genes involved in immunity. This finding suggested that having high levels of vitamin D and VDR went hand in hand with reduced cancer growth and improved immune response. This could potentially explain why these patients had improved survival over those who had lower levels of vitamin D and VDR. To test if this ‘cause-effect’ relationship was true, I performed laboratory experiments where I artificially increased the production of VDR in melanoma cells grown in the lab. This experiment was meant to mimic the comparison of two sets of patients whose melanomas had either very high or very low VDR expression. The melanoma cells with high VDR production grew slower and were less aggressive compared to cells with low VDR production. This finding was very similar to the effects observed in patients. This led me to conclude that vitamin D levels in the blood and VDR production in melanomas affect the growth of the cancer by forcing it to grow slower and to attract more cancer-killing immune cells. My research could have an impact on how melanoma patients will be treated in the future, taking into account the vitamin D levels in their blood.

Publications

Nsengimana J, Laye J, Filia A, O’Shea S, Muralidhar S, Poźniak J, Droop A, Chan M, Walker C, Parkinson L, Gascoyne J, Mell T, Polso M, Jewell R, Randerson-Moor J, Cook GP, Bishop DT, Newton-Bishop J. β-Catenin-mediated immune evasion pathway frequently operates in primary cutaneous melanomas. J Clin Invest. 2018 May 1;128(5):2048-2063. Available at: https://www.jci.org/articles/view/95351

Poźniak J, Nsengimana J, Laye JP, O’Shea SJ, Diaz JMS, Droop AP, Filia A, Harland M, Davies JR, Mell T, Randerson-Moor JA, Muralidhar S, Hogan SA, Freiberger SN, Levesque MP, Cook GP, Bishop DT, Newton-Bishop J. Genetic and Environmental Determinants of Immune Response to Cutaneous Melanoma. Cancer Res. 2019 May 15;79(10):2684-2696. Available at: https://cancerres.aacrjournals.org/content/79/10/2684

Muralidhar S, Filia A, Nsengimana J, Poźniak J, O’Shea SJ, Diaz JM, Harland M, Randerson-Moor JA, Reichrath J, Laye JP, van der Weyden L, Adams DJ, Bishop DT, Newton-Bishop J. Vitamin D-VDR signaling inhibits Wnt/beta-catenin-mediated melanoma progression and promotes anti-tumor immunity. Cancer Res. 2019 Dec 1;79(23):5986-5998. Available at: https://cancerres.aacrjournals.org/content/79/23/5986.

Find out about ESR06 – Sathya Muraldihar – based in Leeds's research

ESR07 – Sofia Chen – based in Cambridge

Project Title: Dissecting the role of the immune system and stroma in melanoma development
PhD Awarded: January 2020

I am Swedish, but originally from China, and currently doing my PhD in David Adam’s group at the Wellcome Trust Sanger Institute in Cambridge, UK. Before starting my PhD, I completed a M.Sc in biotechnology engineering at Lund University in Sweden, followed by a 2 year industrial research graduate programme (IMED) at AstraZeneca.

Research is my biggest passion, and I am particularly interested in the connection between cancer and immunology. I want to understand how tumours manage to escape the immune system, and my hope is that this knowledge can lead to development of new drugs for treatment of cancers.

Outside of science, I spend a lot of time in the kitchen, experimenting with new recipes or exploring different combinations.What also gives me lots of energy and positive endorphins is exercising; whether it is the gym, classes, running or spinning.


Research Summary

The primary goal of my project was to understand how genetic alterations (changes in DNA) shape melanoma as a disease: from the biological processes that govern tumour development to how tumours evolve and escape immune regulation. Studying these changes could therefore help refine our understanding of melanoma progression with the added potential to offer new perspectives for disease management.

Due to the significant diversity present across melanomas, it is important to study the genetic differences between different patients’ tumours.  This can be done, for instance, by comparing which genes or biological pathways are commonly mutated, by studying histopathological subtypes or degree of sun exposure. Ultimately, genetic changes could also have a prognostic impact, which could help researchers understand what factors play a role in making a tumour particularly aggressive and assist  doctors in deciding what treatments to prescribe any given patient.

We have learnt a great deal about melanoma genetics through large collaborative studies in the past. However, most of these projects have focused on advanced disease. Thus, genetic alterations that influence the behaviour of early-stage tumours have not been fully explored, and we still have much to learn. Consequently, with my project I have analysed a large collection of 524 tumours, the first large comprehensive study on primary melanomas. Therefore, my work could help us understand how cancers evolve and are regulated in their earliest stages.

With my project, I present the architecture of primary melanomas where most tumours are bursting with mutations, often associated with sun damage as a result of sun exposure. I confirmed and presented new candidate genes potentially important in driving melanoma. I also describe additional genes which could have critical roles in central biological pathways responsible for promoting tumour development.

As an example, most melanomas have an activating BRAF mutation, an alteration that promotes tumour growth. I found that a proportion of melanomas could rely on alternative switches, activating the same biological pathway through mutations in EGFR instead of BRAF. These patients might therefore benefit from existing EGFR inhibitor treatments. A second example proposes a role of PRDM2 in cell-cycle regulation, linking to CDKN2A, an important melanoma tumour suppressor gene and a chief component in controlling tumour development.

When tumours progress, they might develop dependencies on particular genes or pathways in order to survive. In my project, I identified such a dependency, where some melanomas harbour amplifications of the IRF4 gene. Subsequently, when the expression of this gene is lost, these tumours cannot survive. Thus, my results suggest that a subset of melanomas would be sensitive to drugs that block expression of this gene.

My outcomes also suggest that an abundance of genetic alterations, known to be associated with a favourable immunotherapy response, do not in fact have a significant survival benefit for patients not receiving immunotherapy. I therefore conclude that an active immune system is required to combat cancer, emphasising the impact of immunotherapy drugs in the treatment of melanoma.

In my second project, I sought to explore the potential of using our immune system to fight cancer. Checkpoint inhibitors, including anti-PD1 and anti-PD-L1 therapies, have revolutionised melanoma care, yet only a minority of patients respond to these treatments. Our comprehension of how PD-L1 expression on melanoma cells is regulated is still limited. By improving our understanding of these processes, we can envision tumour counteractive mechanisms as well as strive to identify new drug targets.

I employed a genome-wide CRISPR-Cas9 screening approach and identified several pathways encompassing the “life cycle of PD-L1”. A second extensive screen validate these findings in eight cancer cell lines of melanoma, bladder and lung cancer origin, and link novel candidate genes to the control of PD-L1 tumour expression. With these results, we learnt that the expression levels of PD-L1 on the surface of tumour cells are carefully monitored through central processes including TAF transcriptional regulators, N-linked glycosylation mediated by an array of ALG proteins and the OST complex, as well as intracellular transport processes governed by TRAPP, COG and HOPS complexes. In addition, my results also suggest a role for epigenetic and cell-cycle regulators in controlling PD-L1 expression, highlighting possible new drug combinations that could be explored in melanoma. Regulation through SPNS1 was also a major finding, providing a novel therapeutic approach by simultaneously targeting PD-L1 expression as well as autophagic processes.

In conclusion, I hope that the knowledge gained through the results of my project will further our comprehension of melanoma progression and diversity, as well as bringing forth novel insights and possible treatment modalities.

Publications

Currently in preparation…

Find out about ESR07 – Sofia Chen – based in Cambridge's research

ESR08 – Catarina Salgado – based in Leiden

Project Title: Examining tumour cell heterogeneity in melanoma metastasis using single cell genomic technologies.
PhD Award Expected: Summer 2020

I was born in Porto, Portugal’s 2nd largest city, best known for its unique historical landscape, exquisite cuisine and world-renowned wines.

Back when I was 8-years old, my teacher brought a book with biographies of historical characters. That day, my homework was to do a summary of Marie Curie’s life. This homework brought to life my dream of becoming a scientist in awe of Curie’s perseverance. Eventually, the dream came true, and I applied for a First Degree in Biology and afterwards to a Master Degree in Molecular Oncology granted by University of Porto. In 2011, I joined Ipatimup as a trainee, to develop my master’s thesis, focused on familial thyroid carcinoma. Alongside I have also contributed to other oncobiology research projects at Ipatimup. These first steps into scientific research showed me how challenging yet fulfilling this world can be. Consistent with my professional goals, I applied and was accepted into MELGEN network as a PhD student.

Apart from science, spending time with family and friends and finding new and quaint restaurants are my favourite hobbies. I also enjoy travelling and reading. Sports are also an essential part of my life as a way of managing the daily stress.


Research Summary

Catarina Salgado is working at Leiden (LUMC, The Netherlands). Her PhD project focuses on the epigenetics of melanoma.

Schematic representation of the development and progression of melanoma
Figure 1. Schematic representation of the development and progression of melanoma. This figure was built using Servier Medical Art images (https://smart.servier.com/)

Melanoma is the most aggressive and lethal type of skin cancer since it has the ability to spread to other organs in the body making it harder to control the disease.

It is very common to hear about cells, chromosomes, DNA, genes, and alterations when we talk about cancer. The chromosomes are confined to the nucleus of our cells and consist of long strands of DNA containing many genes. The most common alterations described in cancer are related to the DNA sequence, the so-called mutations, changes that impair the correct proteins to be produced. The latter may have different implications for the cell and can for instance predispose to cancer.

What does epigenetics stand for?

Mutations are alterations in the DNA strand itself. However, the components of the DNA strand can stay unchanged but be affected by many modifications triggering a similar impact in the generation of  a new (and defect?) protein. The set of these modifications affecting the “behaviour” of the DNA components is called “epigenetics”. The Greek prefix “epi” means “on top of” or “in addition to” the traditional genetics. The term “epigenetics” was originally proposed by Conrad Waddington in 1942 to describe the molecular mechanisms independent of alterations in the DNA sequence. There are three main epigenetic mechanisms: DNA methylation, histone modification and chromatin remodeling.

In this PhD project we aim to explore the degree to which epigenetics play a role in melanoma progression and survival.

Structure of DNA
Figure 2. Structure of DNA. The chromosomes are confined to the nucleus of the cells and consist of long strands of DNA containing many genes. Chromosomes have proteins called histones that bind to DNA. DNA is made up of four components called nucleotides (yellow, red, blue and purple). DNA methylation (m) and DNA hydroxymethylation (hm) are the epigenetic alterations represented [Adapted from © 2015 Terese Winslow LLC, U.S. Govt. has certain rights and Dictionary of Cancer Terms PDQ Cancer Information Summaries, National Cancer Institute (US)].

Therefore, we explore the above-mentioned DNA methylation (m in Fig.2) and its opposite process, DNA demethylation, also known as hydroxymethylation (hm in Fig.2), in the entire genome of benign (normal naevus) and malignant samples (melanoma) of different patients. The intent is to broaden knowledge of the degree to which methylation modifies key biological mechanisms and survival of cells and which processes are therefore potentially modifiable. In this project, we were able to identify specific sites/regions in the genome that might be valuable indicators to help the diagnosis along with regions localized in genes that might contribute to development of melanoma.

Additionally, we also studied the role of a mutation in melanoma development. This alteration in the DOT1L gene was found in a family with many melanoma-affected cases. We performed many cell-based experiments to see which biological processes were affected by adding the desired alteration in these cells. We conclude based on our and previous results, that the study of this alteration deserves further study, since mutations in DOT1L gene seem to affect UV exposure sensitivity, which may enhance malignant transformation.

Another project aims to assess whether the epigenetic alterations can be the cause of melanoma in a great proportion of the familial cases for which a genetic cause has not been identified. Different epigenetic mechanisms were investigated in the DNA from 2 melanoma-affected family members from 5 families. Our results show that heritable DNA methylation alterations are not likely to be a cause of familial melanoma.

Since both genetic and epigenetics modifications often ‘work’ side-by-side to trigger malignant transformation, in our last project we explored the combined contribution of epigenetics (DNA methylation and chromatin remodelling) and genetic (promoter mutations) mechanisms in regulating an important gene involved in cancer, TERT gene. TERT can prevent cell death and promote uncontroled proliferation, two important characteristics of cancer cells. We could observe a complex interplay among mutations, methylation and chromatin organization that explains the distinct biological origins of healthy/benign samples and tumour samples.

Publications

Salgado C, Kwesi-Maliepaard EM, Jochemsen AG, Visser M, Harland M, van Leeuwen F, van Doorn R, Gruis N. A novel germline variant in the DOT1L gene co-segregating in a Dutch family with a history of melanoma. Melanoma Res. 2019; 29(6):582-589.

Salgado C, Oosting J, Janssen B, Kumar R, Gruis N, van Doorn R. Genome-wide characterization of 5-hydoxymethylcytosine in melanoma reveals major differences with nevus. Genes Chromosomes Cancer. 2020 Feb 3. Available at: https://onlinelibrary.wiley.com/doi/full/10.1002/gcc.22837

Salgado C, Gruis N, BIOS Consortium, Heijmans BT, Oosting J, van Doorn R. Genome-wide analysis of constitutional DNA methylation in familial melanoma. Clinical Epigenetics 2020. Mar 6; 12(43) https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-020-00831-7

Find out about ESR08 – Catarina Salgado – based in Leiden's research

ESR09 – Shamik Mitra – based in Lund

Project Title: Establishing the clinical relevance of gene expression phenotypes identified in melanomas
PhD Award Expected: Summer 2020

I am a Marie Curie ESR fellow in Melanoma Genomics under Prof. Dr. Göran Jönsson at Lund University, Sweden. A bioinformatician by training, my research interest lies in the field of data science and its application to solve clinical research problems. I am particularly interested about investigating links between genetic and epigenetic modifications associated with cancer.

My bachelor studies in Biomedical Engineering familiarised me with the impact of medical science and technology on daily life. At postgraduate level, I wanted to go deep into the cellular and molecular level understanding of clinical research problems from computational perspective. My postgraduate studies in Bioinformatics (a one year diploma and a subsequent master’s programme) have provided me the suitable platform for that.

Apart from my academic activities, I enjoy reading both International and Indian literature (Franz Kafka to Dan Brown, whichever I can get hold of!). Poetry has been a long time love for me, especially poems of John Keats. And last but not the least, I am a gourmet and always try to experiment with dishes from different cuisines!


Research Summary

Cancer is probably one of the oldest known diseases to humanity, however it is nothing more than an umbrella term for a group of diseases that are as diverse as countries inside a continent.

Malignant melanoma is a type of cancer which arises from a group of pigment producing cells in our body known as melanocytes. Majority of melanomas are attributed to skin and termed as cutaneous melanoma, nevertheless melanomas can occur in other unrelated organs such as eye and digestive system [1,2].

My PhD project as ESR 9, focused on understanding the molecular diversity that exists within the cutaneous melanoma and how such information can be utilized to select patients for different available treatment options. Now, when we imagine a tumor, we think of it as a cluster of malignant cells. However, tumors in reality consists different other types of cells and these cells contribute to the responsiveness and aggressiveness of the tumor itself. Thus, when deciding on the treatment for a particular melanoma tumor, it will be important to know which other types of non-cancerous cells are present there and how they assist or oppose a particular type of therapy. In my research, I primarily aimed to understand the roles of different immune cells present inside melanoma tumors and how such presence translates into better prognosis for the patients.

Figure 1: Combination of mutational and predicted neo- antigen load with immune system activation, associate with better treatment benefit. Adapted from Lauss et al. 2015 [3]

In the first project of my PhD [3], I worked alongside other researchers from our lab and our international collaborators to identify suitable markers for response to adoptive T-cell therapy (ACT). ACT is one of the newer innovative treatments for advanced-stage cancer patients, where patient’s own immune cells are energized externally to fight the tumor. We observed that having more mutations in the tumor not only translates into clinical benefit from the treatment but also prolongs the patient’s survival (Figure 1A). Similarly, it was noticed that tumor’s ability to generate neoantigens which are protein fragments that likely elicit a response from the immune system, associated strongly with treatment benefit and patient survival (Figure 1B). Additionally, we observed the enrichment of immune system associated genes in the tumors of patients who benefited from the therapy (Figure 1C). Overall, we can speculate that a combination of patient tumor mutational and neoantigen count/load along with the immune enrichment would be a good marker for patient selection in ACT.

Figure 2: Distinct immune-methylation clusters reflect the immune-enrichment in metastatic melanoma tumours and associate with the patient survival

In another project where I worked as a main researcher, we sought to explore how different immune cells enrich in the metastatic melanoma tumors and their association with patient prognosis. Using a major epigenetic modification known as DNA methylation (modification without altering the genomic units of DNA), metastatic melanoma tumors were grouped into 3 distinct clusters based on their immune-methylation characteristics (Figure 2A). Using other metastatic melanoma datasets, these clusters were further validated (Figure 2B) and they reported a statistically significant association with the melanoma-specific survival for the patients (Figure 2C). Overall, we can suggest that these immune-methylation clusters hint at a consensus in the immune enrichment of melanoma tumors and might offer useful guidance to therapies.

Treating melanoma has found new directions in the 21st century with emergence of new innovative therapies, such as immunotherapies and targeted treatments. More and more we are being able to treat advanced-stage patients, which was not possible earlier. I hope that our research shall help significantly to uncover the hidden faces of melanoma and shall broaden the horizon of current therapies.

  1. Simons M, Ferreira J, Meunier R, et al: Primary versus Metastatic Gastrointestinal Melanoma: A Rare Case and Review of Current Literature. Case Rep Gastrointest Med 2016:2306180, 2016
  2. Kaliki S, Shields CL: Uveal melanoma: relatively rare but deadly cancer. Eye 31:241-257, 2017
  3. Lauss M, Donia M, Harbst K, et al: Mutational and putative neoantigen load predict clinical benefit of adoptive T cell therapy in melanoma. Nature Communications 8:1738, 2017

Publications

Lauss M, Donia M, Harbst K, Andersen R, Mitra S, Rosengren F, Salim M, Vallon-Christersson J, Törngren T, Kvist A, Ringnér M, Svane IM, Jönsson G. Mutational and putative neoantigen load predict clinical benefit of adoptive T cell therapy in melanoma. Nat Commun. 2017 Nov 23;8(1):1738. Available at: https://www.nature.com/articles/s41467-017-01460-0

Betancourt* LH, Pawlowski* K, Eriksson* J, A. Szasz* M, Mitra S, Pla I, Welinder C, Ekedahl H, Broberg P, Appelqvist R, Yakovleva M, Sugihara Y, Miharada K, Ingvar C, Lundgren L, Baldetorp B, Olsson H, Rezeli M, Wieslander E, Horvatovich P, Malm J, Jönsson G, Marko-Varga G. Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data. Sci Rep. 2019 Mar 26;9(1):5154. * Equal contribution. Available at: https://www.nature.com/articles/s41598-019-41625-z

Sanna A, Harbst K, Johansson I, Christensen G, Lauss M, Mitra S, Rosengren F, Häkkinen J, Vallon-Christersson J, Olsson H, Ingvar Å, Isaksson K, Ingvar C, Nielsen K, Jönsson G. Tumour genetic heterogeneity analysis of chronic sun-damaged melanoma. Pigment Cell Melanoma Res. 2019 Dec 7. Available at: https://onlinelibrary.wiley.com/doi/full/10.1111/pcmr.12851

Cabrita R, Lauss M, Sanna A, Donia M, Skaarup Larsen M, Mitra S, Johansson I, Phung B, Harbst K, Vallon-Christersson J, van Schoiack A, Lövgren K, Warren S, Jirström K, Olsson H, Pietras K, Ingvar C, Isaksson K, Schadendorf D, Schmidt H, Bastholt L, Carneiro A, Wargo JA, Svane IM, Jönsson G. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature. 2020 Jan;577(7791):561-565. Available at: https://www.nature.com/articles/s41586-019-1914-8

Mitra S, Lauss M, Cabrita R, Choi J, Zhang T, Isaksson K, Olsson H, Ingvar C, Carneiro A, Staaf J, Ringnér M, Nielsen K, Brown KM, Jönsson G. Analysis of DNA methylation-based tumour immune microenvironment patterns in metastatic melanoma. Mol Oncol. 2020 Mar 9; doi:10.1002/1878-0261.12663. https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12663

Find out about ESR09 – Shamik Mitra – based in Lund's research

ESR10 – Adriana Sanna – based in Lund

Project Title: Establishing the role of MITF and related genes in melanoma survival and development
PhD Award Expected: Summer 2020

I’m Adriana and I come from the beautiful island of Sardinia, in Italy. I got my bachelor in Biology in Milan, and afterwards I spent some great time in London while applying for universities abroad. In 2013, I was delighted to receive the acceptance at the Leiden University Medical Center, where I’m graduating in Biomedical Sciences Research. As one of the most prestigious universities in The Netherlands, I personally appreciated its diverse and excellent environment, without to mention that is an astonishing country.

For my final thesis I also had the chance to work in the popular city of Los Angeles (US); I had great time in lab as well as visiting California during spare time. Such stimulating environment reinforced my propensity to continue as PhD in a foreign country. So, once back to Europe, I was particularly proud to have been chosen as member of MELGEN, which is giving me the exciting possibility to keep on working in Lund, Sweden, for the next years of my career.

Of course, I couldn’t do anything without the constant support of my loyal travel-buddy, MaryJane; who, after an year of full summer time in California, is still quite in doubt about how Swedish winter looks like..!


Research Summary

Data for this project is currently under embargo pending publication in an open-access peer-reviewed journal and will be updated here following publication.

Publications

Phung B, Cieśla M, Sanna A, Guzzi N, Beneventi G, Cao Thi Ngoc P, Lauss M, Cabrita R, Cordero E, Bosch A, Rosengren F, Häkkinen J, Griewank K, Paschen A, Harbst K, Olsson H, Ingvar C, Carneiro A, Tsao H, Schadendorf D, Pietras K, Bellodi C, Jönsson G. The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma. Cell Rep. 2019 Jun 18;27(12):3573-3586.e7. Available at: https://www.sciencedirect.com/science/article/pii/S2211124719306989

Sanna A, Harbst K, Johansson I, Christensen G, Lauss M, Mitra S, Rosengren F, Häkkinen J, Vallon-Christersson J, Olsson H, Ingvar Å, Isaksson K, Ingvar C, Nielsen K, Jönsson G. Tumour genetic heterogeneity analysis of chronic sun-damaged melanoma. Pigment Cell Melanoma Res. 2019 Dec 7. Available at: https://onlinelibrary.wiley.com/doi/full/10.1111/pcmr.12851

Cabrita R, Lauss M, Sanna A, Donia M, Skaarup Larsen M, Mitra S, Johansson I, Phung B, Harbst K, Vallon-Christersson J, van Schoiack A, Lövgren K, Warren S, Jirström K, Olsson H, Pietras K, Ingvar C, Isaksson K, Schadendorf D, Schmidt H, Bastholt L, Carneiro A, Wargo JA, Svane IM, Jönsson G. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature. 2020 Jan;577(7791):561-565. Available at: https://www.nature.com/articles/s41586-019-1914-8

Find out about ESR10 – Adriana Sanna – based in Lund's research

ESR11 – Rita Cabrita – based in Lund

Project Title: Transcriptional modules exposing related therapeutic vulnerabilities in melanoma
PhD Award Expected: Summer 2020

I am from a country where you can taste the greatest custard pastry, listen to Fado, get crazy about football and enjoy great weather all year round: Portugal.

I did a Masters in Molecular Biology and Genetics in the Faculty of Sciences, University of Lisbon. I developed my thesis in the European Molecular Biology Laboratory in Italy, in chromosomal instability and aneuploidy.

I bumped into science because of my dad, who surrounded me with biology books and scientific magazines, allowing me to realize how amazing this field is. From then onwards my academic and professional career have been developing like a snowball, until I ended up having the great opportunity to join MELGEN network.

I am passionate about almost all kinds of science, but molecular science and particularly cancer amaze me.

But my life is not only about science and I like to enjoy whatever it has to offer. In my free time I like to travel, hang out with my family and friends, play sports, such as basketball and swimming, go to the movies, read, and, coming from Portugal, it would be impossible not to say this part: eat good food!


Research Summary

Melanoma is one of the most immunogenic tumor diseases with known cases of spontaneous tumor regression and frequent presence of tumor infiltrated lymphocytes (TILs). For this reason, melanoma cells provide a suitable model to investigate the molecular crosstalk of cancer cells with cells of the immune system. Advances in this field have allowed the development of efficient therapeutic strategies and a major revolution in the clinical management of metastatic melanoma patients with introduction of immune checkpoint blockade (ICB) as primary choice of therapy. The main ICB modalities used in the clinical setting are targeting immune checkpoint molecules, such as CTLA-4 and PD1, in order to prevent the T cell inhibitory signals mediated by these molecules, and thus boost the immune response against the tumor cells. Although clinical benefit is frequent, still about 60% of patients develop primary resistance, whereas others experience initial clinical response and later on develop secondary resistance.

Considering this, this project aimed to understand the role of different immune cell subsets and of the immune system as a whole, in melanoma, to define immune profiles and their prognostic value in metastatic melanoma tumors and finally, to derive gene expression signatures applicable to tumors from patients receiving targeted therapy and immunotherapy.

Four works were developed, in order to achieve these aims:

In study I we have used a cohort of metastatic melanomas collected prior to checkpoint inhibitor era and found that the presence of intra-tumoral lymphocytes is associated with improved survival. The presence of CD20+ B cells was frequently concomitant with CD3+/CD8+ T cells, indicating the formation of tertiary lymphoid structures (TLS). The TLS gene signature predicted clinical outcome in patients treated with immune checkpoint inhibitors. We were able to identify the presence of activated and immature B cells in these structures, which supports their functional role in melanoma tumors.

In study II we have used immune cell type associated DNA methylation based signatures on a large cohort of metastatic melanoma tumors. We found three distinct immune clusters for metastatic melanoma that significantly associates with patient survival. Additionally, these DNA methylation signatures showed prognostic implications in other solid tumor cohorts.

In study III we aimed to understand the role of PTEN in prognosis, therapy response and immune escape in melanoma. We confirmed in our cohort that PTEN alterations promote immune evasion highlighted by decreased frequency of T cell infiltration in such tumors, resulting in a worse patient survival. More importantly, our results suggest that dedifferentiated PTEN negative melanoma tumors have poor patient outcome, no T cell infiltration and transcriptional properties rendering them resistant to targeted- and immunotherapy.

In study IV, we aimed to identify immune cell subtypes and its associated markers using scRNA-Seq data and used these markers to create a composite score to predict treatment response in the tumor-derived bulk RNA-Seq data. We have used single-cell RNA-Seq derived immune signatures representing several immune cell populations, and applied them to different datasets, confirming their prognostic value in metastatic melanoma.

Overall, these four projects suggest that distinct molecular and transcriptional properties may predict poor clinical benefit of both targeted therapy and immune checkpoint blockade agents in melanoma.

Publications

Phung B, Cieśla M, Sanna A, Guzzi N, Beneventi G, Cao Thi Ngoc P, Lauss M, Cabrita R, Cordero E, Bosch A, Rosengren F, Häkkinen J, Griewank K, Paschen A, Harbst K, Olsson H, Ingvar C, Carneiro A, Tsao H, Schadendorf D, Pietras K, Bellodi C, Jönsson G. The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma. Cell Rep. 2019 Jun 18;27(12):3573-3586.e7. Available at: https://www.sciencedirect.com/science/article/pii/S2211124719306989

Cabrita R, Lauss M, Sanna A, Donia M, Skaarup Larsen M, Mitra S, Johansson I, Phung B, Harbst K, Vallon-Christersson J, van Schoiack A, Lövgren K, Warren S, Jirström K, Olsson H, Pietras K, Ingvar C, Isaksson K, Schadendorf D, Schmidt H, Bastholt L, Carneiro A, Wargo JA, Svane IM, Jönsson G. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature. 2020 Jan;577(7791):561-565. Available at: https://www.nature.com/articles/s41586-019-1914-8

Mitra S, Lauss M, Cabrita R, Choi J, Zhang T, Isaksson K, Olsson H, Ingvar C, Carneiro A, Staaf J, Ringnér M, Nielsen K, Brown KM, Jönsson G. Analysis of DNA methylation-based tumour immune microenvironment patterns in metastatic melanoma. Mol Oncol. 2020 Mar 9; doi:10.1002/1878-0261.12663. https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12663

Find out about ESR11 – Rita Cabrita – based in Lund's research

ESR12 – Rohit Thakur – based in Leeds

Project Title: Developing statistical and bioinformatic analysis of genomic data from tumours
PhD Awarded: January 2019

Deploying the concepts from mathematics and applying to understand biological pathways and expression networks is an interesting concept. This interest grew in me while pursuing the undergraduate course in Bioinformatics at VIT University, India. An aspiring computational biologist also require interdisciplinary knowledge of biochemical concepts as well as, needs to be adept in computational and programming proficiency. Research experiences at Alpha Net Technologies (India), IISER Pune (India) and EMBL-EBI (UK), provided me with the opportunity to acquire this proficiency.

I always look forward to become part of intellectual discussion groups and had the experience of working for an initiative called RSG India that promotes networking amongst computational biologists in India. I am an active sports player and play cricket and basketball. I also seek to help humanity and have been a part of the NGO named “MAD” (Make A Difference) helping underprivileged students by guiding them to pursue their dream. Experiences inside and outside the lab helped me recognize my ‘driving force’ and career aspirations. My research interest lies broadly in the area of cancer genomics. I look forward to a career as a researcher developing algorithms and designing virtual diagnostic systems which would impact healthcare treatments in the long run.


Research Summary

During my PhD, I developed prognostic gene signatures using genomic datasets from one of the largest primary melanoma cohorts to date. Using unsupervised classification approach, I devised a transcriptome-based molecular signature which predicts prognosis, particularly in stage I one tumors. This is an important contribution to the field as 90% of the melanomas are diagnosed early and this signature could be useful in identifying patients who may benefit from receiving early adjuvant therapies. Furthermore, this molecular signature also demonstrated predictive value in identifying patients with metastatic melanoma treated with immunotherapy who are not likely to respond and this work has now been published in Clinical Cancer Research.

During my PhD, I also received training in machine learning and developed classification models that predict high- and low-risk of melanoma relapse after initial diagnosis. The MELGEN program has been instrumental in defining my career path by providing numerous training opportunities to improve my technical and communication skills as well as scientific interactions with world leaders in melanoma research. As part of the MELGEN program, I conducted short research visits to Dr. Goran Jonsson’s group at University of Lund, Sweden, Dr. Will Spooner at Eagle Genomics, Cambridge, UK and Dr. Manolis Kellis’ group at Massachusetts Institute of Technology, USA where I received training in differential gene expression analysis and machine learning using genomic datasets. As a result of this training I was successful in developing a machine learning model that in future would be helpful in stratifying patients into low risk and high risk of relapse and subsequently would inform clinical decisions.

Publications

Thakur R, Laye JP, Lauss M, Diaz JMS, O’Shea SJ, Poźniak J, Filia A, Harland M, Gascoyne J, Randerson-Moor JA, Chan M, Mell T, Jönsson G, Bishop DT, Newton-Bishop J, Barrett JH. Transcriptomic Analysis Reveals Prognostic Molecular Signatures of Stage I Melanoma. Clin Cancer Res. 2019 Dec 15;25(24):7424-7435. Available at: https://clincancerres.aacrjournals.org/content/25/24/7424

Find out about ESR12 – Rohit Thakur – based in Leeds's research

ESR13 – Joey Mark Diaz – based in Leeds

Project Title: Genomic Copy Number Alterations in Melanoma and Patient Survival
PhD Award Expected: Autumn 2020

In 2016, I joined the Leeds Institute of Cancer and Pathology (now Leeds Institute of Medical Research at St. James’s), School of Medicine in the University of Leeds for a PhD study on analysis of genomic copy number alteration and melanoma survival as one of the selected 17 Marie Skłodowska-Curie research fellows under MELGEN.

I finished BSc in Statistics in Central Luzon State University, Philippines in 2010 as a government scholar with an undergraduate work on multiple imputation using Markov Chain Monte Carlo (MCMC) method. I then worked as research assistant in the National Genetic Program Unit of the Philippine Carabao Center in 2010 and as biostatistician in the University of Sto Tomas Center for Drug Research, Evaluation and Studies, Inc in 2011. I was awarded an Accelerated Science and Technology Human Resource Development Program (ASTHRDP) in 2011 by the Philippine Department of Science and Technology (DOST) to study MSc in Health Informatics-Bioinformatics in the University of the Philippines Manila. My graduate work focused on the classification of lung cancer using statistical and machine learning methods applied to transcriptomic data.

Aside from science, I have more than three years of corporate experience working on statistical financial risk models as credit scoring specialist in the RCBC Savings Bank and as retail modelling analytics manager in the Australia and New Zealand Banking Group Limited.

I look forward to taking part in improving patient treatment in my home country through personalised and stratified medicine using genomics.

 


Research Summary

Coming soon…

Publications

Váraljai R., Wistuba-Hamprecht K., Seremet T., Diaz JMS, Nsengimana J., Sucker A., Griewank K., Placke J-M., Horn P.A., von Neuhoff N., Shannan B., Chauvistré H., Vogel F.C.E., Horn S., Becker J.C., Newton-Bishop J., Stang A., Neyns B., Weide B., Schadendorf D., Roesch A. Application of Circulating Cell-Free Tumor DNA Profiles for Therapeutic Monitoring and Outcome Prediction in Genetically Heterogeneous Metastatic Melanoma. JCO Precision Oncology 2019; :3, 1-10. Available at: https://ascopubs.org/doi/10.1200/PO.18.00229

Poźniak J, Nsengimana J, Laye JP, O’Shea SJ, Diaz JMS, Droop AP, Filia A, Harland M, Davies JR, Mell T, Randerson-Moor JA, Muralidhar S, Hogan SA, Freiberger SN, Levesque MP, Cook GP, Bishop DT, Newton-Bishop J. Genetic and Environmental Determinants of Immune Response to Cutaneous Melanoma. Cancer Res. 2019 May 15;79(10):2684-2696. Available at: https://cancerres.aacrjournals.org/content/79/10/2684

Filia A, Droop A, Harland M, Thygesen H, Randerson-Moor J, Snowden H, Taylor C, Diaz JMS, Pozniak J, Nsengimana J, Laye J, Newton-Bishop JA, Bishop DT. High-Resolution Copy Number Patterns From Clinically Relevant FFPE Material. Sci Rep. 2019 Jun 20;9(1):8908. Available at: https://www.nature.com/articles/s41598-019-45210-2

Thakur R, Laye JP, Lauss M, Diaz JMS, O’Shea SJ, Poźniak J, Filia A, Harland M, Gascoyne J, Randerson-Moor JA, Chan M, Mell T, Jönsson G, Bishop DT, Newton-Bishop J, Barrett JH. Transcriptomic Analysis Reveals Prognostic Molecular Signatures of Stage I Melanoma. Clin Cancer Res. 2019 Dec 15;25(24):7424-7435. Available at: https://clincancerres.aacrjournals.org/content/25/24/7424

Muralidhar S, Filia A, Nsengimana J, Poźniak J, O’Shea SJ, Diaz JM, Harland M, Randerson-Moor JA, Reichrath J, Laye JP, van der Weyden L, Adams DJ, Bishop DT, Newton-Bishop J. Vitamin D-VDR signaling inhibits Wnt/beta-catenin-mediated melanoma progression and promotes anti-tumor immunity. Cancer Res. 2019 Dec 1;79(23):5986-5998. Available at: https://cancerres.aacrjournals.org/content/79/23/5986.

 

Find out about ESR13 – Joey Mark Diaz – based in Leeds's research

ESR14 – Sonia Leonardelli – based in Essen

Project Title: Evaluating clinical implications and prognostic value of less frequent somatic mutations in melanoma, and understanding the role of senescence.
PhD Awarded: December 2019

I am an Italian student who graduated from University of Trento (IT) with Master degree in Cellular and Molecular Biotechnology. Since my strong motivation in cancer research, I was selected to be part of this amazing group of young researchers and contribute with my work towards a most extensive knowledge on melanoma.

Apart from studying, I love traveling and seeing the beauty of the world, going to the most beautiful beaches and the most famous cities. I also love good food, therefore I never miss out the possibility to have a good dinner with friends, cooking for everyone or ordering delicious meals. Finally, I enjoy watching movies. In fact, I look forward to every years’ nominations for the Academy Awards, to guess who the winners will be.


Research Summary

Coming soon…

Publications

Scholz SL, Möller I, Reis H, Süßkind D, van de Nes JAP, Leonardelli S, Schilling B, Livingstone E, Schimming T, Paschen A, Sucker A, Murali R, Steuhl KP, Schadendorf D, Westekemper H, Griewank KG. Frequent GNAQ, GNA11 , and EIF1AX Mutations in Iris Melanoma. Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3464-3470. Available at: http://iovs.arvojournals.org/article.aspx?articleid=2644164

Pieper N, Zaremba A, Leonardelli S, Harbers FN, Schwamborn M, Lübcke S, Schrörs B, Baingo J, Schramm A, Haferkamp S, Seifert U, Sucker A, Lennerz V, Wölfel T, Schadendorf D, Schilling B, Paschen A, Zhao F. Evolution of melanoma cross-resistance to CD8+ T cells and MAPK inhibition in the course of BRAFi treatment. Oncoimmunology. 2018 Apr 18;7(8):e1450127. Available at: https://www.tandfonline.com/doi/full/10.1080/2162402X.2018.1450127

Scholz SL, Cosgarea I, Süßkind D, Murali R, Möller I, Reis H, Leonardelli S, Schilling B, Schimming T, Hadaschik E, Franklin C, Paschen A, Sucker A, Steuhl KP, Schadendorf D, Westekemper H, Griewank KG. NF1 mutations in conjunctival melanoma. Br J Cancer. 2018 May;118(9):1243-1247. Available at: https://www.nature.com/articles/s41416-018-0046-5

Horn S, Leonardelli S, Sucker A, Schadendorf D, Griewank KG, Paschen A. Tumor CDKN2A-Associated JAK2 Loss and Susceptibility to Immunotherapy Resistance. J Natl Cancer Inst. 2018 Jun 1;110(6):677-681. Available at: https://academic.oup.com/jnci/article/110/6/677/4780396

 

Find out about ESR14 – Sonia Leonardelli – based in Essen's research

ESR15 – Renata Varaljai – based in Essen

Project Title: Exploring circulating free DNA (‘liquid biopsies’) from melanoma patients for prognostic biomarkers
PhD Awarded: November 2018

I am a young scientist from Hungary. I graduated with an MSc in Biology from the University of Debrecen. My main research interests include cancer biology, cell biology and epigenetics.

I received my cell biology training from the Hungarian Academy of Sciences in Budapest where I was part of the Membrane Research Group. Throughout the last years I have worked at the University of Illinois at Chicago. My research centralized around the role of epigenetic enzymes – KDM5s and EZH2 in multiple cancers. I investigated the requirement of the histone lysine demethylase KDM5A for coordination of mitochondrial metabolism during differentiation. I have published research papers in these topics and presented my results at multiple conferences.

Currently I am pursuing my PhD at the Department of Dermatology of the University Hospital Essen in Germany within the MELGEN ETN, where I am investigating cancer drug resistance in melanoma. My research is focusing on the establishment of prognostic and predictive circulating cell-free DNA profiles of patients with metastatic melanoma.

When I am not researching, I like to spend time outdoors. I love biking and hiking, and find them a great way to connect with nature and decompress. I am interested in homemade natural remedies and cosmetics.


Research Summary

Coming soon…

Publications

Metzenmacher M, Váraljai R, Hegedüs B, Cima I, Forster J, Schramm A, Scheffler B, Horn PA, Klein C, Szarvas T, Reis H, Bielefeld N, Roesch A, Aigner C, Kunzmann V, Wiesweg M, Siveke JT, Schuler M, Lueong SS. Plasma next generation sequencing and droplet digital-qPCR-based quantification of circulating cell-free RNA for noninvasive early detection of cancer. Cancers (Basel). 2020 Feb 4;12(2). pii: E353. Available at: https://www.mdpi.com/2072-6694/12/2/353

Váraljai R., Wistuba-Hamprecht K., Seremet T., Diaz JMS, Nsengimana J., Sucker A., Griewank K., Placke J-M., Horn P.A., von Neuhoff N., Shannan B., Chauvistré H., Vogel F.C.E., Horn S., Becker J.C., Newton-Bishop J., Stang A., Neyns B., Weide B., Schadendorf D., Roesch A. Application of Circulating Cell-Free Tumor DNA Profiles for Therapeutic Monitoring and Outcome Prediction in Genetically Heterogeneous Metastatic Melanoma. JCO Precision Oncology 2019; :3, 1-10. Available at: https://ascopubs.org/doi/10.1200/PO.18.00229

Find out about ESR15 – Renata Varaljai – based in Essen's research

SWISS1 – Sabrina Hogan – based in Zurich

Project Title: Immune profiling of melanoma patients undergoing immunotherapies
PhD Awarded: October 2019

Since my childhood I have always been fascinated by the human body. When I started my Bachelor in Biology at the University of Lausanne (Switzerland) I had the opportunity to deepen my knowledge in a wide variety of subjects. For my Masters I decided to focus on Medical biology, namely Cancer and Immunology. I find this field extremely interesting and motivating, as the end goal of the studies is to help to improve thousands of patient’s lives.

Following my MSc, I had the opportunity to work for 7 months in a large and reputable pharmaceutical research organization (Nestlé Research Center) as a Junior Research Assistant. Then, I moved to the UK where I undertook a MRes in Biomedical Research at the University of Reading and carried out an 8 months research project on the regulation of metastasis in Rhabdomyosarcoma. I am currently pursuing a PhD aimed at profiling immune responses in melanoma patients undergoing immunotherapy at the University of Zürich Hospital (Switzerland). This position will also help improve my level of German as I was born in the west part of Switzerland and my first language is French.

Regarding my extracurricular activities, I enjoy travelling and doing all kinds of sports, which I like to teach as well. I have done a season as a ski/snowboard instructor and taught tennis for many years. I enjoy coaching, I find it very rewarding.


Research Summary

Coming soon…

Publications

Ramelyte E, Schindler SA, Dummer R. The safety of anti PD-1 therapeutics for the treatment of melanoma. Expert Opin Drug Saf. 2017 Jan;16(1):41-53.

Dummer R, Ramelyte E, Schindler S, Thurigen O, Levesque MP, Koelblinger P. MEK inhibition and immune responses in advanced melanoma. Oncoimmunology. 2017 Aug 10;6(8):e1335843. Available at: https://www.tandfonline.com/doi/full/10.1080/2162402X.2017.1335843

Krieg C, Nowicka M, Guglietta S, Schindler S, Hartmann FJ, Weber LM, Dummer R, Robinson MD, Levesque MP, Becher B. High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy. Nat Med. 2018; 24(2):144-153. Available at: https://www.nature.com/articles/nm.4466. Erratum in: Nat Med. 2018; 24(11):1773-1775.

Hogan SA, Levesque MP, Cheng PF. Melanoma Immunotherapy: Next-Generation Biomarkers. Front Oncol. 2018 May 29;8:178. Available at: https://www.frontiersin.org/articles/10.3389/fonc.2018.00178/full

Hogan SA, Courtier A, Cheng PF, Jaberg-Bentele NF, Goldinger SM, Manuel M, Perez S, Plantier N, Mouret JF, Nguyen-Kim TDL, Raaijmakers MIG, Kvistborg P, Pasqual N, Haanen JBAG, Dummer R, Levesque MP. Peripheral blood TCR repertoire profiling may facilitate patient stratification for immunotherapy against melanoma. Cancer Immunol Res. 2019; 7(1):77-85. Available at: https://cancerimmunolres.aacrjournals.org/content/7/1/77

Poźniak J, Nsengimana J, Laye JP, O’Shea SJ, Diaz JMS, Droop AP, Filia A, Harland M, Davies JR, Mell T, Randerson-Moor JA, Muralidhar S, Hogan SA, Freiberger SN, Levesque MP, Cook GP, Bishop DT, Newton-Bishop J. Genetic and Environmental Determinants of Immune Response to Cutaneous Melanoma. Cancer Res. 2019 May 15;79(10):2684-2696. Available at: https://cancerres.aacrjournals.org/content/79/10/2684

Find out about SWISS1 – Sabrina Hogan – based in Zurich's research

SWISS2 – Ishani Banik – based in Zurich

Project Title: Functional in vivo screening of novel oncogenic driver mutations in a zebra fish melanoma model
PhD Awarded: December 2019

I am a PhD student at ETH, Zurich. I am a biotech engineer from the silicon city of India-Bangalore. I went on to pursue my master degree in molecular medicine at the University of Ulm, Germany. I graduated with my thesis on “Epigenetic regulators in normal and malignant development of skin” while working with Prof.Dr.Karin Scharffetter-Kochanek. Having realized my passion for the same and determined about improvising medical treatment for melanoma cancer I am now working on “Functional in vivo screening of novel oncogenic driver mutations in a zebrafish melanoma model” under the guidance of Prof. Levesque Mitchell Paul. When I’m not glued to the bench work in lab I like travelling and am always up for a sensible conversation. I can now be found admiring the beauty of the Alps in Zurich and am only an email away at ishani.banik@gmail.com


Research Summary

Firstly 2 transgenic zebrafish were established to model melanoma. The in vivo transgenic lines Tg(mitf:BRAFV600E)mitf(lf)p53(lf) and Tg(mitf:NRASQ61K)mitf(lf)p53(lf) strongly phenocopied nodular/cutaneous melanoma in humans. The in vivo results obtained from this study coincide with previous studies that have made similar transgenic lines. The time to tumor onset was significantly accelerated in NRAS driven melanoma in comparison to BRAF driven melanoma. The median onset of tumor development was 267 days in Tg(mitf:BRAFV600E)mitf(lf)p53(lf) while 52 days in Tg(mitf:NRASQ61K)mitf(lf)p53(lf). This observation paved way to testing candidate tumor suppressors in NRAS melanoma. In the TCGA cohort of NRAS mutant p53 null patients, MAPK14 is frequently amplified in those that have O.S.≥ 1 year and even lost in some patients that had O.S.≤1 year. This led to the hypothesis that p38α-MAPK14 is a tumor suppressor in NRAS melanoma context. Over-expression of p38α in Tg(mitf:NRASQ61K) mitf(lf)p53(lf) reduced the number of fish developing melanoma by 13% in addition to delaying tumor onset time by 50%. In order to reproduce the results in vitro, 6 patient derived primary melanoma cell lines with NRAS mutations were chosen. Stable transfection of p38α in 2 cell lines with NRAS mutation and p53 wt resulted in reduced cell viability and reduced clonogenicity. Furthermore, it was determined that the anti-tumorigenic effect wad mediated by apoptosis.  Next, in order to verify that over-expression of p38α produced tumor suppressive effects, a different route of p38α activation was used viz by treating cells with anisomycin. Anisomycin was used to activate the p38 pathway while SB203580 was used to inhibit the p38 pathway. It was observed that low dose anisomycin induced apoptosis mediated cell death in all 6 NRAS mutant melanoma cells irrespective of p53 mutation status. Cell viability was dose dependently reduced in all 6 melanoma cell lines upon anisomycin treatment. The effectiveness of anisomycin as a single agent was directly compared to the standard drug used for NRAS mutant melanoma- MEK inhibitor-Trametinib. All cell lines except one were resistant to varying concentrations of trametinib. On the contrary, all cell lines responded with an IC50 of 0.02-1μM to anisomycin treatment. Finally, upon co-treatment with anisomycin and trametinib, a synergistic effect on the IC50 of all cell lines including the ones that were stably transfected to express p38α was observed. To conclude, the use of anisomycin or a similar compound to activate the p38α-MAPK14 pathway can be a suitable therapeutic target for the treatment of NRAS mutant melanoma patients and can be a topic for further investigation. In summary, the results of this study, both in vitro and in vivo strongly are in favor of the hypothesis that p38α-MAPK 14 is a tumor suppressor in NRAS melanoma context.

Publications

Currently in preparation…

Find out about SWISS2 – Ishani Banik – based in Zurich's research