Pilot study evaluating everolimus molecular mechanisms in tuberous sclerosis complex and focal cortical dysplasia

Abstract

Background: Tuberous sclerosis complex (TSC) and some focal cortical dysplasias (FCDs) are associated with dysfunctional mTOR signaling, resulting in increased cell growth and ribosomal S6 protein phosphorylation (phospho-S6). mTOR inhibitors can reduce TSC tumor growth and seizure frequency, and preclinical FCD studies indicate seizure suppression. This pilot study evaluated safety of mTOR inhibitor everolimus in treatment resistant (failure of >2 anti-seizure medications) TSC and FCD patients undergoing surgical resection and to assess mTOR signaling and molecular pathways.

Methods and findings: We evaluated everolimus in 14 treatment resistant epilepsy patients undergoing surgical resection (4.5 mg/m2 daily for 7 days; n = 4 Active, mean age 18.3 years, range 4-26; n = 10, Control, mean age 13.1, range 3-45). Everolimus was well tolerated. Mean plasma everolimus in Active participants were in target range (12.4 ng/ml). Brain phospho-S6 was similar in Active and Control participants with a lower trend in Active participants, with Ser235/236 1.19-fold (p = 0.67) and Ser240/244 1.15-fold lower (p = 0.66). Histologically, Ser235/236 was 1.56-fold (p = 0.37) and Ser240/244 was 5.55-fold lower (p = 0.22). Brain proteomics identified 11 proteins at <15% false discovery rate associated with coagulation system (p = 1.45x10-9) and acute phase response (p = 1.23x10-6) activation. A weighted gene correlation network analysis (WGCNA) of brain proteomics and phospho-S6 identified 5 significant modules. Higher phospho-S6 correlated negatively with cellular respiration and synaptic transmission and positively with organophosphate metabolic process, nuclear mRNA catabolic process, and neuron ensheathment. Brain metabolomics identified 14 increased features in Active participants, including N-acetylaspartylglutamic acid. Plasma proteomics and cytokine analyses revealed no differences.

Conclusions: Short-term everolimus before epilepsy surgery in TSC and FCD resulted in no adverse events and trending lower mTOR signaling (phospho-S6). Future studies should evaluate implications of our findings, including coagulation system activation and everolimus efficacy in FCD, in larger studies with long-term treatment to better understand molecular and clinical effects.

Clinical trials registration: ClinicalTrials.gov NCT02451696.

Fig 1. Ribosomal S6 phosphorylation in whole brain homogenate.

A) Representative western blot images from surgically resected brain tissue of Active participants after taking 4.5 mg/m² everolimus for 7 days and controls. Total S6, phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), and beta-actin as loading control were evaluated in 14 participants. TSC participants are indicated “*”, while all others are FCD participants. B) Quantification of total S6 relative to actin indicates variability in baseline levels from sampled brain tissue, as well as one patient with increased expression of a smaller detected band (NYU-002). C) Percentage of phospho-S6 (Ser235/236) relative to total S6 indicates an average 1.19-fold decrease when comparing all Active participants to controls (p = 0.67). The highest level was seen in NYU-004. D) Percentage of phospho-S6 (Ser240/244) relative to total S6 indicates an average 1.15-fold decrease when comparing all Active participants to controls (p = 0.66). The highest level was seen in NYU-004. Error bars indicate SEM.

Fig 2. Histological quantification of ribosomal S6 phosphorylation in gray matter.

Representative images show total S6, phospho-S6 (Ser235/236), and phospho-S6 (Ser240/244) (red) in resected cortical tissue from FCD participants that were Controls A-C) or Active D-F) and in TSC participants that were Controls G-I) or Active J-L). M) Quantification of total S6 expression in the gray matter indicated no difference in baseline expression between Active and Control participants. N) Quantification of percent phospho-S6 (Ser235/236) relative to total S6 in the gray matter indicated an average 1.56-fold decrease when comparing all Active participants to Controls (p = 0.37). O) Quantification of percent phospho-S6 (Ser240/244) relative to total S6 in the gray matter indicated an average 5.55-fold decrease when comparing all Active participants to Controls (p = 0.22). Histology was performed on all cases with available FFPE, excluding 1 Active FCD case. Error bars indicate SEM. Scale bar represents 100 μm.

Fig 3. Proteomics in whole brain homogenate.

Proteomics was performed in surgically resected brain tissue from Active and Control participants. A) PCA in brain tissue indicated no segregation of Active and Control participants in PCA1 (p = 0.73) and with some segregation in PCA2 (p = 0.016). Clinical diagnoses (FCD, TSC) are noted as well. B) Differential expression analysis in brain tissue indicated that there were 11 significantly altered proteins in Active versus Control participants at an FDR < 15% (dotted line), detailed in S1 Table and S1 Fig. There were no significant proteins at FDR < 5–10%. C) WGCNA of brain proteomics and phospho-S6 evaluated in total brain homogenate by western blot, regardless of everolimus and clinical diagnoses. There were 1109 proteins that correlated with phospho-S6 levels (P235/236, p < 0.05), distributed across 10 modules (all but M-Purple). The top significantly correlated protein with P235/236 was a positive correlation to HSPA2 (p = 1.18 x 10⁻⁶, R² = 0.87) in the M-Brown module. D) The top significantly correlated protein with P240/244 was a negative correlation with ANK2 (p = 7.92 x 10⁻⁵, R² = 0.74) in the M-Brown module. E) Module trait correlation identified 5 significantly associated modules with phospho-S6 (p < 0.05). Modules were clustered by eigenprotein adjacency (relatedness to other modules) on the left. Name of module is indicated by “M-color.” P values are indicated for those modules with p < 0.05 correlation. Positive correlation is indicated in red and negative correlation in blue. Top module GO annotations are noted on the right (FDR < 5%, at least 5 proteins/annotation). Several modules did not have a significant GO annotation (“n.s.”).

Fig 4. Metabolomics in whole brain homogenate.

Global polar metabolomics by LC-MS was performed on surgically resected brain tissue from Active and Control participants. A) PCA indicated distribution of participants, as well as clinical diagnoses (FCD, TSC). There was no segregation of Active and Control participants in PCA1 (p = 0.32) or PCA2 (p = 0.48). B) Differential expression analysis (p < 0.05, log₂ (fold change) > 1) identified 14 metabolite features that were increased in Active participants, associated with the indicated annotations detailed in C).

Fig 5. Plasma proteomics and cytokine levels.

A) Proteomics was performed on plasma obtained at time of surgical resection after 7 days everolimus. PCA indicates no segregation of Active from Control participants, nor by clinical diagnoses (FCD, TSC). B) Differential expression analysis of plasma proteins indicated no altered proteins in Active versus Control participants at FDR < 5%, nor at FDR < 15%. C-K) Plasma cytokine levels were evaluated at time of surgical resection. There were no significant differences in the cytokines evaluated.