Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.

Fig. 1. SNAI2 is highly expressed in RMS and is regulated by a MYOD bound super enhancer.

a Violin plot showing expression (log₂FPKM) of SNAI2 across RMS and normal tissue. FPKM, Fragments Per Kilo base of transcript per Million mapped reads. b Representative western blot (n = 3 biologically independent experiments) of SNAI2 expression in different RMS cell lines. c Representative SNAI2 immunohistochemical staining in RMS primary tumors (14 positive of 19 FN-RMS and 3 positive of 4 FP-RMS) compared to normal muscle and Isotype control antibody. Scale Bar = 100 μM. d Topological interactions (Hi-C data from IMR90)¹⁷ of TAD containing the SNAI2 locus. e H3K27ac ChIP-seq data at SNAI2 TAD showing recurrent super enhancers (SEs) in a panel of FP-RMS (red), FN-RMS (blue) primary tumor samples and cell lines, myoblasts, myotubes, and skeletal muscle cells (yellow). Solid horizontal blocks show location of predicted super enhancers. f SNAI2 promoter and enhancer bound by MYOD and loaded with active histone mark H3K27ac in FN-RMS cell lines. * Previously reported MYOD peak near SNAI2. RRPM, Reference-adjusted Reads Per Million Mapped Reads. g Topological interactions in SMS-CTR (HiChIP of H3K27ac) of SEs in the SNAI2 locus. h siRNA targeting MYOD1 was used to knock down MYOD expression in SMS-CTR cells. MYOD and SNAI2 expression was detected by western blot (top) and qRT-PCR (bottom). Data was normalized to cells treated with scramble siRNA (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). i Targeted disruption of MYOD binding sites in enhancers surrounding the SNAI2 gene in SMS-CTR cells, using sgRNAs to deliver dCas9-KRAB suppressor. Location of enhancers E1–E5 are shown above. Schematic of experimental workflow shown in bottom left. Bar chart of qRT-PCR measurements for SNAI2 expression after dCas9-KRAB perturbation with various guides is shown in the bottom right; p values shown were calculated among biological triplicates using t-test with Welch’s correction. Error bars represent the SD among the 3 biologically independent replicates.

Fig. 2. Suppression of SNAI2 activates myogenic differentiation and suppresses stemness in vitro in FN-RMS.

a The level of SNAI2 knockdown by shSNAI2 in RD cells compared to shScr assessed by western blot (Representative blot, n = 3 biologically independent experiments). b Representative images of immunostaining in RD cells stably expressing shScr or c shSNAI2.1 shRNA stained for Myosin Heavy Chain 1 (MyHC, green), MEF2C (red) and DAPI for nuclei (blue). d Quantitation of immunostaining counts as percentage values to total nuclei per image (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). e qRT-PCR gene expression analysis in RD cells comparing shScr to shSNAI2.1 KD showing early and late myogenic markers (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). f Representative western blot (n = 3 biologically independent experiments) of muscle differentiation genes in RD shScr vs shSNAI2.1 transient KD cells at 3, 5, 7, and 10 days post puromycin selection. g, h Representative images of sphere formation assays in RD cells containing shScr or shSNAI2.1. i Quantitation of sphere counts in RD cells plated at three densities (10,000, 1000, and 100 per well). n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure, Scale Bars, b, g = 100 μM.

Fig. 3. Suppression of SNAI2 reduces tumorigenicity and growth, and induces muscle differentiation in vitro and in vivo in FN-RMS.

a Growth curve analysis of RD cells 3 days post puromycin selection after lentiviral infection with shScr or shSNAI2 shRNAs (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). b, c Single cell colony formation assay in RD cells containing shScr or shSNAI2 knockdown and quantitation values of colony forming units (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). d, e Soft agar colony formation assay comparing RD shScr to shSNAI2 infected cells and quantification of colony numbers in wells (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). f RD cells xenografted subcutaneously in mice with shScr (left) or shSNAI2 (right) and followed for 76 days (3 representative mice of 6 shScr, 3 shSNAI2.1, and 3 shSNAI2.2 tumors each, 1 × 10⁶ cells). g Tumor volume of mice injected with either shScr or shSNAI2 cells assessed weekly by caliper measurement represented as mm (n = 6 shScr, n = 3 shSNAI2.1, and n = 3 shSNAI2.2 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). h Weight measurement of xenograft tumors with either shScr or shSNAI2.1, SNAI2.2 post mortem (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). i Images of shScr and shSNAI2.2 RD tumors taken at 76 days. j–m Hematoxylin and eosin and Immunohistochemistry of MyHC in the same tumors (Representative images of n = 3 biologically independent experiments.) n Growth of vincristine treated (0.5 mg/kg once weekly for 3 weeks) RD tumors expressing shScr or shSNAI2 assessed by Luciferase imaging. o Tumor volume of transplanted RD xenografts with shScr and shSNAI2 + vincristine (VCR) assessed by caliper measurement represented in mm³ followed for 96 days (n = 10 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). p Tumor weight of RD xenografts expressing shScr or shSNAI2 treated with VCR and harvested at 96 days (n = 20 mice, 20 shScr, 10 shSNAI2.1 and 10 shSNAI2.2 tumors from biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). q Images of shScr and shSNAI2.2 tumors extracted from mice post euthanasia. r–u H&E and MyHC immunohistochemistry of tumor sections from RD xenografts expressing shScr or shSNAI2 treated with vincristine (VCR). Representative images of n = 3 biologically independent experiments. Scale Bars in j, l, r, t = 100 μM, Scale bar in b = 100 μM, d = 10 mm.

Fig. 4. SNAI2 binds key enhancers in FN-RMS.

a ChIP-seq signal for SNAI2 and H3K27ac peaks shared in all three cell lines are shown. b Hypergeometric Optimization of Motif EnRichment (HOMER) analysis identified SNAI2 binding motifs (top) as well as bHLH (basic helix-loop-helix) motifs at SNAI2 shared peaks using the HOMER package (homer.salk.edu/homer/ngs/peakMotifs.html). p statistic is calculated using the HOMER statistical comparison against size matched DNA sequences from randomly selected background genomic sequences. c Chromatin states in SMS-CTR cells (left) and abundance of SNAI2 peaks per Gb of each state (right). d Venn diagram (left) and average plot for ChIP-seq signal (right) depicting overlap between SNAI2 (2 or all 3 cell lines) and MYOD (2 cell lines) binding sites. RPM, Reads Per Million Mapped Reads. e Co-occupancy of MYOD and SNAI2 as measured by ChIP-reChIP and qPCR at locations previously identified (by ChIP-seq) as being preferentially bound by MYOD, SNAI2, or both. Single-target ChIP-qPCR controls are shown below (n = 2 biologically independent experiments, data presented as mean values).

Fig. 5. Ablation of SNAI2 enables MYOD to activate myogenic target genes.

a Composite plots showing SNAI2 signal intensities (reads per million mapped reads) at SNAI2 high confidence peaks (n = 1069) in SMS-CTR (top) and RD (bottom). b Heatmaps of SNAI2 peak intensity at SNAI2 high confidence peaks. Each row represents a genomic location and is centered around SNAI2 peaks, extended 4 kb in each direction, and sorted by SNAI2 signal strength. c Composite plots showing MYOD signal intensities at SNAI2 high confidence peaks (left) and at MYOD peaks (right) in SMS-CTR and RD. RPMPR, Reads Per Million Mapped Peak Reads. d Bubble plot depicting Gene Set Enrichment Analysis (GSEA) in SMS-CTR and RD cells (left). The size of the bubble is proportional to the −log10 nominal (NOM) p, and the color of the bubble corresponds to the normalized enrichment score (NES) value. GSEA enrichment plots showing positive enrichment for a set of genes up-regulated during differentiation of human skeletal muscle myoblasts into myotubes (right, top), and a set of myogenically induced super-enhancer genes (right, bottom). p values are determined by the GSEA algorithm relative to the null distribution calculated with 1000 permutations. For each of the enrichment plots shown here, the false discovery rate (FDR) q value and the nominal p value is <0.0005. e Diagram illustrating SNAI2 direct or indirect myogenic target genes through EDEN analysis. SE, Super Enhancer; TAD, Topologically Associated Domain. f Sites of direct SNAI2 mediated gene suppression at MYOG, MYBPH (left), and MEF2A (right), with both H3K27ac HiChIP for 3D chromatin folding and ChIP-seq. Representative ChIP-seq tracks are shown for MYOD, SNAI2, H3K27ac, and delta (Δ) value (shSNAI2.1 minus shScr) in MYOD and SNAI2 and gene expression (RNA-seq) at MYOG, MYBPH, and MEF2A loci in SMS-CTR. Arrows depict SNAI2/MYOD regulation on direct target myogenic genes. RPMPR, Reads Per Million Mapped Peak Reads; RRPM, Reference-adjusted Reads Per Million Mapped Reads; RPM, Reads Per Million Mapped Reads.

Fig. 6. SNAI2 anti-differentiation effects are mediated by the blockade of MYOG, MEF2A/C/D and CDKN1A.

a Schematic representation of role of SNAI2 in RAS-mutated RMS and induction of differentiation after SNAI2 knock down. b, c, e, f, h, i Representative images of RD shScr and shSNAI2 cells transfected with siRNA stained for Myosin Heavy Chain 1 (MyHC, green), MEF2C (red) and DAPI for nuclei (blue). Green numbers bottom of each image represents the average number of percentage of positive cells for three images. Scale Bar in b = 100 μM. d, g Western blot of RD shScr and shSNAI2.1 cells transfected with siRNAs against MEF2A, and MYOG along with Control siRNA (scramble) probed for MEF2A and MYOG. Representative blot, n = 3 biologically independent experiments. j Box plot depicting SNAI2 myogenic direct target gene expressions (RNA-seq) in SMS-CTR cells transduced with shRNAs or treated with trametinib. TPM, Transcripts per Million. Box plots show quartiles, black bar shows the median, and whiskers show the 1.5 × interquartile range. The exact p values are reported in the figure (n = 3 biologically independent experiments). k Scatter plot of log2 fold change (L2FC) of MYOG-activated SE gene expression (RNA-seq) (top) and shSNAI2/shScr transduced vs trametinib/DMSO treated cells for RAS-dependent SE gene expression (RNA-seq) (bottom) in SMS-CTR cells. l Representative ChIP-seq tracks for SNAI2 (blue), MYOD (green), H3K27ac (yellow) (top) and gene expression (RNA-seq) (bottom) in SMS-CTR. Δ, trametinib minus DMSO; TPM, Transcripts per Million. m Representative ChIP-seq tracks for SNAI2 (blue), MYOD (green), H3K27ac (yellow) (top) and gene expression (RNA-seq) (bottom) in SMS-CTR. Δ, shSNAI2.1 minus shScr; TPM, Transcripts per Million. n Western blot of RD, JR1, and SMS-CTR cells treated with 10 nM trametinib compared to vehicle control (DMSO), probed for p-ERK (phosphorylated), ERK, SNAI2, MEF2C, and actin (loading control). Representative blot, n = 3 biologically independent experiments. o Schematic model: SNAI2 expression in FN-RMS is regulated by MYOD and SNAI2 binding is able to dampen MYOD binding at myogenic transcription factor genes, which contributes to maintenance of a myogenic differentiation block (left). MEK inhibition with trametinib and/or SNAI2 knockdown in FN-RMS releases SNAI2, allowing MYOD to activate genes important for myogenic differentiation (MYOG, MEFs, TNNTs) thus inducing muscle differentiation (middle). Silencing of myogenic SNAI2 target genes (like MYOG or MEF2A) blocks differentiation downstream of SNAI2. Bubbles depict transcriptional co-activators in inactive (gray) or active (yellow) status.