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. 2025 Jun;45(6):702-708.
doi: 10.1002/cac2.70015. Epub 2025 Mar 15.

Comprehensive DSRCT multi-omics analyses unveil CACNA2D2 as a diagnostic hallmark and super-enhancer-driven EWSR1::WT1 signature gene

Florian Henning Geyer  1   2   3   4 , Alina Ritter  1   2   3   4 , Seneca Kinn-Gurzo  5 , Tobias Faehling  1   2   3   4 , Jing Li  1   2   3 , Armin Jarosch  6   7 , Carine Ngo  8 , Endrit Vinca  1   2   3 , Karim Aljakouch  9 , Azhar Orynbek  9 , Shunya Ohmura  1   2   3 , Thomas Kirchner  10 , Roland Imle  1   2   11   12 , Laura Romero-Pérez  13   14 , Juan Díaz-Martín  13 , Stefanie Bertram  15 , Enrique de Álava  13   16   17 , Clémence Henon  18 , Sophie Postel-Vilnay  18   19 , Ana Banito  1   2   12 , Martin Sill  1   2   20 , Yvonne Versleijen-Jonkers  21 , Benjamin Friedrich Berthold Mayer  22 , Martin Ebinger  23 , Monika Sparber-Sauer  24   25 , Sabine Stegmaier  24 , Daniel Baumhoer  26 , Wolfgang Hartmann  27 , Jeroen Krijgsveld  9 , David Horst  6   7 , Olivier Delattre  28 , Patrick Joseph Grohar  5 , Thomas Georg Phillip Grünewald  1   2   3   29 , Florencia Cidre-Aranaz  1   2   3
Affiliations

Comprehensive DSRCT multi-omics analyses unveil CACNA2D2 as a diagnostic hallmark and super-enhancer-driven EWSR1::WT1 signature gene

Florian Henning Geyer et al. Cancer Commun (Lond). 2025 Jun.
No abstract available

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Conflict of interest statement

The authors declare no competing interests.

Figures

FIGURE 1
FIGURE 1
CACNA2D2 is a diagnostic hallmark and super‐enhancer‐driven EWSR1::WT1 signature gene. (A) Left: Venn diagram showing the overlap of genes significantly overexpressed (log2FC > 2.5; Padj < 0.01) in DSRCT compared to all other tumor entities (as in Supplementary Figure S1A) with genes or proteins potentially regulated by EWSR1::WT1, as determined by ChIP‐seq and Mass spectrometry analyses. Right: Violin plot depicting CACNA2D2 and IQCG mRNA expression in 32 DSRCT patient samples. The black dotted line indicates the median, while blue dotted lines indicate quartiles. Statistical analysis: unpaired two‐sided Mann‐Whitney test. (B) EWSR1::WT1 de novo binding motifs and the epigenetic profile of the CACNA2D2 locus (chromosome 3:50,506,517‐50,548,707, hg19) from four DSRCT tumor samples (DSRCT_1‐4), JN‐DSRCT‐1 wild‐type, and JN‐DSRCT‐1 cells carrying an shRNA against GFP (control) or EWSR1::WT1. ChIP‐seq profiles from GSE156277 and GSE212977 are depicted for EWSR1::WT1 (blue), RNA polymerase II (green), H3K4me3 (red), H3K4me1 (yellow), H3K27ac (orange), and H3K9ac (salmon). Rectangles highlight the CACNA2D2 promoter and enhancer regions. (C) Scatter plot showing spatial chromatin interactions between genomic loci in chromosome 3, captured by Hi‐ChIP (GSE212978). Dot density reflects the frequency of interactions. Highlighted dots represent interactions between the CACNA2D2 promoter and enhancer loci. The CACNA2D2 promoter region is marked in red, and the enhancer region is marked in blue. (D) Graph depicting H3K27ac signal density at active enhancer sites in the JN‐DSRCT‐1 cell line (GSE212977), ranked by normalized intensity. Red lines indicate the cutoff for super‐enhancer identification (n = 859). (E) Bar plot showing relative mRNA expression levels of EWSR1::WT1 and CACNA2D2, quantified by qRT‐PCR in JN‐DSRCT‐1 cell line expressing a DOX‐inducible shRNA‐mediated KD of EWSR1::WT1 for 96 hours. n = 4 biologically independent experiments. Horizontal bars represent mean expression levels, and whiskers indicate SEM. The number of analyzed samples is given in parentheses. Statistical analysis: unpaired two‐sided Mann‐Whitney test. (F) Western blot using antibodies against CACNA2D2, (EWSR1::)WT1, and GAPDH (loading control) in the SK‐DSRCT2‐endo‐WT1‐HaloTag cell line, which carries a knock‐in of the HaloTag‐HiBiT tag at the endogenous locus of EWSR1::WT1 C‐terminus. Cells were cultured with 1 µmol/L PROTAC or DMSO as control for 24 hours. (G) x‐y scatter plots illustrating the correlation of ssGSEA enrichment scores between (sc)EWSR1::WT1 and (sc)CACNA2D2 signatures of orthotopically xenografted single DSRCT cells. The numbers and colors in the correlation matrix represent Pearson correlation values for each signature enrichment in single cells from orthotopically xenografted DSRCT (top) or 32 bulk DSRCT patient samples (bottom). (H) Heatmap depicting ssGSEA signature enrichment scores for (sc)EWSR1::WT1 and (sc)CACNA2D2 signatures, along with log2 expression values for CACNA2D2 across 32 DSRCT patient samples and 20 additional cancer entities relevant for DSRCT differential diagnosis. (I) UMAP plot of scRNA‐seq analysis of merged and integrated data from eleven samples, comprising tumor‐derived and normal cells from four DSRCT patients (GSE263523), using the scCACNA2D2 signature. The color gradient indicates the ssGSEA score for scCACNA2D2 signature enrichment. (J) Representative histological images of CACNA2D2 IHC in a DSRCT murine orthotopic xenograft from a primary site and micrometastases in inner organs (muscle, liver, and kidney). DAB (brown chromogen) was used. (K) Bar plot showing individual IRS scores for CACNA2D2 staining in DSRCT and other morphological mimics. The number of analyzed samples is given in parentheses. Bars represent mean IRS values, and whiskers indicate SEM. DSRCT samples are highlighted in blue. Statistical analysis: unpaired two‐sided Mann‐Whitney test. (L) Left: Representative histological image of CACNA2D2 IHC in DSRCT primary tumor tissue. Middle: Representative histological image of CACNA2D2 IHC in a lung metastasis of DSRCT. Right: Representative histological image of CACNA2D2 IHC in a tumor sample with residual DSRCT cells after treatment. DAB (brown chromogen) was used. (M) Graph showing sensitivity (blue solid line) and specificity (red dashed line) of CACNA2D2 IHC, both expressed as percentages. (N) Diagram illustrating the proposed workflow for establishing a robust diagnosis of DSRCT. Abbreviations: AML, acute myeloid leukaemia; ARMS, alveolar rhabdomyosarcoma; ASPS, alveolar soft part sarcoma; BCOR::CCNB3, BCOR::CCNB3 rearranged sarcomas; CACNA2D2, Calcium voltage‐gated channel auxiliary subunit alpha2delta 2 gene; CAFs, cancer‐associated fibroblasts; CDS, coding sequence; ChIP‐seq, chromatin immunoprecipitation followed by sequencing; Chrom, chromosome; CIC::DUX4, CIC::DUX4 rearranged sarcomas; CNS‐PNET, primitive neuroectodermal tumor arising in the central nervous system; DAB, 3,3'‐diaminobenzidine; DEG, differential gene expression; DLBCL, diffuse large B‐cell lymphoma; DMSO, dimethyl sulfoxide; DOX, doxycycline; DSRCT, Desmoplastic small round cell tumor; DSRCT_1 – 4, DSRCT patient sample data; ERMS, embryonal rhabdomyosarcoma; EWS::WT1‐HaloTag‐HiBiT, EWSR1::WT1 protein fused to HaloTag‐HiBiT protein; EWSR1::NFATC2, EWSR1::NFATC2 rearranged sarcomas; EWSR1::WT1, fusion protein of EWSR1 and WT1 proteins; FISH, Fluorescent in situ hybridization; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase; GFP, Green fluorescent protein; GIST, gastrointestinal stromal tumor; H&E, hematoxylin and Eosin; H3K27ac, histone H3 lysine 27 acetylation; H3K4me1, histone H3 lysine 4 aminomethylation; H3K4me3, histone H3 lysine 4 trimethylation; H3K9ac, histone H3 lysine 9 acetylation; hg19, human genome version 19; Hi‐ChIP, high‐throughput chromatin immunoprecipitation followed by sequencing; IHC, immunohistochemistry; IQCG, IQ motif containing G gene; IRS, immune reactive score; JN‐DSRCT‐1/TR/shWT1, JN‐DSRCT‐1 cell line stably expressing a doxycycline inducible shRNA expression cassette targeting EWSR1::WT1 mRNA; kDa, kilodalton; log2FC, log2 fold change; MALT, mucosa‐associated lymphoid tissue lymphoma; MFH, malignant fibrous histiocytoma; MPNST, malignant peripheral nerve sheath tumor; NGS, next generation sequencing; Padj, adjusted P‐value; PROTAC, proteolysis targeting chimera; qRT‐PCR, quantitative real‐time polymerase chain reaction; RMS, rhabdomyosarcoma; RNA pol II, RNA polymerase II; rpm/bp, reads er million per base pair; scCACNA2D2, single‐cell data derived CACNA2D2 signature; scEWSR1::WT1, single‐cell data derived EWSR1::WT1 signature; scRNA‐seq, single‐cell RNA‐sequencing; SEM, standard error of the mean; shGFP, shRNA targeting GFP mRNA; shRNA, short hairpin RNA; shWT1, shRNA targeting EWSR1::WT1 mRNA; SK‐DSRCT2 endo‐WT1‐HaloTag, SK‐DSRCT2 cell line expressing endogeneous CACNA2D2 protein fused to HaloTag protein; SRCSs, small round cell sarcomas; ssGSEA, single sample gene set enrichment analysis; UMAP, uniform manifold approximation and projection; UTR, untranslated region.
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