Fig 1.
Clinical characterization of Type 2 Rothmund-Thomson Syndrome patients in this study.
(A) Pedigrees of the two families with RTS Type 2 and RECQL4 pathogenic variants reported in this study. (B) Photos show the classic poikiloderma on the face, ears, and arms of individual RTS-A at age 2 years (left panels, top and bottom). Note the sparing of the trunk and abdomen as well as sparse scalp hair and absence of eyebrows and presence of gastrostomy feeding tube. Middle panels illustrate variability in the severity of poikiloderma on the lower extremities in RTS-A (upper) and RTS-B (lower). Note hypoplastic thumb and dystrophic nails in RTS-A. Both individuals developed osteosarcoma. The right top panel shows an x-ray of the skeletal defect (radiohumeral synostosis, age 2 years) in RTS-A, and the bottom panel shows an MRI image of osteosarcoma that developed in the right proximal radius of RTS-A at age 10 years. This T1 fat-saturated, coronal, post-contrast view demonstrates diffuse solid enhancement of the tumor with some central areas of non-enhancement. (C) Sanger sequencing verifies biallelic RECQL4 (c. 2719C>T/Q907X) pathogenic variant in RTS-A fibroblasts (upper panel) and RECQL4 (1568G>C;1573delT and VS11+32del24) in RTS-B fibroblasts (middle and lower panels). (D) Immunoblotting indicates the loss of RECQL4 protein in RTS-A fibroblasts and the truncated RECQL4 protein in RTS-B fibroblasts. *, non-specific band.
Table 1.
Clinical characteristics of RTS Type 2 individuals with RECQL4 mutations.
Fig 2.
Generation of RTS iPSCs and iPSC-derived osteoblasts.
(A) RTS and FC iPSCs exhibit hESC morphology and express pluripotency transcription factors NANOG and OCT4 as well as hESC surface antigens TRA-1-81 and SSEA44 ubiquitously. Scale bar, 100 μm. (B) qRT-PCR assay for pluripotency genes NANOG, OCT4, and SOX2 in RTS and FC iPSCs. Error bars indicate SEM of triplicates. n = 3 biological replicates. (C) Immunostaining demonstrates that iPSC-derived MSCs exhibit swirling morphology (phase contrast image) and express MSC surface markers CD44, CD73, and CD105. Scale bar, 100 μm. (D) ARS staining reveals attenuated mineral deposition ability in RTS osteoblasts. Scale bar, 100 μm. (E) qRT-PCR assay for osteoblast lineage genes COL1A1, IGF2, and CLEC3B in RTS and FC osteoblasts. Error bars indicate SEM of triplicates. n = 3 biological replicates. (F) AIG assay for in vitro tumorigenicity demonstrates that RTS osteoblasts present an increased clonal growth ability in soft agar in comparison with FC and WT osteoblasts. Positive colonies after 1.5-month growth of differentiated osteoblasts in osteoblast differentiation medium are those larger than 50 μm (scale bar, 50 μm). n = 3 biological replicates.
Fig 3.
Transcriptional analysis reveals increased OXPHOS in RTS osteoblasts.
(A) GSEA analyses identify gene ontology biological processes (GO_BP) from a collection of 7525 genes sets (left) enriched in RTS or FC osteoblasts. GO_BP gene sets enriched (orange, corresponding to a positive normalized enrichment score (NES)) or depleted (green, corresponding to a negative NES) in the transcriptome of RTS compared to FC osteoblasts are shown. Enriched gene sets are selected based on statistical significance (normalized p-value < 0.05 and FDR q-value < 0.25). Middle, heatmap of significantly altered GO_BPs in RTS MSCs (D0), pre-osteoblasts (D15), and osteoblasts (D24) compared to FC counterparts. Right, GSEA leading edge analysis demonstrates the overlap between gene sets enriched in RTS and FC osteoblasts. (B) GSEA GO_BP results for mitochondrial electron transfer NADH to ubiquinone and bone morphogenesis. (C) KEGG pathway analyses identify enriched pathways in RTS and FC osteoblasts. Left, 183 total pathways are included in KEGG pathway analysis. Pathways enriched in the transcriptome of FC osteoblasts have a negative normalized enrichment score (NES) (blue), while pathways with a positive NES (shown in red) are enriched in the transcriptome of RTS osteoblasts. Enriched pathways are selected based on statistical significance (normalized p-value < 0.05 and FDR q-value < 0.25). Right, heatmap showing significantly altered pathways in RTS MSCs (D0), pre-osteoblasts (D15), and osteoblasts (D24) compared with FC counterparts. (D) Representative KEGG pathways differentially regulated in RTS osteoblasts, including OXPHOS and Wnt signaling pathway. (E) Individual genes (NDUFA7, NDUFB1, NDUFB2, and NDUFS8) that compose complex I of the electron transport chain are significantly upregulated in RTS osteoblasts. n = 3 biological replicates. (F) Immunoblotting indicates that NDUFA7 and NDUFS8 are upregulated in RTS osteoblasts compared to FC osteoblasts.
Fig 4.
Metabolic differences between RTS and FC osteoblasts demonstrate the role of OXPHOS in increasing ATP production in RTS osteoblasts.
(A) Enzyme activity of complex I is increased in RTS osteoblasts. n = 3 biological replicates. (B) Seahorse assays indicate increased oxygen consumption rate, a measure of OXPHOS, in RTS osteoblasts. n = 3 biological replicates. (C-H) Individual calculations of basal respiration (C), proton leak (D), ATP-linked respiration (E), maximal respiration (F), and spare capacity (G) in RTS osteoblasts as measured by seahorse assay, all of which are significantly upregulated. Non-mitochondrial respiration (H), which is not attributed to OXPHOS, is comparable between RTS and parental osteoblasts. n = 3 biological replicates. (I) Seahorse assays show decreased extracellular acidification rate, a measure of glycolysis, in RTS osteoblasts. n = 3 biological replicates. (J-K) Significantly decreased basal extracellular acidification rate (J) and maximal acidification rate (K) in RTS osteoblasts indicate RTS osteoblast reliance on OXPHOS for ATP production. n = 3 biological replicates.
Fig 5.
Inhibition of mitochondrial respiratory complex I specifically impairs ATP respiration, reliance on OXPHOS, and survival in RTS osteoblasts.
(A-B) RTS osteoblasts treated by 100 nM of IACS-010759 show reduced ATP respiration, maximal respiration, and spare capacity. Differences in OCR with seahorse assay are most apparent after the addition of FCCP, which measures the maximum potential of ATP production through the electron transport chain. n = 3 biological replicates. (C) IACS-010759 alters RTS osteoblast cell morphology from a spindle shape to an irregular shape. Scale bar, 100 μm. (D) IACS-010759 selectively inhibits cell proliferation of RTS osteoblasts. Cell proliferation assays after treatment with 100 nM of IACS-010759 show growth inhibition of RTS osteoblasts but little effect on FC osteoblasts. n = 4 biological replicates. (E) IACS-010759 induces the cellular senescence of RTS osteoblasts. The SA-β-Gal+ areas are measured using ImageJ. n = 3 biological replicates. (F) Scatter plot indicates a marked difference of transcripts between DMSO and 100 nM of IACS-010759-treated RTS osteoblasts. H19 and numerous ribosomal protein genes are upregulated but MAPK pathway and cell cycle-associated genes are downregulated upon IACS-010759 treatment. (G) GO_BP analysis indicates dysregulated pathways in RTS osteoblasts upon IACS-010759 treatment. IACS-010759 activates protein targeting, T cell-mediated toxicity, and protein translation related biological processes but inhibited MAPK and cell cycle processes in RTS osteoblasts. (H) Immunoblotting indicates a decrease MAPK pathway related STK9 and EREG in RTS osteoblasts upon IACS-010759 treatment. (I) IACS-010759 impairs ERK1/2 activity in RTS osteoblasts. (J) Model for the elevated mitochondrial respiratory complex I function in RTS osteoblasts. In RTS patients, impaired RECQL4 function leads to an increase of mitochondrial respiratory complex I gene expression and function, and thereby induces premalignant phenotypes. Pharmacological inhibition of mitochondrial respiratory complex I function by IACS-010759 can be applied to prevent and/or treat RTS patient associated osteosarcomas.