Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours

Abstract

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies.

Fig. 1. MRT are phylogenetically closely related to neural crest-derived Schwann cells.

A Phylogenetic tree representing the somatic genetic relation of a renal MRT and normal tissues. Percentages: clone size in tissues. Numbers inside circles: mutation burden within cluster. Red or white coloured rectangles: SMARCB1 mutations status (red = mutant; white = wild type). LOH: loss of heterozygosity. H&E (B) staining of biopsies and INI1 (C) immunostaining, showing INI1 negative Schwann cells in hilum biopsy 2. Scale bars = 100 µm. D Pattern of positive INI1 staining in Schwann cells of normal nerve sheath from control hilar regions of two independent donors. Scale bars = 100 µm. E Phylogenetic tree representing the somatic genetic relation of an extradural (spinal) MRT and normal tissues. Embryonic clusters of mutations are denoted (a–d). The annotation otherwise follows A. H&E staining (F), clone size of the different mutational clusters (a–d, G), and INI1 immunostaining (H) of dorsal nerve root, ventral nerve root, tumour and (I) bone marrow of the same donor. The latter showing positive INI1 staining. Scale bars = 100 µm.

Fig. 2. SMARCB1 reconstitution drives MRT differentiation.

A Schematic representation of SMARCB1 reconstitution in patient-derived MRT organoids and subsequent single-cell transcriptome comparison to fetal mouse neural tube and neural crest cell types. Branching tree represents differentiation trajectories of mouse neural crest. Abbreviations are indicated. B Representative immunofluorescence images of MRT control (C) and SMARCB1+ (S) organoids. White: DAPI (nuclei), red: phalloidin (membranes), green: MMP2 (mesenchymal marker). Scale bars equal 50 µm. C UMAP representation of single cells from MRT control (grey) or SMARCB1+ (green) organoid lines (60T control/SMARCB1+: 8059/425 cells, 78T control/SMARCB1+: 3195/806 cells, 103T control/SMARCB1+: 2694/953 cells). D Dot plots represent similarity of MRT control (circles) or SMARCB1+ (squares) cells to neural crest differentiation trajectories. Colours represent the average probability (prob) that the MRT cells are similar to the indicated neural crest cell type (predicted similarity score estimated by logistic regression). Changes in similarity score between control and SMARCB1+ cells were assessed for cell types with average similarity score >0.5. P values were calculated using an unpaired Student’s t test (two-tailed): *<1e−3, **<1e−9, ***<1e−15 (−log10 (p value): 60T D = 45, S = 27, M2 = 66, ME = 3.7; 78T NT = 9, D = 54, M1 = 14, S = 22, M2 = 4.4; 103T D = 198, EM = 40, S = 7.8, M2 = 314, ME = 3.2). E Stacked bar plot represents relative frequencies of single-cell annotations for MRT control (−) and SMARCB1+ (+) organoids, showing a consistent conversion of neural to mesenchymal signals. Cell type annotation was assigned for each single-cell based on the highest similarity score. Colours represent neural crest cell types depicted in Fig. 2a. Cell type migratory2 (M2) was assigned as either migratory mesenchyme (ME(M2)) or migratory autonomic (A(M2)) based on the highest similarity score. The relative frequency of the mesenchymal/autonomic (ME/A) branch was compared between control and SMARCB1+ organoids for each patient line. P values were calculated using a chi-square test: *<0.01, ***<1e−15 (p value: 60T = 0.0048; 78T = 4.9e−48; 103T = 1.0e−32). F Dot plot shows expression levels (exp) of mesenchymal marker MMP2 for MRT control (−) and SMARCB1+ (+) organoids for each patient line. Colour-code from grey to red refers to average MMP2 transcript levels (unique molecular identifier (UMI)). Dot size refers to the percentage of cells (pct) showing MMP2 expression. G Box plot representation of gene module scores for MRT control (grey) and SMARCB1+ (green) single cells (n = 60T control/SMARCB1+: 8059/425 cells; 78T control/SMARCB1+: 3195/806 cells; 103T control/SMARCB1+: 2694/953 cells), showing consistent upregulation of mesenchymal/autonomic differentiation genes for SMARCB1+ cells. Box plots indicate median (middle line), 25th and 75th percentile (box). Whiskers represent the range excluding outliers (dot). Module scores were generated by averaging gene expression levels per set of genes. Gene sets include marker genes for either sensory (S) or mesenchymal/autonomic (ME/A) differentiation branches, distinguishing early and late differentiation genes. Module scores were assessed by comparing control and SMARCB1+ cells. P values were calculated using an unpaired Student’s t test (two-tailed): *<1e−3, **<1e−9, ***<1e−15 (−log10 (p value) ME/A late 60T = 28, 78T = 64, 103T = Inf; ME/A early 60T = 77, 78T = 134, 103T = Inf; S early 60T = 5.6, 78T = 72, 103T = 54; S late 60T = 28, 78T = 16, 103T = 11).

Fig. 3. Combined HDAC/mTOR inhibition mirrors SMARCB1 reconstitution.

A Overview of methodology used for discovery of potential differentiation therapeutics. B Representative immunofluorescence images of MRT organoids treated with DMSO control or a combination of vorinostat (HDACi, 1 µM) and sirolimus (mTORi, 2 nM). White: DAPI (nuclei), red: phalloidin (membranes), green: MMP2 (mesenchymal marker). Scale bars equal 50 µm. C Heatmaps represent gene expression values (n = 2 independent experiments) of MRT control or SMARCB1+ organoids, or MRT organoids treated with vorinostat (HDACi, 1 µM) or both vorinostat and sirolimus (combination,1 µM/2 nM). Heatmaps are subset for genes differentially expressed upon SMARCB1 re-expression (Supplementary Data 3). Genes are ordered by the average mRNA changes induced by SMARCB1 re-expression and treatment. Colour-code represents gene expression values scaled by gene. Pearson correlation coefficients (corr.) were generated by comparing mRNA changes induced by either SMARCB1 re-expression or HDACi/combination treatment. P values are indicated for Pearson’s correlation tests (two-tailed): ***<1e−15 (−log10 (p value): Combi 60T = 217, 78T = Inf, 103T = 221; HDACi 60T = 268, 78T = 306, 103T = 192). D Schematic overview of the dose-response matrix setup to find synergy between HDAC (vorinostat) and mTOR (sirolimus) inhibitors in MRTs. E Graphs show zero interaction potency (ZIP) scores that indicate either synergistic (red) or antagonistic (blue) effects of combination treatment. ZIP scores are generated by calculating the observed deviation from a reference model that assumes drugs are non-interacting (synergy when ZIP > 10%). The dashed rectangles highlight the drug concentration ranges where synergy between the two drugs is the strongest. Source data are provided as a Source Data file. F Schematic overview of the regrowth assay. G Bar graphs represent cell viability values normalised to timepoint 1 (T1) DMSO controls for each MRT or normal kidney organoid line. Mean and SD (error bars) of independent experiments (dot) are indicated (n = 60T/103T: 3, 78T mTOR/HDAC 1 µM/Combi 2 nM 1 µM: 6, 78T HDAC 3 µM/Combi 2 nM 3 µM: 4. normal kidney: 7). Each independent experiment is an average of four technical replicates. Source data are provided as a Source Data file. Additional effect of combination treatment on cell viability was determined by comparing combination (T2) with HDACi (T2) treatment. Regrowth capability was assessed by comparing T2 to T1. P values were calculated using a paired ratio Student’s t test (two-tailed): *<0.05, **<0.01, ***<0.001 (p value: Combi 1 µM T1 vs. HDACi 1 µM T1 60T = 0.020, 78T = 0.012; Combi 3 µM T2 vs. Combi 3 µM T1 78T = 0.013, normal kidney donor 1 = 2.5e−5, donor 2 = 1.8e−5).