Cancer Research UK: London Edition

THE INSTITUTION FOR CANCER RESEARCH (ICR) LOCATED IN LONDON, ENGLAND IS CURRENTLY RANKED AS THE UK’S LEADING ACADEMIC RESEARCH CENTER AND LEADS

THE WORLD IN ISOLATING CANCER-RELATED GENES AND DISCOVERING NEW, TARGETED CANCER DRUGS. DURING MY TIME AT ICR I WORKED SPECIFICALLY IN THE MOLECULAR PATHOLOGY AND CANCER THERAPEUTICS DEPARTMENT UNDER DR. CHRIS JONES WITH HIS BREAK THROUGH RESEARCH IN GENOME WIDE PROFILING OF PEDIATRIC TUMORS

LOCATED PRIMARILY IN THE BRAINSTEM AND CEREBRAL CORTEX AREA.

JONES’ LAB IS FILLED WITH SEVERAL DIVERSE PROJECTS FROM MODEL EXPERIMENTAL MICE, DRUG TREATMENTS, AND IDENTIFYING STRUCTURAL VARIANTS IN DNA THAT LEAD

TO BREAKTHROUGH MUTATIONS. OVER MY TWO MONTHS IN THE LABORATORY I HAVE LEARNED TECHNIQUES INCLUDING FISH, PCR, CELL CULTURE, PREPARING CLONOGENIC

ASSAYS, DNA AND RNA EXTRACTION, WESTERN BLOTS, SIRNA KNOCKDOWNS, AND CULTURING STEM CELLS. IN ADDITION TO LEARNING BASIC LAB PROCEDURE AND GAINING

INVALUABLE EXPERIENCE, I WORKED DIRECTLY UNDER A DISSERTATION PROJECT IN STRUCTURAL REARRANGEMENTS OCCURRING IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG).

Overview

THE OVERALL AIM OF THE PROJECT WAS TO DISCOVER NOVEL FUSION GENES AND OTHER CHROMOSOMAL REARRANGEMENTS AVAILABLE IN SAMPLES OF PHGG. THE

MAIN PROCEDURES BY WHICH THE LAB USED TO ACHIEVE THIS GOAL WAS THROUGH CHARACTERIZATION OF THE CELL LINES AND STRUCTURAL VARIATION IN PHGG, IN ORDER TO DETERMINE RECURRENCE OF NOVEL FUSIONS IN PATIENT SAMPLES, AND ASSESS ROLE OF NOVEL FUSION IN VITRO. THE LAB IS UTILIZED THREE SPECIFIC CELL LINES: KNS42, SF188, AND UW479. PRIMARILY, I WORKED DIRECTLY WITH SF188 CELL LINE AND ANALYZED THE EFFECT OF THESE NOVEL

FUSION GENES IN VITRO.

Research TO REPRESENT THE SEQUENCED GENOME A SF188 CIRCOS PLOT WAS

FORMED (FIGURE 1). FROM THIS WE BEGIN TO DISSECT THE GENE. EVERY CHROMOSOME IS SHOWN AND THIS ALLOWS US TO SENSE

CHANGES WITHIN THE GENE. THE OUTERMOST CIRCLE SHOWS ANY MUTATIONS THAT OCCUR AND THEIR LOCATION ON WHICH SPECIFIC

CHROMOSOME. THE MIDDLE CIRCLE, DEPENDING ON THE COLOR, TELLS THE VIEWER WHETHER A DELETION OR ADDITION HAS

OCCURRED (GREEN- ADDITION, RED- DELETION), AND THE INNERMOST CIRCLE CONTAINS BLUE LINES THAT DEPICT INTER-CHROMOSOMAL TRANSLOCATIONS AND INTRA-CHROMOSOMAL TRANSLOCATIONS

DEPENDING ON WHETHER THE TRANSLOCATION OCCURS WITHIN A CHROMOSOME OR BETWEEN CHROMOSOMES.

IN ORDER, TO PROVE THIS FUSION AND FINDS IT’S FUNCTIONALITY, SEVERAL TECHNIQUES WERE PERFORMED. TECHNIQUES INCLUDED, BUT WERE NOT LIMITED TO FISH, PCR, WESTERN BLOTS, AND EXTRACTIONS.

RESULTS WERE OBTAINED AND FOLLOWED BY DRUG TESTING TO OBTAIN FURTHER RESULTS. THE FINAL GOAL WAS TO VALIDATE THE FUSION

BETWEEN RPTOR AND TULP 4 AND THE FURTHER ANALYZE THE FUNCTIONALITY OF THIS SPECIFIC GENE AND DETERMINE DRUG

TREATMENTS AND ACTIONS.

FOR THE SIX WELL PLATE IT WAS DECIDED THAT APPROXIMATELY 35,000 CELLS SHOULD BE SEEDED IN ORDER TO PROPERLY PERFORM THE DRUG TREATMENT. FURTHERMORE, FOR THE NINETY-SIX WELL PLATE

APPROXIMATELY 2,000 CELLS WERE FOUND TO BE THE BEST SUIT FOR THE INTERVAL OF 80-90% CONFLUENCY NEEDED FOR THE TREATMENTS.

AFTER THE PROPER CELL OPTIMIZATIONS WERE OBTAINED IT WAS DETERMINED THAT THE DRUG OF CHOICE WAS PI-103 (A DRUG CURRENTLY IN STAGE THREE OF CLINICAL TRIALS IN ENGLAND). DIFFERENT ALIQUOTS OF THE DRUG WERE MADE AND TESTED IN DIFFERENT CONCENTRATIONS ACROSS THE PLATE.

THE LAYOUT IS PICTURED BELOW (FIGURE 5) FOR THE DRUG TREATMENT.

FROM THESE DIFFERENT CONCENTRATIONS A GRAPH WAS PLOTTED AND AN IC50 WAS FOUND. THE IC50 OCCURRED AT .156ΜM. THE CURVE TURNED OUT AS EXPECTED AND ALL GOALS WERE MET. IN

ORDER TO VALIDATE THESE RESULTS THE TREATMENT WILL BE REPEATED.

FISH IS A BASIC LAB EXPERIMENT THAT ALLOWS THE VISUALIZATION OF GENETIC MATERIAL IN SPECIFIC IDENTIFIED CELLS. THIS WAS VERY IMPORTANT IN ALLOWING RESEARCHERS TO VISUALIZE SPECIFIC GENETIC MUTATIONS

(DELETIONS, TRANSLOCATIONS, ETC.) AND CHROMOSOMAL ABNORMALITIES. IN THE SPECIFIC EXAMPLE OF FISH UTILIZED WITH SF188, THE AIM WAS TO FIND A MUTATION IN THE RPTOR AND TULP4. IN THE BEGINNING SPECIFIC PROBES WERE DESIGNED IN ORDER TO BIND SPECIFICALLY TO THE GENES THAT WE ARE HOPING TO ANALYZE

THROUGH THIS EXPERIMENT.RPTOR IS A GENE ON CHROMOSOME 17. IT IS A PROTEIN ENCODING A GENE ASSOCIATED WITH THE REGULATION OF

CELL GROWTH IN THE MTOR PATHWAY IN RESPONSE TO NUTRIENT AND INSULIN LEVELS. WHEN MAKING THE SPECIFIC PROBES THE BAC USED BEFORE THE BREAKPOINT WAS RP11-317F05 (RED) AND THE BAC USED AFTER THE

BREAKPOINT WAS RP11-812H19 (GREEN). USUALLY THESE PROBES WOULD HYBRIDIZE TO REGIONS NEXT TO EACH OTHER ON THE NORMAL GENE. IN THE

PARTICULAR IMAGES SHOWN ABOVE IT IS EVIDENT THAT THE RED AND GREEN PROBES HAVE HYBRIDIZED TO THE SAME REGION, THUS PROVING A NORMAL GENE (TWO YELLOW SPOTS). HOWEVER, IF THERE IS A TRANSLOCATION IN

THIS REGION (LIKE EXPECTED), THE PROBES WILL BE SEPARATED (FIGURE 9). IN SF188 A TRANSLOCATION WAS SHOWN, SO THE SIGNALS ARE SPLIT.

IN ADDITION TO SPENDING TIME IN THE CANCER THERAPEUTICS AND MOLECULAR PATHOLOGY LABS, I SPENT TIME ON THE PEDIATRIC

ONCOLOGY WARD AT THE ROYAL MARSDEN HOSPITAL. DURING MY TIME ON THE WARD I WAS ALLOWED TO ACT AS A RESIDENT AND LEARN THE DAILY ROUTINES OF A PRACTICING ONCOLOGIST. I PERFORMED DAILY

ROUNDS, ATTENDED MDT MEETINGS, PERFORMED BASIC PATIENT CARE, AND SAT IN ON BRAIN TUMOR RESECTIONS. THROUGH THIS PROCESS I

WAS ABLE TO FURTHER CONFIRM MY FUTURE CAREER PATH OF A PEDIATRIC ONCOLOGIST.

WHILE IN THE LAB I WORKED ALONG SIDE PHD STUDENTS, POST-DOCS, AND LAB TECHNICIANS LEARNING THE TECHNIQUES OF A SCIENTIST.

SOME BASIC PROCEDURES INCLUDED, BUT WERE NOT LIMITED TO, PCR, FISH, STEM CELL CULTURE, CLONOGENIC ESSAYS, WESTERN BLOTS, AND EVEN UTILIZING EXPERIMENTAL MICE. ALSO, I WAS ABLE TO SPEND A FEW

HOURS EVERY DAY WORKING WITH PATHOPHYSIOLOGY ON THE HUMAN SAMPLES THAT WERE CULTURED IN THE BRAIN TUMOR RESECTIONS

PREVIOUSLY OBSERVED AT THE ROYAL MARSDEN HOSPITAL. THIS LAB WORK ALLOWED ME TO GAIN FURTHER EXPERIENCE IN A LAB SETTING,

WHILE ALSO SPARKING AN INTEREST IN CANCER RESEARCH THAT I HOPE TO PURSUE IN THE FUTURE.

ALTHOUGH MY TIME IN LONDON WAS VERY BUSY WITH WORK IN THE LAB, I WAS ABLE TO EXPLORE THE WONDERFUL COUNTRY OF

ENGLAND IN MY SPARE TIME. I WAS ABLE TO VISIT SEVERAL CASTLE RUINS, BIG BEN, PARLIAMENT, TOWER OF LONDON, MANY

CATHEDRALS, WATCHING THE TROOPING OF THE COLOURS, AND EVEN SPENT TIME VISITING SPAIN. THE CULTURE IMPACTS OF THIS PROJECT WERE MUCH MORE THAN I HAD EVER EXPECTED. I, LIKE MANY OTHER, THOUGHT THAT LONDON WOULD BE QUITE LIKE THE USA, HOWEVER I

WAS QUITE WRONG IN MY ASSUMPTIONS. THE ATMOSPHERE AND CULTURE OF LONDON WAS ONE THAT IMMERSED MYSELF IN

COMPLETELY AND ONE THAT I GREW TO LOVE AND APPRECIATE THROUGHOUT MY TIME AS A LONDONER.