Proteasomal pathway inhibition as a potential therapy for NF2-associated meningioma and schwannoma

Abstract

Background: Neurofibromatosis 2 (NF2) is an inherited disorder caused by bi-allelic inactivation of the NF2 tumor suppressor gene. NF2-associated tumors, including schwannoma and meningioma, are resistant to chemotherapy, often recurring despite surgery and/or radiation, and have generally shown cytostatic response to signal transduction pathway inhibitors, highlighting the need for improved cytotoxic therapies.

Methods: Leveraging data from our previous high-throughput drug screening in NF2 preclinical models, we identified a class of compounds targeting the ubiquitin-proteasome pathway (UPP), and undertook studies using candidate UPP inhibitors, ixazomib/MLN9708, pevonedistat/MLN4924, and TAK-243/MLN7243. Employing human primary and immortalized meningioma (MN) cell lines, CRISPR-modified Schwann cells (SCs), and mouse Nf2-/- SCs, we performed dose response testing, flow cytometry-based Annexin V and cell cycle analyses, and RNA-sequencing to identify potential underlying mechanisms of apoptosis. In vivo efficacy was also assessed in orthotopic NF2-deficient meningioma and schwannoma tumor models.

Results: Testing of three UPP inhibitors demonstrated potent reduction in cell viability and induction of apoptosis for ixazomib or TAK-243, but not pevonedistat. In vitro analyses revealed that ixazomib or TAK-243 downregulates expression of c-KIT and PDGFRα, as well as the E3 ubiquitin ligase SKP2 while upregulating genes associated with endoplasmic reticulum stress-mediated activation of the unfolded protein response (UPR). In vivo treatment of mouse models revealed delayed tumor growth, suggesting a therapeutic potential.

Conclusions: This study demonstrates the efficacy of proteasomal pathway inhibitors in meningioma and schwannoma preclinical models and lays the groundwork for use of these drugs as a promising novel treatment strategy for NF2 patients.

Keywords: NF2; apoptosis; meningioma; schwannoma; ubiquitin–proteasome pathway inhibitors.

Graphical abstract

Figure 1.

Treatment with ubiquitin-proteasome pathway inhibitors leads to reduced cell viability and apoptosis in NF2-deficient MN cell lines. (A) Dose-response curves (DRCs), representing percent cell viability relative to DMSO, are shown for human NF2-null MN lines, including immortalized Ben-Men-1 and 5 primary MN lines treated with TAK-243, ixazomib, or pevonedistat. Cell lines were treated with 9 dosage points (1.5 nM–10 μM, 1:3 serial dilution) for each drug representing 3 biological replicates (performed in triplicate) ± SEM. (B–E) Assessment of apoptosis was carried out by flow cytometry (B and D, numbers shown in upper and lower right quandrants for each plot represent percentage) and immunoblotting for cleaved-PARP (C and E). Treatment with TAK-243 or ixazomib (24 h) led to increased apoptosis in Ben-Men-1 and MN1-LF cells (B and C) and primary MN lines MN597 and MN647D (D and E).

Figure 2.

Transcriptome analysis reveals differentially expressed genes in NF2-deficient MN lines treated with UPP inhibitors. (A) Volcano plots of differentially expressed genes (DEGs) from drug-treated cell lines vs DMSO controls with the top 5% DEGs at Bonferroni-adjusted P-value (Bf) < .05 (maroon), the remaining DEGs at P-value (Bf) < .05 (dark blue), and the DEGs at P-value (Bf) > .05 (grey) shown. BH, Benjamini–Hochberg adjusted P-value. (B) Venn diagrams represent numbers of top 5% DEGs shared between Ben-Men-1 and MN1-LF upon treatment with TAK-243 (left) or ixazomib (right). Numbers within parenthesis denote DEGs in the same direction of dysregulation (up or down). (C and D) GO terms enriched for molecular function upon TAK-243 (C) or ixazomib (D) treatment that are upregulated (right-shifted from 0) or downregulated (left-shifted from 0) are shown, along with corresponding numbers of DEGs from the RNA-seq dataset.

Figure 3.

Treatment with proteasome pathway inhibitors leads to decreased expression of proto-oncogenes in NF2-null MN lines. (A) Quantification of qRT-PCR for KIT, PDGFRA, and SKP2 of Ben-Men-1 and MN1-LF cell lines demonstrated significant decrease in the expression levels of all three genes upon 24 h treatment with 100 nM TAK-243 or ixazomib. (B) Immunoblotting of Ben-Men-1 and MN1-LF cells treated with TAK-243 or ixazomib (12 and 24 h, 100 nM) for c-KIT and PDGFRα revealed decreased expression. β-actin serves as a loading control. (C) Immunoblotting of treated MN1-LF cells demonstrated decreased expression of SKP2 by 48 h or 72 h with 100 nM of TAK-243 or ixazomib, respectively. (D) Immunoblotting revealed increased p21, p27, and cyclinD1 in Ben-Men-1 and MN1-LF cells treated for 12 and 24 h with 100 nM TAK-243 or ixazomib. (E and F) Cell cycle data for Ben-Men-1 (n = 4) and MN1-LF (n = 2) treated for 24 h with TAK-243 or ixazomib are shown (F as a representative). Quantification for percentage of cells in cell cycle phases G0/G1, S, and G2/M is shown ± SEM (F). *P < .05, **P < .01, ***P < .001 (for A).

Figure 4.

UPP inhibition leads to activation of the unfolded protein response in NF2-null MN cells. (A and C) Immunoblotting confirmed increased expression of unfolded protein response (UPR) markers GRP78, spliced XBP1 (XBP1s), and ATF3 in Ben-Men-1 (A) and MN1-LF (C) cell lines following 12 and 24 h treatment with 100 nM of TAK-243 or ixazomib. (B and D) Quantitation of 24 h treatment in Ben-Men-1 (B) and MN1-LF (D) using ImageJ/Fiji is shown representing the average of 3 replicates (DMSO, normalized to 1) ± SEM. (E) Cell viability assays, in MN1-LF cells pretreated for 24 h with 5 µM of IREα inhibitor 4µ8C, followed by 48 h treatment with ~41 nM or ~124 nM of ixazomib (4µ8C+Ixa) showed significant increase in percent viability compared to DMSO pretreatment (DMSO+Ixa). (DMSO alone, normalized to 100). Each bar represents average of 3 replicates ± SEM; *P < .05; **P < .01; ns, not significant.

Figure 5.

UPP inhibitor treatment leads to tumor shrinkage in mouse models of NF2-related meningioma and schwannoma. (A) SchematicFigure 5. Continuedrepresentation of drug treatment timeline for in vivo NF2-null Grade III meningioma mouse model. (B) Treatment with TAK-243 (left) or ixazomib (right) suppressed tumor growth in the KT21-MG1-Luc5D skull-base xenograft model. Relative tumor-emitted bioluminescent signals are calculated as the percentage of total photon flux during treatment relative to the pretreatment total flux, denoted as 1. Data are shown as mean ± SD for each treatment group. (C) Schematic representation of ixazomib treatment timeline for in vivo schwannoma mouse models. (D) Tumor growth curves of mouse Nf2^−/− sciatic nerve model (left) and patient-derived VS4 sciatic nerve model (right). Schwannoma mouse model studies presented are representative of at least three independent experiments, n = 8 per group in each experiment and data presented as mean ± SEM. P-values are shown in the figure.