Targeting AKT and CK2 represents a novel therapeutic strategy for SMO constitutive activation-driven medulloblastoma

Abstract

Aims: Sonic hedgehog subtype medulloblastoma is featured with overactivation of hedgehog pathway and can be targeted by SMO-specific inhibitors. However, the resistance is frequently developed leading to treatment failure of SMO inhibitors. W535L mutation of SMO (SMO^W535L ) is thought to be an oncogenic driver for Sonic hedgehog subtype MB and confer resistance to SMO inhibitors. The regulation network of SMO^W535L remains to be explored in comparison with wild-type SMO (SMO^WT ).

Methods: In this study, we profiled transcriptomes, methylomes, and interactomes of MB cells expression SMOWT or SMOW535L in the treatment of DMSO or SMO inhibitor, respectively.

Results: Analysis of transcriptomic data indicated that SMO inhibitor disrupted processes of endocytosis and cilium organization in MB cells with SMO^WT , which are necessary for SMO activation. In MB cells with SMO^W535L , however, SMO inhibitor did not affect the two processes-related genes, implying resistance of SMO^W535L toward SMO inhibitor. Moreover, we noticed that SMO inhibitor significantly inhibited metabolism-related pathways. Our metabolic analysis indicated that nicotinate and nicotinamide metabolism, glycerolipid metabolism, beta-alanine metabolism, and synthesis and degradation of ketone bodies might be involved in SMO^W535L function maintenance. Interactomic analysis revealed casein kinase II (CK2) as an important SMO-associated protein. Finally, we linked CK2 and AKT together and found combination of inhibitors targeting CK2 and AKT showed synergetic effects to inhibit the growth of MB cells with SMO constitutive activation mutation.

Conclusions: Taken together, our work described SMO-related transcriptomes, metabolomes, and interactomes under different SMO status and treatment conditions, identifying CK2 and AKT as therapeutic targets for SHH-subtype MB cells with SMO inhibitor resistance.

Keywords: CK2; SMO; W535L mutation; constitutive activation; medulloblastoma.

FIGURE 1

Transcriptomic analysis on MB cells with SMO^WT or SMO^W535L. (A) Western blotting of MB cells expressing flag tagged SMO^WT treated with indicated reagents. (B) Western blotting of MB cells expressing flag tagged SMO^W535L treated with indicated reagents. (C) IC₅₀ of LDE225 in MB cells with SMO^WT or SMO^W535L. (D) PCA on transcriptomes of SMO^WT or SMO^W535L MB cells treated with indicated reagents. (E) Volcano graph of altered genes by LDE225 vs. DMSO in MB cells with SMO^WT (Upper panel) or SMO^W535L (Lower panel). (F) Geneset enrichment in the context of GO and KEGG terms for genes altered by LDE225 vs. DMSO in MB cells with SMO^WT. (G) Venn diagram of significantly downregulated genes by LDE225 vs. DMSO and gene enrichment in the context of GO and KEGG terms

FIGURE 2

Metabolomic analysis on MB cells with SMO^WT or SMO^W535L. (A) PCA on metabolomes of SMO^WT or SMO^W535L MB cells treated with indicated reagents. (B)–(E) KEGG analysis on significantly changed metabolites in different comparison groups

FIGURE 3

Interactomic analysis on MB cells with SMO^WT or SMO^W535L. (A) IP‐western confirmation of SMO expression subjected to MASS‐Spectrum. (B) Geneset enrichment in the context of KEGG term for proteins interacted with SMO^WT or SMO^W535L in the indicated treatment condition. (C) Venn diagram of proteins interacted with SMO^WT or SMO^W535L in the indicated treatment condition. (D) Heatmap cluster analysis on transcriptomes of high and low expression of SMO, AKT, and CK2, respectively, using SHH‐subtype MB from GSE85217 dataset

FIGURE 4

Synergetic effects of CX4945 and MK2206 for MB cells with SMO^W535L. (A) Measurement of combination index of CX4945 plusMK2206 in three MB cells with SMO^W535L. (B) Colony formation of three MB cells with SMO^W535L in treatment of CX4945 or/and MK2206. The data are shown as mean ± SD; ***p < 0.001; **p < 0.01; n = 4 per group. (C) Western blotting of MB cells expressing flag tagged SMO^W535L treated with indicated reagents. (D) Representative images of orthotopic growth of ONS76 cells with SMO^W535L treated with vehicle, CX4945, MK2206, or MK+CX). (E) Statistic graph of tumor size using bioluminescence signal intensity. The data are shown as mean ± SD; ***p < 0.001; **p < 0.01; n = 8 per group. (F) Kaplan–Meier survival analysis of mice treated with indicated conditions. n = 8 per group