Dual targeting of the mitochondrial Lon peptidase 1 and the chymotrypsin-like proteasome activity as a potential therapeutic strategy in malignant astrocytoma models.

Malignant astrocytomas are aggressive primary brain tumors characterized by extensive hypoxia-induced, mitochondria-dependent changes such as altered respiration, increased chymotrypsin-like (CT-L) proteasome activity, decreased apoptosis, drug resistance, stemness, and increased invasiveness. Mitochondrial Lon Peptidase 1 (LonP1) overexpression and increased CT-L proteasome activity are biomarkers of an aggressive high-grade phenotype and found to be associated with recurrence and poor patient survival. In preclinical models, small molecule agents targeting either LonP1 or the proteasome CT-L activity have anti-astrocytoma activity. Here, we present evidence that the dual inhibition of LonP1 and CT-L proteasome activity effectively induces ROS production, leading to apoptosis in malignant astrocytoma established cell lines and patient-derived glioma stem cell-like cultures. We also evaluated a novel small molecule, BT317, derived from the coumarinic compound 4 (CC4) using structure-activity modeling, which we found to inhibit both LonP1 and CT-L proteasome activity. Using gain- and loss-of-function genetic models, we discovered that LonP1 is both necessary and sufficient to drive BT317 drug sensitivity in established and patient-derived glioma stem-like cells by generating ROS and inducing apoptosis. In vitro, BT317 had activity as a single agent but, more importantly, enhanced synergy with the standard of care commonly used chemotherapeutic temozolomide (TMZ). In an orthotopic xenograft astrocytoma model, BT317 crossed the blood-brain barrier, showed selective activity at the tumor site, and demonstrated therapeutic efficacy as a single agent and combined with TMZ. BT317 defines an emerging class of LonP1 and CT-L inhibitors that exhibited promising anti-tumor activity and could be a potential candidate for malignant astrocytoma therapeutics. SIMPLE SUMMARY: Malignant astrocytoma patients have poor clinical outcomes, and novel treatments are needed to limit tumor recurrence and improve their overall survival. These tumors have a malignant phenotype mediated by altered mitochondrial metabolism, abnormal protein processing, and adaptation to hypoxia. We have previously published that astrocytomas are especially vulnerable to proteasome inhibitors as well as to inhibitors of the mitochondrial Lon Peptidase 1 (LonP1), but the effect of combining the two strategies has not been reported. Here, we present evidence that the dual inhibition of LonP1 and Chymotrypsin-like (CT-L) proteasome activity effectively induces cellular reactive oxygen species (ROS) production, leading to apoptosis in malignant astrocytoma established cell lines and patient-derived glioma stem cell-like cultures. We developed BT317, a small molecule dual inhibitor, which crosses the blood-brain barrier and shows strong synergy with the standard of care, temozolomide (TMZ), in the astrocytoma cell lines independent of their isocitrate dehydrogenase (IDH) profile and in an orthotopic glioma murine model. This preclinical study demonstrated the potential of dual LonP1 and CT-L proteasome inhibition as a novel therapeutic strategy for malignant astrocytoma and provides insight for future clinical translational studies alone or in combination with other chemotherapies.