Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma

Abstract

Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

Keywords: PAX3-FOXO1; chimeric RNA; gene fusion; myogenesis; rhabdomyosarcoma.

Fig. 1.

Fusion transcriptome profiling grouped samples of same or similar tissues together. (A) A large number of fusions were identified from RNA-seq of 27 nonneoplastic human tissues. The majority of fusions are seen in one or two tissue types, and a small fraction is seen in more than five tissue types. (B) Unsupervised clustering of fusion RNA expression (presence or absence) grouped samples from the same tissue type together, as in the case of all placental samples. This analysis used binary format with 1 indicating that the fusion was present (red) and 0 indicating that the fusion was absent (green). We used a variance filter value of 50. Heat maps of the “sample tree” were obtained by “complete lineage clustering” of filtered data. The Pearson correlation matrix was used in this analysis. (C) Similarly, unsupervised clustering with traditional transcriptome expression data was performed.

Fig. S1.

Pie chart demonstrating the distribution of fusion RNAs in various tissues.

Fig. 2.

More than 100 novel chimeric RNAs throughout myogenesis were revealed by RNA-seq. (A) Myogenic differentiation was induced in MSCs by differentiation medium (DM). RNA was extracted from samples collected at four time points. RT-PCR detected PAX3–FOXO1 and GAPDH. (B) Circos plot depicting chimeric RNAs discovered across the genome. Lines connect parental genes. Half lines are due to the close proximity between the two parental genes. Outer ring, chromosomes; innermost ring, lines denote the chimeric RNAs connecting two parental genes; middle two rings, heat map and histogram depicting gene expressions. Arrows point to the five chimeras common to T3 and RH30 samples. (C) RT-PCR and Sanger sequencing validation. Representative examples of fusion RNAs amplified by RT-PCR and fractionated by gel electrophoresis are shown. Bands were excised from the gel and processed for Sanger sequencing. The asterisks (*) indicate the correct band when multiple bands were seen. Vertical dashed red lines in Sanger sequencing results mark the fusion junction site.

Fig. S2.

Additional Sanger sequencing results of validated chimeric RNAs. A vertical red line marks the fusion junction site.

Fig. S3.

Expression levels of the parental genes are plotted according to their FPKM. The 5′ parental genes and 3′ genes are located on the left and right sides, respectively.

Fig. 3.

Similar chimeric fusion RNA profiles between T3 myogenesis sample and ARMS cell line RH30. (A) Fusion RNA profiling analysis revealed the closest similarity between T3 and RH30. This analysis used binary format, with 1 indicating that the fusion was present (red) and 0 indicating that the fusion was absent (black). (B) Whole-transcriptome profiling failed to uncover the connection between T3 and RH30. (C) Venn diagram showing the overlap between 1,075 up-regulated genes in T3 and RH30 compared with T1, and 1,072 PAX3–FOXO1 downstream target genes derived from published ChIP-seq experiments (10). (D) Gene Set Enrichment Analysis (26) confirmed the enrichment of PAX3–FOXO1 targets in the commonly up-regulated genes in T3 and RH30.

Fig. 4.

Eighteen chimeric RNAs shared by RH30 and PAX3–FOXO1-expressing myogenic cells. (A) Myogenic differentiation was induced in hES-MSCs. Samples were collected every other day. All validated chimeric RNAs found in RH30 cells by RNA-seq were tested by RT-PCR in myogenesis samples. All were detected at day 2. All except one were detected at day 6. (B) qRT-PCR analyses of six chimeric RNAs that were almost exclusively detected at the same time points as PAX3–FOXO1. Expression levels of these chimeric transcripts were normalized to GAPDH and then to the level of RH30. (C) Hierarchical clustering of the expression data for these 18 fusion RNAs at various time points. Days 2 and 6 clustered closest to RH30. (D) Stable MSCs expressing PAX3–FOXO1 (MSC-P3F) were established. RT-PCR results for representative fusions in MSC-P3F, parental MSC, and RH30 are shown here. Only one of the assayed chimeric RNAs was induced by PAX3–FOXO1.

Fig. S4.

PAX3 –FOXO1, PAX3, and FOXO1 expression throughout muscle differentiation. Experiments were conducted as described in Fig. 4. Expression levels of these transcripts were normalized to GAPDH and then normalized to that in RH30.

Fig. S5.

Myogenic differentiation was induced in MSC-7043L cells. Shown are the qRT-PCR analyses for the seven chimeras found to have the same expression pattern as PAX3–FOXO1 seen during hES-MSC myogenesis. Expression levels of these chimeric transcripts were normalized to GAPDH and then normalized to that in RH30.

Fig. S6.

MSCs stably express a transduced PAX3–FOXO1 construct. qRT-PCR analysis shows comparable PAX3–FOXO1 RNA expression in MSC-P3F and RH30 (Upper). Western blot analysis using a FOXO1 antibody shows comparable PAX3–FOXO1 protein expression in MSC-P3F and RH30 (Lower).

Fig. 5.

Chimeric RNAs in sarcoma cell lines and clinical RMS samples. (A) A total of 18 chimeric RNAs were examined by RT-PCR in four additional rhabdomyosarcoma cell lines and eight non-RMS sarcoma cell lines. A673, TTC-466, and TC32 are Ewing sarcomas; 402-91, myxoid liposarcomas; JN-DSRCT, desmoplastic small round cell sarcoma; SUCCS-1, clear cell sarcoma; OsACL, osteosarcoma; and A2243, synovial sarcoma. (B) qRT-PCR analysis shows 10 chimeras that are specific to RMS cell lines. Of note, all seven chimeras that have the same wavering expression pattern as PAX3–FOXO1 during MSC myogenesis are specific to RMS. (C) A total of 18 chimeric RNAs were measured by qRT-PCR in 14 clinical rhabdomyosarcoma samples, 2 noncancer muscle biopsies, and RH30. In addition to the RMS cases, two synovial sarcoma (SS), two liposarcoma (LS), two desmoplastic small round cell sarcoma (DS), two osteosarcoma (OS), and one Ewing sarcoma (EWS) clinical samples were used as controls. Three RMS-specific examples are shown here.

Fig. S7.

Fourteen clinical RMS samples, including six fusion-negative, five P3F-positive, and three P7F-positive cases. (Upper) qRT-PCR measuring PAX3– and PAX7–FOXO1 level normalized against GAPDH. (Lower) Gel images of PAX3–, PAX7–FOXO1, and GAPDH RT-PCR.

Fig. S8.

Additional qRT-PCR analyses of clinical cases. Shown are results for ROBO1–XPC and LINC00545–MEDAG. Expression levels of these chimeric transcripts were normalized to GAPDH and then normalized to that in RH30. ROBO1–XPC displayed higher expression in RH30, as well as in some RMS cases; expression was nearly absent in the fetal or adolescent muscle sample. In contrast, LINC00545–MEDAG had similar levels of expression in the fetal muscle sample and the clinical samples.