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

Abstract

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.

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

“…We next examined if NK cell sensitivity to scaffolding LSD1 inhibitors was dose and time dependent, and we found higher doses and longer incubation times amplified the cytotoxic effect ( Figure 1E, q < 0.001). Our previous publication also found metabolic suppression unique to scaffolding LSD1 inhibitors in NK cells (7). We were able to replicate these findings using numerous unique NK cell donors, observing that scaffolding LSD1 inhibitors abolish all oxidative phosphorylation in NK cells ( Figure 1F, * q < 0.01) and reduce OXPHOS to a much lesser degree in T-cells ( Figure 1G, * q < 0.01).…”

“…Our group has previously published that the scaffolding LSD1 inhibitor SP-2509 and its clinical successor SP-2577, or seclidemstat, potently suppress the viability and metabolism of NK cells (7). The NK cells in our previous report and this current study are expanded from healthy human donors into a cell therapy-grade product.…”

“…Scaffolding inhibitor SP-2509 acts through a potential allosteric mechanism ( 21 ) and can disrupt LSD1 in complex with CoREST ( 8 ) in addition to the demethylation function ( Figure 1A ). We previously observed that scaffolding LSD1 inhibitors were more potent against NK cells compared to T-cells, into the nanomolar range for NK cells, using the AlamarBlue assay ( 7 ). Here we replicated doses of LSD1 inhibitors we previously characterized to induce NK reactivity in pediatric brain tumors without exerting direct tumor cytotoxicity, as a model of co-administration, and measured viability using amine-reactive dyes and flow cytometry.…”

“…Among tumors with low mutational burdens, it has been proposed that epigenetic inhibitors can make these cancers more visible to the immune system by activating gene expression programs (4). Recently, it has been demonstrated that LSD1 inhibition can accomplish this by stimulating T-cell immunity in epithelial cancers (5,6) and innate immunity in pediatric brain tumors (7).…”

Histone-Mutant Glioma: Molecular Mechanisms, Preclinical Models, and Implications for Therapy

“…A more expansive in vitro followed by in vivo screen of the combinatorial druggable DMG landscape, with clinically relevant factors such as potency and blood–brain-barrier penetrance taken into account, revealed the proteasome inhibitor marizomib combined with panobinostat as the most promising pairing [ 88 ]. Additional in vivo studies have suggested inhibition of lysine demethylase [ 89 , 90 ], DNA methylation [ 63 ], AXL kinase [ 91 ], and PI3K [ 92 ] as promising combination strategies with panobinostat, several of which are now in clinical trials.…”