Small Molecule 20S Proteasome Enhancer Regulates MYC Protein Stability and Exhibits Antitumor Activity in Multiple Myeloma

Abstract

Despite the addition of several new agents to the armamentarium for the treatment of multiple myeloma (MM) in the last decade and improvements in outcomes, the refractory and relapsing disease continues to take a great toll, limiting overall survival. Therefore, additional novel approaches are needed to improve outcomes for MM patients. The oncogenic transcription factor MYC drives cell growth, differentiation and tumor development in many cancers. MYC protein levels are tightly regulated by the proteasome and an increase in MYC protein expression is found in more than 70% of all human cancers, including MM. In addition to the ubiquitin-dependent degradation of MYC by the 26S proteasome, MYC levels are also regulated in a ubiquitin-independent manner through the REGγ activation of the 20S proteasome. Here, we demonstrate that a small molecule activator of the 20S proteasome, TCH-165, decreases MYC protein levels, in a manner that parallels REGγ protein-mediated MYC degradation. TCH-165 enhances MYC degradation and reduces cancer cell growth in vitro and in vivo models of multiple myeloma by enhancing apoptotic signaling, as assessed by targeted gene expression analysis of cancer pathways. Furthermore, 20S proteasome enhancement is well tolerated in mice and dogs. These data support the therapeutic potential of small molecule-driven 20S proteasome activation for the treatments of MYC-driven cancers, especially MM.

Keywords: MYC; chemotherapy; multiple myeloma; novel treatment; protein degradation; proteosome.

Figure 1

(A) Structure of TCH-165 and its proposed model of α-ring binding, allowing the gate to open for easy access of disordered proteins to the internal proteolytic sites; (B) EC₂₀₀ values of TCH-165 and maximum fold enhancement of 20S activities for each of the three catalytic sites.

Figure 2

(A) Multiple Myeloma (RPMI-8226) cells treated with vehicle or TCH-165 (4 h, 5 mM) in the presence and absence of proteasome inhibitor BTZ (5 μM); (B) percent MYC remaining in L363 cells treated with various concentrations of TCH-165 (n = 3); (C) percent MYC remaining in H929 cells treated with various concentrations of TCH-165 (n = 4). * p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 3

MYC-luciferase reporter assay: (A) MYC-luciferase reporter assay in stable transfected HCT-116 cells treated with various concentrations of TCH-165 (EC₅₀ 2.57 μM; 95% CI 2.46–2.95); (B) MYC-luciferase reporter assay in stably transfected HCT-116 cells treated with vehicle, TCH-165 (3 μM), bortezomib (BTZ, 10 nM) and bortezomib (2 h pre-incubation, 10 nM) followed by TCH-165 (3 μM). *** p < 0.001.

Figure 4

(A) Volcano scatter plots of genes in cancer pathways analyzed by the PanCancer multiplexed transcript detection assay. Dotted vertical lines indicate a 1.5-fold change in normalized gene expression, with overexpressed genes in green and under-expressed genes in purple. Labels indicate genes showing over 2-fold change; (B) Changes in gene expression observed over three independent experiments indicating transcriptional misregulation are presented in the heatmap. Consistent with the upregulation of the DNA damage response, we demonstrated induction of apoptosis in all three MM cell lines (Figure S1).

Figure 5

In vivo efficacy and tolerance of the proteasome activator, TCH-165. (A) Comparative analysis AUC, Tmax and Cmax of TCH-165 bid in mice (n = 5) and dogs (n = 3), calculated using PKSolver [45]. (B) RPMI-8226 subcutaneous xenograft model in SCID mice treated with vehicle (3:7 (v/v) propylene glycol/5% dextrose), and TCH-165 (100 mg/kg, bid with oral gavage), bortezomib (at 0.375 mg/kg day 0, 0.18 mg/kg day 2, 0.09 mg/kg day 5 and beyond) was given 3X per week intravenously. Tumor volumes show a significant difference between TCH-165 vs. control as well as TCH-165 vs. bortezomib groups at FDR of 0.0014 and 0.0127, respectively, using the pairwise Wilcoxon rank-sum test with the Benjamini–Hochberg adjustment, after application of Kruskal–Wallis test showing significant p-value of 0.0286 across all treatments, calculated using R Stats v3.6.2.