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Principal Investigators

David Ashley, Duke University
Jerry Shay, University of Texas Southwestern

Title

6-thio-2-deoxyguanosine: A Novel Immunogenic Telomerase-Mediated Therapy in Glioblastoma - A Duke and UTSW Collaboration

Project Information

Project 1 - Gliomas are the most common primary malignant brain tumor in adults and account for over 14,000 deaths annually in the United States. The most common type of glioma, glioblastoma (GBM) has a median overall survival of less than 21 months in spite of aggressive therapy. GBMs, like other human cancers, have activated an enzyme called telomerase that rebuilds the ends of the chromosomes – regions known as telomeres – to enable the cell’s replicative immortality. Indeed, roughly 90% of GBM cases harbor genetic alterations in the TERT gene that activate telomerase. Unfortunately, efforts to directly target telomerase activity to date have been hindered by lack of effective small molecules that cross the blood-brain-barrier, demonstrate on-target effects, and show efficacy and specificity in GBMs. Therefore, there is a critical need to develop safe and efficacious telomerase-targeted therapies for patients with GBM whose tumors harbor telomerase activating genetic alterations. We previously used the purine analog pro-drug 6-thio-2’-deoxyguanosine (6-thio-dG), which was used in human clinical trials in the 1970s, to develop a strategy for rapidly inducing telomerase-mediated cytotoxic DNA damage at telomeres. Rather than inhibiting telomerase and allowing telomeres to get progressively shorter, 6- thio-dG is taken up by cancer cells and converted into 6-thio-dGTP, which is then incorporated into newly synthesized telomeric repeats. Once these modified segments accumulate in the telomeres, telomeric DNA damage rapidly results, ultimately leading to cell death. In pre-clinical models of lung, colon, and melanoma, treatment with 6-thio-dG led to rapid killing of the cancer cells with little toxicity to normal cells and tissues. Importantly, telomeric DNA damage induced by 6-thio-dG also enhanced anti-tumor innate immunity. Building on these data, we have extended our pre-clinical analysis to GBMs and obtained evidence that 6-thio-dG crosses the blood-brain-barrier. The overall objective for Project 1 is to advance 6-thio-dG toward a clinical trial to be conducted by Project 2. We propose the following Specific Aims: 1) Characterize the pre-clinical efficacy and pharmacodynamic biomarkers of 6-thio-dG treatment alone or in combination with Temozolomide (TMZ) in an extended panel of patient-derived cell lines, PDX and organoid models; 2) Test the anti-tumor efficacy and inflammatory potential of 6-thio-dG alone and in combination with TMZ or immune checkpoint blockade (ICB) therapies in immune competent murine models of GBM; and 3) Define cell toxicity and innate inflammatory potential of 6-thio-dG in an ex vivo glioma tissue framework and patient-derived organoids. These studies will determine the pre-clinical efficacy of 6-thio-dG in GBM and confirm biomarkers of efficacy that will guide the design of clinical trials including enrollment criteria. This Project will work closely with the proposed Administrative Core, Molecular Biomarker Core Resource, and Project 2 to achieve our shared goal of advancing 6-thio-dG toward clinical application in GBM.

Project 2 - Glioblastoma (GBM) remains uniformly lethal, with an overall survival of <21 months despite standard of care therapies. Immunotherapy has remarkable efficacy in many cancers, but has been less successful in GBM due in part to the tumor’s immunosuppressive effects and high levels of intratumoral heterogeneity. The Duke/UT Southwestern Glioblastoma Therapeutics Network (GTN) team will complete pre-clinical development of a novel treatment for patients with glioblastoma (GBM) and investigate the biologic activity of this agent in an early-phase clinical trial. Project 2 will contribute to this goal by investigating biomarkers of response to the telomerase- targeted agent 6-thio-dG in pre-clinical models and by conducting a Phase 0 clinical trial to examine these biomarkers in humans. 6-thio-dG is a blood brain barrier (BBB)-penetrant purine analog pro-drug that is preferentially incorporated into newly synthesized telomeres under the control of telomerase. Incorporation of 6- thio-dG into telomeres induces DNA damage and activates innate immune signaling, resulting in cell death. Because roughly 90% of GBM express telomerase resulting from early and highly clonal TERT-promoter mutations, 6-thio-dG represents an exciting mechanism to overcome tumor heterogeneity and activate anti-tumor immune responses. Although we have shown that treatment with 6-thio-dG induces telomeric DNA damage and elicits immune-mediated cytotoxicity in telomerase-positive cells, the optimal time-point for measuring 6-thio-dG- induced DNA damage and innate immune activation as pharmacodynamic (PD) endpoints is unknown and will be rigorously determined using patient-derived xenograft models (Aim 1). Additionally, most GBM patients receive first-line treatment with temozolomide (TMZ), which can induce hypermutation and loss of telomerase activity at recurrence. We will therefore evaluate the efficacy of 6-thio-dG following prior TMZ treatment and identify mechanisms of therapy resistance in vivo (Aim 1). Based on experiments conducted in Aim 1 and in Project 1, we will establish preliminary biomarkers of sensitivity and response to 6-thio-dG treatment in a Phase 0 window-of-opportunity trial in adults with newly diagnosed, telomerase-positive GBM (Aim 2). Following a 2- day pre-surgical course of 6-thio-dG, we will examine GBM tissues for detectable increases in DNA damage (primary endpoint) and activation of immune responses. These studies will enable the design and conduct of a Phase 0 trial of 6-thio-dG, identify patient populations likeliest to benefit from therapy, and assess biomarkers of sensitivity and response to 6-thio-dG among newly diagnosed patients with telomerase-positive GBM. The GBM clinical trials infrastructures of Duke and UTSW, which treat a diverse patient population representing ~10% of all U.S. patients with GBM, is an excellent setting for this trial. Project 2 thus contributes to this GTN U19’s overall goal and to the NCI’s goal to develop novel therapies to improve treatment for adults with GBM.