Comparison of pharmacological inhibitors of lysine-specific demethylase 1 in glioblastoma stem cells reveals inhibitor-specific efficacy profiles.

Introduction: Improved therapies for glioblastoma (GBM) are desperately needed and require preclinical evaluation in models that capture tumor heterogeneity and intrinsic resistance seen in patients. Epigenetic alterations have been well documented in GBM and lysine-specific demethylase 1 (LSD1/KDM1A) is amongst the chromatin modifiers implicated in stem cell maintenance, growth and differentiation. Pharmacological inhibition of LSD1 is clinically relevant, with numerous compounds in various phases of preclinical and clinical development, but an evaluation and comparison of LSD1 inhibitors in patient-derived GBM models is lacking. Methods: To assess concordance between knockdown of LSD1 and inhibition of LSD1 using a prototype inhibitor in GBM, we performed RNA-seq to identify genes and biological processes associated with inhibition. Efficacy of various LSD1 inhibitors was assessed in nine patient-derived glioblastoma stem cell (GSC) lines and an orthotopic xenograft mouse model. Results: LSD1 inhibitors had cytotoxic and selective effects regardless of GSC radiosensitivity or molecular subtype. In vivo, LSD1 inhibition via GSK-LSD1 led to a delayed reduction in tumor burden; however, tumor regrowth occurred. Comparison of GBM lines by RNA-seq was used to identify genes that may predict resistance to LSD1 inhibitors. We identified five genes that correlate with resistance to LSD1 inhibition in treatment resistant GSCs, in GSK-LSD1 treated mice, and in GBM patients with low LSD1 expression. Conclusion: Collectively, the growth inhibitory effects of LSD1 inhibition across a panel of GSC models and identification of genes that may predict resistance has potential to guide future combination therapies.

NKTR-255 is a polymer-conjugated IL-15 with unique mechanisms of action on T and natural killer cells.

NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8+ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8+ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα-/- mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα-/- OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα-/- OT-I cell responses were partially impaired, suggesting that naive CD8+ T cells are more dependent on cis presentation of NKTR-255 than memory CD8+ T cells. In bone marrow chimera studies, IL-15Rα-/- and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rß agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαß agonist on CD8+ T cells.

Scaffolding LSD1 Inhibitors Impair NK Cell Metabolism and Cytotoxic Function Through Depletion of Glutathione.

Cell therapies such as chimeric-antigen receptor (CAR) T-cells and NK cells are cutting-edge methods for treating cancer and other diseases. There is high interest in optimizing drug treatment regimens to best work together with emerging cell therapies, such as targeting epigenetic enzymes to stimulate recognition of tumor cells by immune cells. Herein, we uncover new mechanisms of the histone demethylase LSD1, and various inhibitors targeting unique domains of LSD1, in the function of NK cells grown for cell therapy. Catalytic inhibitors (tranylcypromine and the structural derivatives GSK LSD1 and RN-1) can irreversibly block the demethylase activity of LSD1, while scaffolding inhibitors (SP-2509 and clinical successor SP-2577, also known as seclidemstat) disrupt epigenetic complexes that include LSD1. Relevant combinations of LSD1 inhibitors with cell therapy infusions and immune checkpoint blockade have shown efficacy in pre-clinical solid tumor models, reinforcing a need to understand how these drugs would impact T- and NK cells. We find that scaffolding LSD1 inhibitors potently reduce oxidative phosphorylation and glycolysis of NK cells, and higher doses induce mitochondrial reactive oxygen species and depletion of the antioxidant glutathione. These effects are unique to scaffolding inhibitors compared to catalytic, to NK cells compared to T-cells, and importantly, can fully ablate the lytic capacity of NK cells. Supplementation with biologically achievable levels of glutathione rescues NK cell cytolytic function but not NK cell metabolism. Our results suggest glutathione supplementation may reverse NK cell activity suppression in patients treated with seclidemstat.

Pharmacologic inhibition of lysine-specific demethylase 1 as a therapeutic and immune-sensitization strategy in pediatric high-grade glioma.

BACKGROUND: Diffuse midline gliomas (DMG), including brainstem diffuse intrinsic pontine glioma (DIPG), are incurable pediatric high-grade gliomas (pHGG). Mutations in the H3 histone tail (H3.1/3.3-K27M) are a feature of DIPG, rendering them therapeutically sensitive to small-molecule inhibition of chromatin modifiers. Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) is clinically relevant but has not been carefully investigated in pHGG or DIPG. METHODS: Patient-derived DIPG cell lines, orthotopic mouse models, and pHGG datasets were used to evaluate effects of LSD1 inhibitors on cytotoxicity and immune gene expression. Immune cell cytotoxicity was assessed in DIPG cells pretreated with LSD1 inhibitors, and informatics platforms were used to determine immune infiltration of pHGG. RESULTS: Selective cytotoxicity and an immunogenic gene signature were established in DIPG cell lines using clinically relevant LSD1 inhibitors. Pediatric HGG patient sequencing data demonstrated survival benefit of this LSD1-dependent gene signature. Pretreatment of DIPG with these inhibitors increased lysis by natural killer (NK) cells. Catalytic LSD1 inhibitors induced tumor regression and augmented NK cell infusion in vivo to reduce tumor burden. CIBERSORT analysis of patient data confirmed NK infiltration is beneficial to patient survival, while CD8 T cells are negatively prognostic. Catalytic LSD1 inhibitors are nonperturbing to NK cells, while scaffolding LSD1 inhibitors are toxic to NK cells and do not induce the gene signature in DIPG cells. CONCLUSIONS: LSD1 inhibition using catalytic inhibitors is selectively cytotoxic and promotes an immune gene signature that increases NK cell killing in vitro and in vivo, representing a therapeutic opportunity for pHGG. KEY POINTS: 1. LSD1 inhibition using several clinically relevant compounds is selectively cytotoxic in DIPG and shows in vivo efficacy as a single agent.2. An LSD1-controlled gene signature predicts survival in pHGG patients and is seen in neural tissue from LSD1 inhibitor-treated mice.3. LSD1 inhibition enhances NK cell cytotoxicity against DIPG in vivo and in vitro with correlative genetic biomarkers.

IL-15 is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses.

Previous studies have provided evidence that IL-15 expression within human tumors is crucial for optimal antitumor responses; however, the regulation of IL-15 within the tumor microenvironment (TME) is unclear. We report herein, in analyses of mice implanted with various tumor cell lines, soluble IL-15/IL-15Rα complexes (sIL-15 complexes) are abundant in the interstitial fluid of tumors with expression preceding the infiltration of tumor-infiltrating lymphocytes. Moreover, IL-15 as well as type I IFN, which regulates IL-15, was required for establishing normal numbers of CD8 T cells and natural killer cells in tumors. Depending on tumor type, both the tumor and the stroma are sources of sIL-15 complexes. In analyses of IL-15 reporter mice, most myeloid cells in the TME express IL-15 with CD11b+Ly6Chi cells being the most abundant, indicating there is a large source of IL-15 protein in tumors that lies sequestered within the tumor stroma. Despite the abundance of IL-15-expressing cells, the relative levels of sIL-15 complexes are low in advanced tumors but can be up-regulated by local stimulator of IFN genes (STING) activation. Furthermore, while treatment of tumors with STING agonists leads to tumor regression, optimal STING-mediated immunity and regression of distant secondary tumors required IL-15 expression. Overall, our study reveals the dynamic regulation of IL-15 in the TME and its importance in antitumor immunity. These findings provide insight into an unappreciated attribute of the tumor landscape that contributes to antitumor immunity, which can be manipulated therapeutically to enhance antitumor responses.