Author ORCID Identifier
https://orcid.org/0000-0001-9936-2658
Date of Graduation
12-2024
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Jason T. Huse
Committee Member
Ahsan S. Farooqi
Committee Member
Jian Hu
Committee Member
Alexander J. Lazar
Committee Member
William K. Plunkett, Jr.
Committee Member
Kunal Rai
Abstract
Mutational inactivation of histone chaperone ATRX (a-thalassaemia/mental retardation X-linked) represents a defining molecular feature in several cancers, including malignant glioma. As standard of care only leads to transient responses and poor outcomes, there is an unmet clinical need for developing new therapies that target ATRX-deficient glioma. Loss of ATRX gives rise to abnormal G-quadruplex DNA secondary structures at GC-rich sites of the genome, such as telomeric and pericentromeric regions, enhancing replication stress and genomic instability. These mutations are mutually exclusive with TERT promoter mutations, promoting the alternative lengthening of telomeres (ALT) pathway as a telomere maintenance mechanism in ATRX-deficient glioma. Recently, a class of agents known as G4 stabilizers demonstrated strong therapeutic promise, however, the genomic consequences and efficacy of this treatment are poorly understood. Studying the molecular changes induced by and efficacy of G4 stabilizers in the treatment of ATRX-deficient glioma will advance the development of novel targeted therapies for this invariably fatal cancer.
Building upon earlier work, we evaluated the mechanisms of action and efficacy of the G4 stabilizer CX-5461 as both a single agent and in combination with ionizing radiation (IR), a mainstay in the current standard of care, using patient-derived glioma stem cell (GSC) preclinical models. We found that ATRX-deficient GSCs demonstrate dose-sensitive lethality to CX-5461, relative to ATRX-intact controls. Mechanistic studies revealed that CX-5461 disrupted histone variant H3.3 deposition, enhanced replication stress and DNA damage, activated p53-independent apoptosis, and induced G2/M arrest selectively in ATRX-deficient GSCs. These data were corroborated in vivo, where combinational treatment profoundly delayed tumor growth and prolonged survival exclusively in mice bearing ATRX-deficient GSC flank xenografts. Histopathological analyses revealed decreased proliferation, increased apoptosis, and significant induction of G4s, replication stress, and DNA damage in CX-5461-treated tumors, both alone and in combination with IR. Furthermore, systemic CX-5461 treatment induced tangible pharmacodynamic effects in ATRX-deficient intracranial GSC models, despite suboptimal central nervous system (CNS) penetration. Additionally, we conducted proof-of-concept studies in sarcoma models and found that CX-5461 induces dose-sensitive lethality, enhances replication stress and DNA damage, and induces G2/M cell cycle arrest in ATRX-deficient sarcoma cells, relative to ATRX-intact controls. These data implicate G4 stabilization as an effective treatment strategy with direct applications to other ATRX-deficient malignancies. Lastly, as DNA damage at telomeres is thought to drive the ALT pathway, we investigated the impact of G4 stabilization on ALT and found that CX-5461 does not inhibit ALT activity in ATRX-deficient glioma and sarcoma models. In its totality, this dissertation demonstrates efficacy and defines mechanisms of action for G-quadruplex stabilization as a novel therapeutic strategy targeting ATRX-deficient cancer, laying the groundwork for clinical translation.
Keywords
ATRX, glioma, G-quadruplex, CX-5461, radiation