ABSTRACT: Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. GBM growth and therapeutic resistance are driven by a combination of self-renewing cancer stem cells (CSCs) and an aging- induced tumor-supportive microenvironment. CSCs are regulated by cell intrinsic genetic and epigenetic networks, extrinsic cellular interactions with the surrounding microenvironment, and the interaction between those intrinsic and extrinsic regulatory programs. While multiple molecular mechanisms that drive self-renewal have been identified, the effects of advanced age on CSC maintenance has yet to be explored. Specifically, it is unclear how advanced age alters CSC maintenance and GBM growth. A recently recognized hallmark of advanced age is the shift in sulfur amino acid metabolism that suppresses enzyme-dependent hydrogen sulfide (H2S) generation, signaling, and bioavailability. H2S is a redox-active metabolite that signals through protein S- sulfhydration (R-SSnH) and impacts metabolism, immune activation, and longevity. Its enzymatic production by cystathionine γ-lyase (CGL) is repressed by thyroid hormone (TH). H2S has both pro- and anti-tumorigenic functions that are tumor-type dependent. However, there is limited information on cell intrinsic and tumor microenvironment functions of H2S in GBM. Recently, we modulated H2S levels through dietary and pharmacological interventions and found that H2S functions as a tumor suppressor in GBM and attenuates CSC self-renewal and tumor growth in pre-clinical models. H2S generation and sulfhydration were decreased in human GBM specimens as compared to non-tumor controls. While the data support the hypothesis that H2S functions as a tumor suppressor in GBM, the effects of aging-induced H2S declines on GBM progression and CSCs, and how to reverse this for clinical use, are unexplored. Based on our published findings and new preliminary data, we hypothesize that decreased H2S production during aging promotes CSC initiation, immune suppression, and drives GBM growth but can ultimately be reversed by anti-TH based H2S boosting approaches. We will test this hypothesis through the following aims by integrating our newly developed H2S and sulfhydration detection assays in combination with in vitro and in vivo GBM models that manipulate H2S production via genetic and pharmacological interventions with human samples to provide clinical relevance. Aim 1 tests the hypothesis that aging-induced suppression of CGL-derived H2S accelerates GBM progression. Aim 2 tests the hypothesis that chemically-induced hypothyroidism stimulates CGL to increase systemic and neural H2S production resulting in reduced GBM progression, CSC enrichment, and improved survival in preclinical GBM models. The long-term goal of this project is to interrogate the function of H2S as a GBM tumor suppressor that is lost during aging, while also studying the therapeutic effects of hypothyroid-induced H2S production and signaling to reduce CSC maintenance and immune suppression. Leveraging this axis represents a new strategy for the management of GBM that may synergize with standard of care chemo-, radio-, and immunotherapies.

PROJECT NARRATIVE Glioblastoma (GBM) is an incurable primary brain cancer with a median age at diagnosis of 65 years old and the mechanisms through which advanced age suppress cell intrinsic and extrinsic factors allowing for maintenance and outgrowth of GBM cancer stem cells (CSCs) is unexplored. We recently discovered that the redox gasotransmitter, hydrogen sulfide (H2S), which decreases during advanced age and is suppressed by thyroid hormone activity, enhances immune surveillance and functions as a tumor suppressor in subjects with GBM. By integrating our team’s collective expertise in GBM, CSCs, and H2S, we will interrogate how advanced age changes the brain microenvironment to favor CSC survival and outgrowth through deficiencies in H2S signaling while also leveraging anti-thyroid hormone approaches to reverse H2S declines and improve GBM outcomes in pre-clinical models.