Research > Focus A > Work Packages A04
Summary
Our project goal is to understand epigenetic and genetic mechanisms associated with temozolomide induced resistance in isocitrate dehydrogenase (IDH) mutant lower grade gliomas that exhibit promoter MGMT methylation. Our long-term aim is to reveal molecular targets that can be used in combination with alkylating agents to prevent the emergence of acquired resistance. The visual abstract below shows the aims and planned tasks of the work package.
Task 1
Characterize the evolution of molecular alterations during TMZ treatment
Steps / Workflow
- Generate TMZ resistant IDH mutant tumorsphere lines
- Analyze molecular state of cells generated in (1) during start of treatment, stress phase, and acquired resistance using next-gen sequencing (Whole exome, RNA-seq, and bisulfite-seq)
- Integrate sequencing data from (2) to identify candidate genes for Task 2
- Direct comparison of findings to N2M2 and AMPLIFY-NEOVAC cohort
Temozolomide (TMZ) treatment and Sequencing
RNA-seq (detect transcripts, isoforms) HumanMethylation EPIC arrays (detect aberrant methylation) Whole genome sequencing (variant calling, mutational burden and mutational signatures)
Task 2
Determine the functional roles of the candidate drivers of resistance in vitro and in vivo
Steps / Workflow
- Generate cell lines with either stable knockout or overexpression of candidates identified in Task 1
- Generate inducible lines of select candidates to test efficacy in preventing resistance
- Intracranial implantation of select cell lines in NSG mice for vehicle/TMZ treatment
Stable cell line generation Intracranial implantation Longitudinal tumor phenotype assessment
Task 3
Investigate the evolution of clonal substructure during TMZ treatment by single cell RNA-seq
Steps / Workflow
- Single cell copy number and transcriptional analysis of naïve and resistant tumorspheres, and matched primary and recurrent IDH mutated tumors
- Data analysis to determine clonal heterogeneity due to TMZ treatment
Single cell sequencing and Data analysis
PRINCIPLE INVESTIGATOR
TURCAN, SEVIN, PH.D
Junior Research Group Leader
University Hospital Heidelberg, Department of Neurology, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany