2019 Fiscal Year Research-status Report
Study of ATRX mediated telomere maintenance in neuroblastoma by using genome editing technology (CRISPR/Cas9)
| Project/Area Number |
18K15256
|
|---|
| Research Institution | Research Institute for Clinical Oncology, Saitama Cancer Center |
|---|
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)
|
|---|
| Project Period (FY) |
2018-04-01 – 2022-03-31
|
| Keywords | Neuroblastoma / ATRX / ALT / ATM/Chk2/p53 |
|---|
| Outline of Annual Research Achievements |
The chromatin remodeler ATRX is a tumor suppressor gene, and its recurrent somatic mutations associated with stage 4 NB patients. But our knowledge about ATRX mutation in NB tumorigenecity is still limited. Previously, ATRX knockout (KO) clones in NB cells by CRISPR/Cas9 system was established and interestingly, the KO clones proliferate slowly compared to control group and become differentiated.
Microarray based GSEA analysis showed that gene-set related to DNA double-strand break (DSBs) repair, DNA damage response, negative cell cycle regulation, G2M checkpoint and, p53 pathway activation were induced in ATRX KO cells. By in vitro analysis, ATRX loss results in augmentation of gamma-H2AX, a canonical marker for DSBs, indicate the accumulation of endogenous DNA damage in ATRX KO cells. Furthermore, accumulation of DNA damage results in activation of the ATM/Chk2/p53 pathway, leading to cell cycle arrest. Moreover, lentiviral-mediated inactivation of p53 rescued the decreased cell growth levels in ATRX KO cells, suggesting that ATRX plays a role in maintaining DNA integrity and ATM/p53 pathway inactivation is required for NB tumorigenesis. We also studied the correlation between ATRX-mutation and ATM/p53 pathway inactivation by multiomics database analysis, indicating that ATM/p53 pathway inactivation is required for ATRX mutation-related NB tumorigenesis.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Current year, we have established some other advance techniques for CRISPR/Cas9 system. So, we got the knock out clone in other neuroblastoma (NB) cell lines, that was necessary for our study. We also compared the biological
characteristics of the knock out clones in different NB cells. That means experimental work is going well.
|
| Strategy for Future Research Activity |
1.Characterizing the biological function of ATRX knockout clones in different NB cells in terms of telomere maintenance.
2.Identification of synthetic lethal or synthetic essential gene for ATRX mutant NB.
3.Characterizing the mechanism, how ATRX deletion related with DNA damage response in the NB cells.
4.Writing the manuscript for this project
|
| Causes of Carryover |
This year we have submitted our abstract in overseas international meeting. Because of novel corona virus, we couldn't present our study. And also some experiment materials, we couldn't buy last fiscal time.
This year, we are going to do some more experiment and will add the remaining
amount in new fiscal year to analyze the telomere maintenance mechanism in ATRX knock out cells. And also attend the national and international conference to present our data.
|
