Modelling specific chromosome aneuploidies in cancer and the immune response

1: Develop novel, CRISPR-based approach to creating specific aneuploidies in human cells
2: Characterise cellular responses to specific aneuploidies, including T-cell responses
3: Mathematical modelling of generation and selection of cancer-specific aneuploidies

A major hallmark of cancer is aneuploidy (deviation from a normal complement of chromosomes), which we recently showed to be due in part to replication stress (Burrell and McClelland et al., Nature 20131). However, the reasons for specific aneuploidy patterns often observed in cancer remain unclear due to the difficulties in studying the effects of single chromosome aneuploidy in mammalian cells. We have devised a strategy to induce the missegregation of individual specific chromosomes using a novel, CRISPR-directed approach. Kinetochores are large proteinaceous structures formed at centromeres that attach chromosomes to the mitotic spindle (McClelland and Borusu et al., EMBO J, 20072). Recent work has elucidated a single small protein domain (CENP-T) that is capable of recruiting downstream components to create a functioning kinetochore at an ectopic location3. We have created a nuclease-dead CRISPR-CENP-T fusion protein and can guide this to specific chromosomal locations using guide RNA sequences. This will allow the specific and transient creation of a second kinetochore (and thus a dicentric chromosome), leading to the missegregation of specific chromosomes during mitosis (collaboration: Przewloka laboratory4). We will couple this strategy to an existing experimental system developed in our laboratory to isolate cells aneuploid for a single specific chromosome. This will allow us to understand the effects of single specific aneuploidies in cancer cells for the first time. In addition to gene expression or RNA-Seq analyses we will analyse the ability of cancer cells to tolerate specific aneuploidies, and couple this to existing data from our laboratory to allow us to model rates of incidence, tolerance, and selection of specific aneuploidies using mathematical approaches (collaboration:Graham laboratory). The establishment of this system will then allow us to analyse the immune response of specific aneuploidies by analysing cell surface antigens and eventually the T-cell and dendritic cell responses to specific aneuploidies, in collaboration with immunology groups at Southampton University (to be established during the course of the studentship, with help from our Southampton collaborator, Dr Przewloka).

Rotation project:
We have recently demonstrated, using a new, high throughput method to analyse aneuploidy rates in single cells, that specific chromosomes are more prone to missegregation during mitosis than others (Worrall and McClelland, unpublished data). Using this system the student will determine the landscape of aneuploidy induced by alkylating agents, commonly used to treat primary cancers, and implicated in the development of secondary haematological cancers, particularly Acute Myeloid Leukaemia (AML) exhibiting monosomy 7. This project will introduce high through-put fluorescence-activated cell sorting (FACS), fluorescence In-Situ hybridisation (FISH), high resolution and live cell microscopy, and statistical analysis. Depending on time constraints we may also be able to apply a mathematical modelling approach (in collaboration with Dr Graham) to combine our data with existing aneuploidy landscapes observed in therapy-induced AML to estimate the possible contribution of alkylating agents to the genetic changes observed in this disease.

Skills Priority Alignment: Advanced Therapeutics, Quantitative Biology