13 research outputs found
MODELING CANCER USING LI-FRAUMENI SYNDROME PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS
Li-Fraumeni syndrome (LFS) is an autosomal dominant disease caused by germline mutations in the gene TP53, which predispose individuals to a wide range of malignancies, including osteosarcoma and breast cancer. In the previous study, our group developed a novel disease model platform by reprograming LFS patients\u27 fibroblasts to induced pluripotent stem cells (iPSCs), and further differentiate these iPSCs into mesenchymal stem cells (MSCs) then to osteoblasts (OBs), the cells from which osteosarcomas originate. Interestingly, LFS iPSC-derived osteoblasts recapitulated the osteosarcoma phenotype, creating “a bone tumor in a dish”. This “tumor in a dish” platform proved that LFS is an ideal model system to study and modeling LFS associated malignancies.
In this study, we applied whole exome deep sequencing in LFS iPSCs derived samples carrying different tumorigenic potential (MSCs, OBs, OB derived tumors) to identify cancer drivers that contribute to LFS associated osteosarcomagenesis. We found that LFS patient derived OBs exhibit both in vitro and in vivo oncogenic properties. We also observed increased somatic mutation prevalence in LFS OBs derived tumors compare to LFS OBs. Genes that are commonly mutated between LFS OBs derived tumors and genes carrying truncating or frameshift mutations in LFS OBs derived tumors were identified, including USP34, ANAPC1, ESPL1, MYLK, SLC35G2, FAM160A2, SLC25A32, SYNE2, RPL8, and FAM20A. These genes are potential candidate driver genes during early osteosarcoma development.
Breast cancer is the most common tumor among women with germline TP53 mutations. In this study, we also generated iPSC lines from LFS breast cancer patient and healthy family member. Using precise genome editing tools, we created TP53 mutation (delG) in unaffected relative derived the iPSCs, generating isogenic controls to facilitate studying of mutant p53 related phenotypic differences. We also demonstrated differentiation of LFS iPSCs to non-neural ectoderm using a chemical based protocol. Further establishment of mammary organoids differentiation protocol will provide in vitro platform in modeling LFS associated breast cancer.
In addition, we successfully corrected TP53 mutation (Y205C) in LFS patient derived iPSCs using TALEN-mediated precise gene editing. Similar approach was used to generate two H1 human embryonic stem cells (hESCs) carrying homozygous TP53 R282W and TP53 R248W mutation. These engineered iPSCs/hESCs offers exciting opportunities for studying mechanisms of mutant p53 associated malignancies and testing existing or potential compounds targeting mutant p53-associated pathway.
In summary, our studies demonstrated the potential of LFS patient derived iPSCs in cancer modeling
Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome
Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies
Generation of human embryonic stem cell line with heterozygous RB1 deletion by CRIPSR/Cas9 nickase
The Retinoblastoma 1 (RB1) tumor suppressor, a member of the Retinoblastoma gene family, functions as a pocket protein for the functional binding of E2F transcription factors. About 1/3 of retinoblastoma patients harbor a germline RB1 mutation or deletion, leading to the development of retinoblastoma. Here, we demonstrate generation of a heterozygous deletion of the RB1 gene in the H1 human embryonic stem cell line using CRISPR/Cas9 nickase genome editing. The RB1 heterozygous knockout H1 cell line shows a normal karyotype, maintains a pluripotent state, and is capable of differentiation to the three germline layers
Generation of a heterozygous p53 R249S mutant human embryonic stem cell line by TALEN-mediated genome editing
Abstract: As one of the most essential genome guardians, p53 and its mutants have been suggested associated with many types of cancers. Many p53 mutants function induce unique phenotypes, including carcinogenesis, metastasis, and drug resistance. The p53(R249S) mutation is the most prevalent and specific mutation associated with liver cancer development. Here, we demonstrate the generation of a heterozygous p53(R249S) mutation in the H9 human embryonic stem cell line using TALEN-mediated genome editing. The generated cell line maintains a normal karyotype, a pluripotent state and the in vivo capacity to develop a teratoma containing all three germ layer tissues
Generation of human embryonic stem cell line with heterozygous RB1 deletion by CRIPSR/Cas9 nickase
A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing
A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing
The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers
Generation of an induced pluripotent stem cell line from an individual with a heterozygous RECQL4 mutation
The DNA helicase RECQL4 is known for its roles in DNA replication and repair. RECQL4 mutations cause several genetic disorders including Rothmund-Thomson syndrome (RTS), characterized by developmental defects and predisposition to osteosarcoma. Here we reprogrammed fibroblasts with a heterozygous RECQL4 mutation (c.1878 + 32_1878 + 55del24) to induced pluripotent stem cells (iPSCs). These iPSCs are pluripotent and are able to be differentiated into all three germ layers, providing a novel tool to further interrogate the role of RECQL4 DNA helicase in vitro
Establishment of a human embryonic stem cell line with homozygous TP53 R248W mutant by TALEN mediated gene editing
Genetic mutations in TP53 contribute to multiple human cancers. Here we report the generation of a H1-p53(R248W/R248W) human embryonic stem cell line harboring a homozygous TP53 R248W mutation created by TALEN-mediated precise gene editing. The H1-p53(R248W/R248W) cell line maintains a normal karyotype, robust pluripotency gene expression, and the potential to differentiate to the three germ layers