Neuronal UV-Initiated Apoptosis is Prevented By 5-Bromo-2’-Deoxyuridine (BrdU) Or A Deficiency in Cockayne Syndrome B Or Xeroderma Pigmentosum A

This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.MAS

Investigating the Role of Wnt Signalling and Identifying of Novel Fitness Genes in Glioblastoma

Glioblastoma (GBM) is the most common malignant tumour of the central nervous system and in spite of aggressive treatment, recurrence remains high. Glioblastoma stem cells (GSCs) have the ability to self-renew and differentiate into the different cell types observed in the tumour and have decreased sensitivity to chemotherapy and radiotherapy. Thus, characterization of GSCs can provide insights into targeted therapy that are tailored to each individual and that will prevent relapse. The Wnt signalling pathway is essential during embryonic development and tissue homeostasis to control stem cell renewal, proliferation and differentiation and is involved in maintaining cancer stem cells in many organs and tissues. Using an orthotopic Wnt reporter model we showed that GBM cells with higher Wnt activity have a higher self-renewing ability relative to cells with lower Wnt activity. In my work I identified a subset of GSCs that are dependent on Wnt signalling for self-renewal and a subset that is independent. Characterization of these subtypes discovered that the Wnt-dependent subset had a gene signature similar to the proneural subtype of GBM and relies on the canonical Wnt signalling pathway whereas the Wnt-independent subtype was similar to the mesenchymal subtype. Xenograft models also demonstrated that inhibition of Wnt signalling reduced tumour burden using Wnt-dependent GSCs but not in Wnt-independent GSCs. We also showed that dual inhibition of Wnt/-catenin and Notch signalling pathways in Wnt-dependent GSCs increased neuronal differentiation and reduced self-renewal. This first project in my thesis identified new contexts for Wnt modulation in GBM specifically its role in self-renewal and differentiation. In my second thesis project we identified context-specific genetic vulnerabilities in GSCs using CRISPR-Cas9 approaches. Specifically we characterized members of the SOX transcription factor family and cholesterol biosynthesis pathway as essential for GSC proliferation. This study provided a large data set that remains to be explored for additional therapeutic targets. In summary, this thesis identified and characterized a subset of GSCs dependent on Wnt signalling for survival and discovered novel genetic vulnerabilities in a panel of GSCs using CRISPR-Cas9 screens.Ph.D

Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells

Summary: Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance. : MacLeod et al. describe genome-wide CRISPR-Cas9 screens identifying genetic vulnerabilities across a panel of patient-derived glioblastoma stem cell cultures. Regulators of stemness (SOX2, SOX9, DOT1L, and SOCS3) and stress response (ufmylation and ERAD pathways) govern the growth of glioblastoma stem cells. Chemogenomic screens using temozolomide identify modulators of sensitivity to chemotherapy. Keywords: glioblastoma, glioblastoma stem cells, CRISPR-Cas9, fitness genes, functional genomic

Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation