Vulnerability to G - Quadruplexes in BRCA2 - Null Medulloblastoma: A Protective Role for the PIF1 Helicase

Author

Danielle L. Keahi

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Smogorzewska Laboratory

Abstract

The reasons why cancer arises in children are not fully understood. Children with biallelic mutations in the essential DNA repair factor BRCA2 are highly predisposed to develop the most common pediatric brain cancer medulloblastoma (MB) before three years of age. Medulloblastoma is often the first malignancy in these children who have a high likelihood of developing several other cancers in their lifetime. MBs have a complex mutational landscape characterized by chromothripsis in p53-deficient tumors and a high level of somatic mutation in many other cases.We utilized a mouse model of spontaneous medulloblastoma development driven by BRCA2 loss in the central nervous system coupled with global inactivation of p53 to study the roles of BRCA2 in protecting from childhood cancer development.We endeavored to address how mutagenesis can occur in tumor-initiating cells in such a short period of time by identifying sources of DNA damage in the cell of origin of BRCA2-null MB–the cerebellar granule cell progenitors(GCPs).GCPs undergo a period of massive expansion in the postnatal cerebellum, stimulated by Sonic hedgehog (Shh) signaling as well as other mitogens and can be isolated from the cerebella of mice during this developmental window that peaks at P7. After multiple symmetric divisions, GCPs exit the cell cycle, migrate into the inner granule layer of the cerebellum, and differentiate into granule cells, the most numerous single neuron type in the brain. The critical developmental window of GCP expansion represents a state where highly proliferative GCPs are vulnerable to mutagenesis. G4-containing genomic regions are vulnerable to mutation and that the demands of tumor growth may cause positive selection of mutations in functionally relevant genes.From gene expression analysis ofBrca2-/-MBs, we identified that several G4-resolving helicases are upregulated in tumor. Of these helicases, the PIF1 helicase was present in tumor and absent from normal cerebellum, indicating its potential as a therapeutic target that could selectively affect tumor cells. To assess the role of PIF1 inBrca2-/-MB cells, we generated primary tumor cell lines and knocked out Pif1 with CRISPR/Cas9. PIF1 loss led to replication stress inBrca2-/-MB cells and downstream genomic instability. Treatment with PDS exacerbated the phenotypes of PIF1deficiency, indicating that G4s that are unable to be resolved can lead to deleterious outcomes such as the development of micronuclei and aberrant mitoses.InBrca2-/-cells, G-quadruplexes represent a hurdle to normal replication that must be resolved by helicases like PIF1 to prevent mutagenesis. Down regulation of PIF1 could sensitizeBrca2-/-cells to treatment with PDS and other G4-ligands and could represent a new vulnerability to exploit inBrca2-/-medulloblastoma.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Keahi, Danielle L., "Vulnerability to G - Quadruplexes in BRCA2 - Null Medulloblastoma: A Protective Role for the PIF1 Helicase" (2023). Student Theses and Dissertations. 761.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/761