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 firstname.lastname@example.org
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.
Currently in preparation…