The epidermal growth factor receptor (EGFR) is genetically altered in nearly 60% of glioblastoma (GBM) tumors; however, tyrosine kinase inhibitors (TKIs) against EGFR have failed to show efficacy for patients with these lethal brain tumors. This failure has been attributed to the inability of clinically tested EGFR TKIs (e.g. erlotinib, gefitinib, lapatinib, afatinib) to effectively penetrate the blood-brain barrier (BBB) and achieve adequate pharmacological levels to inhibit the oncogenic forms of EGFR that drive GBM to induce a tumor response. Hence, there is a highly unmet medical need for effective therapeutics for GBM. In these studies, we detail the identification, development, and evaluation of brain-penetrant, small molecule inhibitors of EGFR to a clinical compound. This dissertation begins with a structure-activity relationship (SAR) to develop JCN037 as an early pre-clinical lead molecule. JCN037 was developed from a 4-anilinoquinazoline scaffold by ring fusion of the 6,7-dialkoxy groups to reduce the number of rotatable bonds and polar surface area, and by introduction of an ortho-fluorine and meta-bromine on the aniline ring for improved potency and BBB penetration. Relative to the conventional EGFR TKIs erlotinib and lapatinib, JCN037 displayed potent activity against EGFR amplified/mutant patient-derived cell cultures, significant BBB penetration (2:1 brain-to-plasma ratio), and superior efficacy in an EGFR-driven orthotopic glioblastoma xenograft model. However, JCN037 was limited by a poor in vivo half life and a quick metabolism. Further SAR analysis lead to the development of JCN068, an EGFR TKI that potently inhibits oncogenic forms of EGFR with improved BBB penetration (>3:1 brain-to-plasma ratio). Compared to clinically tested EGFR TKIs, JCN068 demonstrates improved potency activity against EGFR amplified/mutant patient-derived cell cultures, significantly higher BBB, ideal clinical candidate in vivo pharmacology, and superior efficacy in multiple EGFR-driven orthotopic glioblastoma xenograft models. Additionally, rapid changes in tumor 18F-fluorodeoxyglucose (18F-FDG) uptake using non-invasive positron emission tomography (PET) was utilized as an effective predictive biomarker of response to JCN068 therapy in vivo. JCN068 is currently advancing in IND-enabling studies as a new potential therapeutic for EGFR-driven GBM.

Alterations in lipid metabolism are a hallmark of cancer and provide potential for therapeutic exploitation in the universally lethal brain tumor glioblastoma (GBM). However, an understanding of how distinct molecular features impact lipid metabolism is required for the development of effective targeted therapies. Importantly, extrinsic factors within the tumor microenvironment shape lipid metabolism, necessitating investigation within physiologic contexts. In these studies, we have performed a comprehensive characterization of the GBM lipidome through the unbiased analysis of transcriptomic, genomic, and lipidomic datasets spanning diverse tumor microenvironments and encompassing the intertumoral molecular heterogeneity observed in GBM. In the first portion of this work, we discuss the many tumor intrinsic and extrinsic factors that lead to metabolic vulnerabilities in GBM. In the second portion of this work, we identify a novel regulator of the GBM lipidome and demonstrate the resulting metabolic vulnerabilities that occur as a result of this altered lipid metabolism.

Effective treatments for cancer require the engagement of the apoptotic (cell death) machinery. These pathways have been implicated in the therapeutic efficacy of almost every chemotherapy, targeted agent, and even in some forms of immunotherapy. Inability to sufficiently engage the apoptotic machinery results in tumor survival and resistance. Glioblastoma (GBM), the deadliest form of brain cancer, has poor clinical response to almost every therapy, suggesting a heightened anti-apoptotic signature. In these studies, we are the first to comprehensively characterize the apoptotic machinery in patient-derived GBM samples through genetic, molecular, and functional measures. In the first portion of this work, we demonstrate how GBM are capable of utilizing multiple apoptotic blocks and how this versatility endows them with intrinsic resistance to standard of care. By understanding the intricacies in GBM apoptotic machinery, we are able to rationally design therapeutic strategies to exploit these dependencies, as highlighted by the second portion of this work.

Cellular metabolism is tightly regulated by growth factor signaling, which promotes metabolic rewiring to support growth and proliferation. While growth factor-induced transcriptional and post-translational modes of metabolic regulation have been well-defined, whether post-transcriptional mechanisms impacting mRNA stability regulate this process is less clear. Here, we present the ZFP36/L1/L2 family of RNA-binding proteins and mRNA decay factors as key drivers of metabolic regulation downstream of acute growth factor signaling. We quantitatively catalogue metabolic enzyme and nutrient transporter mRNAs directly bound by ZFP36 following growth factor stimulation – many of which encode rate-limiting steps in metabolic pathways. Further, we show that ZFP36 directly promotes the mRNA decay of Enolase 2 (Eno2), altering Eno2 protein expression and enzymatic activity, and provide evidence of a ZFP36/Eno2 axis during VEGF-stimulated developmental retinal angiogenesis. Thus, ZFP36-mediated mRNA decay serves as an important mode of metabolic regulation downstream of growth factor signaling within dynamic cell and tissue states.