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COMAL:COhesin Mutations in Acute Leukemia: from modeling and mechanisms to novel therapeutics

Periodic Reporting for period 3 - COMAL (COMAL:COhesin Mutations in Acute Leukemia: from modeling and mechanisms to novel therapeutics)

Reporting period: 2018-09-01 to 2020-02-29

The aims and objectives of this project are to broadly determine the mechanisms whereby alterations in the structural packaging of DNA and the communication between different DNA sequence elements, over long distances, control gene expression, subsequent protein manufacture and cellular behaviour. In particular, we are interested in the mechanisms whereby these processes are altered in cancer. As an examplar of this pathological regulation we have chosen the tractable tumour type Acute Myeloid Leukaemia (AML) and are focusing on the AML subtype with mutation of the Cohesin complex, a global regulator of interactions between DNA segments over long distances through the looping out of intervening DNA. Although specifically looking at one type of mutation in one tumour type, mutations in Cohesin complex members occur frequently in other forms of cancer, we suspect that many other mutations interact with the Cohesin complex members to alter this process and alterations of gene expression are present and drive almost every type of cancer.

• What is the problem/issue being addressed?
We know that all forms of Cancer are associated with abnormal gene expression patterns and from experimental studies and the successful use of therapeutics that interfere with these abnormally expressed genes that cancers are dependent upon these programmes. The issue being addressed here is therefore to better understand these processes. This will identify potential therapeutic targets to improve cancer outcomes and in an ideal world how they might be prevented from developing into the aggressive and often fatal forms that we commonly see at presentation.
• Why is it important for society?
Cancer is currently the second most common cause of death amongst western populations, after cardiovascular disease. However, with improvements in the treatment of vascular disease and with the increasing age of the population, deaths from malignant disease are predicted to overtake cardiovascular disease within the decade. A better knowledge of the causes of cancer and improved therapies are therefore key in preventing or detecting developing cases earlier and in treating established cases.
• What are the overall objectives?
The overall objectives are as outlined above – we will model the loss/reduction of Cohesin complex members in cell lines and mice and determine how their loss alters gene expression, and the interaction and activity of DNA elements that control gene expression. We will also determine the consequences for cellular behaviour following these changes, will map the proteins that interact with the Cohesin complex to help us works its proper function and will use all of this information to identify targets and potential therapies to treat AML and other malignancies.
Within the reporting period we have set up and established the project. This has involved identifying staff members with the correct skill mix for the planned experiments and setting up and/or optimising a number of techniques required within the project. The predominant “new” technique has been the setting up of the promoter based capture HiC technique and the subsequent bioinformatics analysis of this technique and the two staff members have achieved this. We have also generated critical reagents for the project including the murine Smc3 conditional knock out strain that we are currently ageing to determine its phenotypic effects. In addition, we have generated multiple isogenic haematopoietic stem and progenitor cell lines with inducible knockdown of multiple cohesin complex family members (Smc3, Smc1a, Rad21 and Stag2). This has allowed us to perform the quantitative proteomic experiments from objective 3. This analysis, taking the high confidence overlap of multiple replicates and utilising knockdown of multiple cohesion proteins as controls, has provided the first documented protein interactome of the cohesin complex within the cellular context of haematopoietic stem and progenitor cells. These cell lines have also allowed us to perform functional experiments to determine the effects on self-renewal and differentiation following loss of Cohesin function and to compare this across multiple Cohesin family members, as well as genomic studies looking at the changes in the genomewide localisation of cohesion members, chromatin marks and the associated genome structural protein Ctcf following cohesion loss. We are currently analysing this data to determine pre-leukaemic mechanisms that occur following Cohesin family member loss and are hoping to put together a manuscript around this soon. In addition, we are interrogating a capture panel based sequencing dataset from a very large AML patient cohort, for mutations within the extended Cohesin protein interactome described above to further inform the identity of interacting proteins whose function is critical for Cohesin function and whose mutation may contribute to AML pathogenesis. We speculate that these proteins will also provide potential therapeutic targets and this hypothesis will feed into the therapeutic aims of Objective 4.
As the project is at a relatively early juncture, our current findings are preliminary, however we have already generated novel reagents and interesting unpublished data related to the biology of the Cohesin complex, including the first protein interactome within the haematopoietic context. Our data also has potential clinical implications in that it has identified potential therapeutic targets. As the project matures we will expect the research findings to progress beyond the state of the art and to impact on wider society.