Mapping the initiating steps of aneuploidy and aneuploidy tolerization in tumorigenesis

• Applicant: Sahil Gupta, Ph.D.
• Subject Area: Hematology, Oncology ; Cell Biology
• Term: from 2019 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420142384

Aneuploidy reduces cellular fitness, still it impacts the majority of cancers, suggesting that aneuploid cancer cells have engaged mechanisms to cope with aneuploidy-related stress. This project plans to explore which upstream mutations can trigger carcinogenesis in an aneuploid background and whether these occur early or late during aneuploid tumor formation. We investigate this by employing two models, 1) a sporadic Chromosome instability (CIN)- model of tumorigenesis in mice and 2) CRISPR screens in various primary aneuploid cells. We will specifically track aneuploidy-corroborating mutations in early stages of tumor formation combining in vivo transposon mutagenesis with lineage tracing. For this, we will cross mice that harbour alleles for tissue-specific transposon mobilization and aneuploidization with mice carrying a GFP reporter that will be activated specifically in the cells that become aneuploid. As a next step we sort GFP+ cells at early and later stages of tumor formation; end-point tumor latencies are already known for the aneuploid transposon mice, making this approach feasible. Early and late tumor GFP+ cells will be assessed for aneuploidy and transposon integration sites and assessed by single cell RNA-sequencing to establish heterogeneity between early tumour cells. In parallel, we will perform CRISPR screens in primary aneuploid (Mad2f/f) cell types to identify mechanisms that help primary cells cope with aneuploidy-imposed stress. Both the models will be validated with downstream functional and mechanistic assays as defined underneath.Our work is highly relevant for cancer research, as it will contribute to a better understanding of a key feature of cancer that affects to majority of cancers: aneuploidy. We lineage specifically track aneuploid cancer cells that provides selection to aneuploidy tolerization in early stages of tumor development. Using state of art genomics such as single cell RNA sequencing in transgenic sporadic mouse models and single cell DNA sequencing in CRISPR screens in primary cells or NSCs, we will investigate which lesions are the first to arise with aneuploidy during the process of malignant transformation. This will be the first study to identify factors that provide an overall fitness to aneuploid chromosomes after CIN induced mis segregation and define the mechanisms of how aneuploidy is tolerated in tumors. Most of our work addresses fundamental questions on aneuploidy, however we are exploring our findings into the clinic that will benefit patients in parallel projects.

• DFG Programme: Research Fellowships
• International Connection: Netherlands
• Host: Professor Floris Foijer, Ph.D.