Discovery and Validation of Novel LonP1 and Proteasome Inhibitor in IDH1-R132H Malignant Astrocytoma Models

Abstract

AbstractMalignant astrocytomas are aggressive glioma tumors that have poor prognosis and limited treatments available following recurrence. These tumors are characterized by extensive hypoxia-induced, mitochondria-dependent changes such as glycolytic respiration, increased chymotrypsin-like (CT-L) proteasome activity, decreased apoptosis, and increased invasiveness. Mitochondrial Lon Peptidase 1 (LonP1) is an ATP-dependent protease directly upregulated by hypoxia-inducible factor 1 alpha (HIF-1α). Both LonP1 expression and CT-L proteasome activities are increased in gliomas and are associated with a high tumor grade and poor patient survival. Recently, dual LonP1 and CT-L inhibition has been found to exhibit synergy against multiple myeloma cancer lines.We now report that dual LonP1 and CT-L inhibition has synergistic toxicity in IDH mutant astrocytoma when compared to IDH wildtype glioma, due to increased reactive oxygen species (ROS) generation and autophagy. A novel small molecule, BT317, was derived from coumarinic compound 4 (CC4) using structure-activity modeling and was found to inhibit both LonP1 and CT-L proteasome activity and subsequently induce ROS accumulation and autophagy-dependent cell death in high-grade IDH1 mutated astrocytoma lines. In vitro, BT317 also had enhanced synergy with the commonly used chemotherapeutic temozolomide (TMZ), which blocked BT317-induced autophagy. This novel dual inhibitor was selective to the tumor microenvironment and demonstrated therapeutic efficacy both as a single agent and in combination TMZ in IDH mutant astrocytoma models. We show that BT317, a dual LonP1, and CT-L proteasome inhibitor exhibited promising anti-tumor activity and could be a promising candidate for clinical translation in the space of IDH mutant malignant astrocytoma therapeutics.Data Access StatementResearch data supporting this publication are as presented in the manuscript.HighlightsThe novel compound BT317 acts as a LonP1 and chymotrypsin-like proteasome inhibitorLonP1 and CT-L proteasome inhibition drives ROS production in IDH mutant astrocytomaLonP1 and CT-L proteasome inhibition drives autophagy in IDH mutant astrocytomaBT317 shows blood-brain barrier permeability and has low normal tissue toxicityBT317 synergizes with the first-line chemotherapy agent TMZImportance of StudyMalignant astrocytomas (IDH mutant astrocytomas grade 4 and IDH wildtype glioblastoma) have poor clinical outcomes, and novel treatments are needed to limit recurrence and improve overall survival. These tumors have a malignant phenotype that is mediated by altered mitochondrial metabolism and adaptation to hypoxia. Here, we present evidence that the small-molecule inhibitor BT317, with a dual Lon Peptidase 1 (LonP1) and chymotrypsin-like (CT-L) inhibition profile, can be effectively used to induce increased ROS production and autophagy-dependent cell death in clinically relevant, IDH mutant malignant astrocytoma, patient-derived orthotopic models. BT317 showed strong synergy with the standard of care, temozolomide (TMZ), in the IDH mutant astrocytoma models. This could lead to the development of dual LonP1 and CT-L proteasome inhibitors as novel therapeutic strategies for IDH mutant astrocytoma and provide insight for future clinical translation studies in combination with the standard of care.Graphical AbstractGraphical Abstract. BT317, a novel dual Chymotrypsin-like proteasome and LonP1 inhibitor drives autophagy-dependent cell death and exhibits synergy with TMZ through induction of the mTOR signaling pathway