Glioblastoma (GBM) remains the most aggressive and prevalent malignant primary brain tumor in adults. Unchanged since 2005, standard of care (SoC) consists of surgical resection, followed by radiation therapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). Despite these therapeutic efforts, patients succumb to recurrent disease with a median overall survival of 14.6 months and a five-year survival rate of 5.5-6.8%. Therapeutic failure is largely explained by ITH and the presence of treatment-resistant GBM stem-like cells (GSCs). Given the lack of understanding of recurrent GBM and absence of second line therapies patients, I hypothesize that genome-scale functional genetic interrogation will unravel recurrent GBM-specific tumor biology and inform development of novel therapeutics.
First, I compared primary and recurrent GBM at the genetic, transcriptomic, proteomic and functional genetic levels. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 repress axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and exploit a genetic dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition.
Given the genetic dependency on ROBO signaling and enrichment of ROBO1 expression in GBM tissues, I undertook a campaign to evaluate ROBO1 as a therapeutic target in recurrent GBM and develop anti-ROBO1 chimeric antigen receptor T (CAR-T) cells using camelid single-domain antibodies targeting human ROBO1. I optimized the design of anti-ROBO1 CAR-T cells and tested the anti-tumor activity of these modalities in in vitro using patient-derived recurrent GBM lines and orthotopic patient-derived xenograft models. I present data to expand the repertoire of GBM-enriched antigens suitable for effective CAR-T cell therapy. Given that resistance to SoC and disease relapse are inevitable for GBM patients, pre-clinical and clinical advancement of immunotherapeutic modalities, combined with recent insights into the tumor immune microenvironment, are poised to improve clinical outcomes for this patient population. / Thesis / Doctor of Philosophy (PhD) / Glioblastoma remains the most lethal and prevalent primary brain tumor in adults. Standard of care for patients remains unchanged since 2005, consisting of surgery to remove visible tumor at diagnosis (primary tumor), followed by radiation therapy and chemotherapy to treat remaining tumor cells. Despite these therapeutic efforts, tumor relapse (recurrent tumor) is inevitable with no standardized second-line therapy. Patients succumb to recurrent disease with a median overall survival of 14.6 months and only 5.5-6.8% of patients survive five years post diagnosis.
Therapy failure and tumor relapse are explained by immense diversity among tumor cells at the DNA and protein levels, giving rise to a subset of tumor cells with abilities to resist therapy and seed the recurrent tumor. Previous studies have presented evolution of tumor cells through therapy, with recurrent tumor cells harboring novel changes at the DNA and protein levels. However, the impact of these changes on tumor cell function has not been evaluated.
In this thesis, we developed and applied a genetic screening technique to determine the functional role of thousands of genes in primary and recurrent tumor cells from the same patient. This analysis revealed numerous genes that exhibit differential effects on survival of primary and recurrent tumor cells, including genes that drive recurrent tumor cell growth but are dispensable in primary tumor cells.
Functional remodeling of these genes and pathways revealed a new functional role of multiple proteins belonging to a process called axonal guidance in recurrent tumor cells. To evaluate the therapeutic potential of these findings, we deeply interrogated the mechanism by which axonal guidance drives recurrent tumor cells and targeted crucial molecular players using chemical and immunological therapies. Using models that predict clinical effectiveness, we engineered and tested a novel therapy that redirects immune cells to target recurrent tumor cells driven by dysfunctional axonal guidance activity. The goal of this thesis was to discover the functional differences between primary and recurrent tumor cells, thereby leveraging this information to engineer candidate therapies for treatment of recurrent glioblastoma.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/27965 |
| Date | January 2022 |
| Creators | Chokshi, Chirayu R |
| Contributors | Singh, Sheila K, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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