Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation

Abstract

The N⁶-methyladenosine (m⁶A) RNA modification is an important regulator of gene expression. m⁶A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m⁶A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m⁶A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Keywords: CP: Cancer; differentiation; epitranscriptome; gene expression regulation; m(6)A; neuroblastoma; therapeutic target; tumor growth.

Figure 1.. METTL3/METTL14 expression correlates with survival in patients with neuroblastoma, and METTL3 siRNA knockdown induces neuroblastoma cell differentiation

(A and B) Event-free survival (A) and overall survival (B) are significantly inferior for patients in the SEQC-NB cohort (n = 498) with neuroblastomas that express high vs. low levels of METTL3/METTL14 expression by log-rank test (p < 0.05). (C) Violin plot showing high-risk patients in the SEQC-NB cohort had significantly higher METTL3/METTL14 expression compared to non-high-risk patients (p < 2.2 × 10⁻¹⁶, unpaired t test). (D) Western blot analysis shows heterogeneous levels of METTL3, METTL14, and MYCN expression among 11 neuroblastoma cell lines with gel activation image as loading reference. (E) METTL3 siRNA decreased METTL3 protein expression in Kelly and NGP neuroblastoma cells compared to control. (F) Global levels of m⁶A, quantified by triple-quadrupole liquid chromatography mass spectrometry (LC-MS QQQ), are decreased in neuroblastoma cells with siRNA-mediated METTL3 knockdown compared to control cells (n = 3, p < 0.05, unpaired t test). (G) Cell viability is decreased in neuroblastoma cells with siRNA-mediated METTL3 knockdown compared to controls (n = 3, p < 0.05, unpaired t test). (H) Up-regulated genes in Kelly cells with siRNA-mediated METTL3 knockdown were enriched for Gene Ontology (GO) pathways of synapse organization and axon development (n = 3 biological replicates). Bar graphs show mean and SD of data from 3 independent experiments.

Figure 2.. STM2457 treatment decreases neuroblastoma cell proliferation and global levels of m⁶A in vitro and impairs the growth of neuroblastoma xenografts in vivo

(A) STM2457 dose-response curves for 8 neuroblastoma cell lines (mean curve of 3 biological replicates). (B) The viability of SK-N-BE2 cells with overexpression of METTL3 treated with 16 μM STM2457 is higher relative to control cells (n = 3, p < 0.05, unpaired t test). (C) Quantitative m⁶A analysis by LC-MS QQQ shows that global m⁶A levels are significantly decreased in Kelly cells treated with 8 μM STM2457 compared to cells treated with DMSO control (n = 3, p < 0.05, unpaired t test). (D) Cell cycle analysis shows that treatment with 16 μM STM2457 increases the distribution of Kelly cells in G0/G1 compared to DMSO (n = 3, p < 0.001, unpaired t test). (E) Western blot analysis shows that cleaved PARP expression is decreased in SK-N-BE2 cells with METTL3 overexpression treated with 16 μM STM2457 compared to control cells treated with STM2457. (F) STM2457 treatment (50 mg/kg/day × 14 days) suppresses the growth of subcutaneous neuroblastoma xenografts (mm³) comprised of Kelly cells (p = 0.036, unpaired t test) or NGP cells (p = 0.044) compared to vehicle control. (G) STM2457 treatment improves overall survival for mice compared to vehicle control in neuroblastoma xenografts comprised of Kelly cells (p = 0.0007, log-rank test). Bar graphs show mean and SD of data from 3 independent experiments.

Figure 3.. STM2457 treatment promotes neurite outgrowths in neuroblastoma cells and increases expression of neuronal differentiation genes

(A) Phase contrast images of Kelly and NGP cells analyzed by the ImageJ processing program show an increase in the length (NGP: p < 0.005 and Kelly: p < 0.001, unpaired t test) and the number of neurite outgrowths (NGP: p < 0.08 and Kelly: p < 0.001, unpaired t test) following 6 days of STM2457 treatment (8 μM) compared to control cells treated with DMSO (n = 3). (B) TRKA protein expression is increased in Kelly and NGP cells treated for 3 days with 8 μM STM2457. (C) Volcano plot showing differentially expressed genes in Kelly cells treated for 6 days with 8 μM STM2457. (D) Venn diagram showing overlap of up-regulated genes in SK-N-BE2, Kelly, and NGP cells treated with STM2457 for 6 days (12, 8, and 1 μM, respectively). (E) Up-regulated genes that are shared between SK-N-BE2, Kelly, and NGP cells treated with STM2457 for 6 days (12, 8, and 1 μM, respectively) were enriched for GO pathways of nervous system development and cell differentiation. (F and G) Event-free survival (F) and overall survival (G) are significantly improved for patients in the SEQC-NB cohort (n = 220) with high (n = 220) vs. low (n = 273) (p < 0.0001 for both, log-rank test) METTL3 inhibitor response signature scores. The signature is comprised of 73 overlapping genes that were up-regulated in NGP, Kelly, and SK-N-BE2 cells following STM2457 treatment and enriched for nervous system development.

Figure 4.. STM2457 treatment decreases m⁶A mRNA deposition in networks enriched for neuronal differentiation and up-regulates expression of transcripts by increasing stability

(A) Representative metagene analysis showing m⁶A distribution in control- (blue) and STM2457-treated (red) Kelly and NGP cells and m⁶A located at consensus “DRACH” motif in Kelly cells. (B) Heatmap showing expression value (Z scored expression) of genes with m⁶A loss in Kelly cells treated with DMSO or STM2457 (p < 0.05, unpaired t test). (C) STM2457 treatment of Kelly cells promotes loss of mRNA m⁶A deposition (x axis) assessed by methylated RNA immunoprecipitation sequencing and increased gene expression assessed by RNA sequencing (y axis) compared to DMSO. (D) GO enrichment analysis of transcripts with decreased m⁶A and increased expression in Kelly cells at 24 h. (E) Tracks of m⁶A enrichment across CHGA in Kelly cells in DMSO- (top) and STM2457-treated cells (bottom). (F) Western blot analysis shows overexpression (OE) of METTL3 in SK-N-BE2 cells transfected with the pkmyc vector cloned with METTL3 compared to control cells. (G) The increase in CHGA and NTRK1 expression assessed by RT-qPCR in SK-N-BE2 cells treated with 16 μM STM2457 is not observed in SK-N-BE2 cells with METTL3 OE (p < 0.05, Student’s t test). (H) Half-life of mRNA (y axis) plotted against change in m⁶A level (x axis) in neuroblastoma cells treated with 8 μM STM2457 or DMSO. Bar graphs show mean and SD of data from 3 independent experiments.