2018 Fiscal Year Research-status Report
Study of ATRX mediated telomere maintenance in neuroblastoma by using genome editing technology (CRISPR/Cas9)
| Project/Area Number |
18K15256
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| Research Institution | Research Institute for Clinical Oncology, Saitama Cancer Center |
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Principal Investigator |
AKTER JESMIN 埼玉県立がんセンター(臨床腫瘍研究所), 臨床腫瘍研究所, 技師 (70795830)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | Neuroblastoma / ATRX / ALT |
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| Outline of Annual Research Achievements |
The chromatin remodeler ATRX is frequently mutated in neuroblastoma (NB) and in other pediatric tumors. Recent study confirmed that some NB tumors appear to maintain telomere length by activating the ALT pathway which is a telomerase independent mechanism of telomere length maintenance, and ALT activity was associated with significantly reduced overall survival. A unifying feature of these tumors is mutations in ATRX. Unfortunately, the molecular mechanism of ALT activity is unknown. Therefore, our primary objectives is to analyzing the link between ATRX loss of function and ALT pathway in NB, which will be remarkable clue for the development of ALT-related targets, and may contribute to the individualized treatment for high risk NB cases.
To date, we used the CRISPR/Cas9 system, which is the robust genome editing tool for genetic loss-of-function (LoF) studies of gene-of-interest in a wide variety of organisms. First, We have developed the ATRX knockout (KO) clones in MYCN amplified (NGP) and MYCN single copy (NB-69) NB cells using CRISPR-Cas9 technology. The clones proliferate slowly compared to control and become differentiated.
Interestingly, our qPCR assay showed that KO clone has the lower telomere content than ALT positive SK-N-FI cells. Furthermore, microarray gene expression analysis also revealed that KO clones have the different gene expression signature than ALT+ ve cell lines. In conclusion, our data indicate that loss of ATRX alone is not enough to activate ALT in NB cells. Further studies are aimed at understanding how ATRX affects the ALT mechanism in NB.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In our lab CRISPR/Cas9 system is working well. SO, we could get the knock out clone as our planned time. Also we started different biological experiment to characterize the knock out clones. Some of these results is exciting and experimental work is going well.
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| Strategy for Future Research Activity |
1. Establishment of gene editing system (Lentiviral)for ATRX by CRISPR/Cas9 in neuroblastoma (NB) cells as well as neural crest cells(NCC).
2. Exploring the biological function of ATRX knockout clones in NB cells and NCC cells. Investigation of telomere maintenance mechanism in ATRX knockout clones.
3. Identification of other novel genes and their role in telomere maintainace in NB cells.
4. Generation of p53 mutant NCC cells. After getting the p53 mutant NCC cells, start to analyze the biological status of those kinds of mutant NCC cells.
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| Causes of Carryover |
We have done some minor change in our research plan. Initially we have decided to use CRISPR/ Cas9 system in some more cells. Because of cells unavailability, we couldn't do. And also some experiment materials (example) we couldn't buy last fiscal time because of unavailability.
This year, we are going to use more advanced CRISPR/ Cas9 system for more efficiently knock out the targeted gene. And we will add the remaining amount in new fiscal year to analyze the biological properties of ATRX knock out cells.
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[Journal Article] EZH2 regulates neuroblastoma cell differentiation via NTRK1 promoter epigenetic modifications2018
Author(s)
Zhenghao Li, Hisanori Takenobu, Amallia Nuggetsiana Setyawati, Nobuhiro Akita, Masayuki Haruta, Shunpei Satoh, Yoshitaka Shinno, Koji Chikaraishi, Kyosuke Mukae, Jesmin Akter, Ryuichi P. Sugino, Atsuko Nakazawa, Akira Nakagawara, Hiroyuki Aburatani, Miki Ohira & Takehiko Kamijo
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Journal Title
Oncogene
Volume: 37(20)
Pages: 2714-2727
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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