OTHR-13. Combination LP-184 and spironolactone induce apoptosis in medulloblastoma and diffuse midline glioma

Abstract Medulloblastoma is the most common malignant brain tumor in children. Cure rates reach as high as 60% with the standard treatment of radiation and chemotherapy, however treatment-related neurological deficits persist long-term. Diffuse midline glioma (DMG) is a malignant pediatric brain tumor with fatal outcomes and currently no successful treatment options. LP-184 is a novel acylfulvene that is currently in phase I clinical trials for glioblastoma, brain metastases, and other solid tumors. LP-184 alkylates DNA and activates nucleotide excision repair (NER). The ERCC3 protein is involved in NER and may be a potential target in this pathway. Spironolactone is a commonly used diuretic drug which binds to ERCC3 and targets it for ubiquination and proteolysis. Both medulloblastoma and DMG exhibit markers of replicative stress, which may make them susceptible to LP-184 and spironolactone treatment. We hypothesized that LP-184 and spironolactone in medulloblastoma and DMG would exacerbate replicative stress, leading to increased apoptosis. We determined the IC50 of LP-184 in two medulloblastoma cell lines (D283 and MED211) and two DMG cell lines (BT245 and HSJD007). We performed western blots to measure ERCC3 knockdown. In all cell lines, spironolactone decreased ERCC3 protein levels by at least 50 percent compared to DMSO treated samples. LP-184 and spironolactone induced apoptosis as measured by cleaved PARP western blot and cleaved caspase 3 (CC3) immunofluorescence (DMSO vs. combination D283 p 0.0001, DMSO vs. combination BT245 p 0.0001, DMSO vs. combination HSJD007 p 0.0001 by ANOVA and Tukey’s multiple comparisons test). In vivo experiments are currently being planned. The combination of LP-184 and spironolactone has the potential to serve as a therapy for medulloblastoma and DMG. Our data may inform a planned phase 1 clinical trial of LP-184 in patients with pediatric brain tumors.