ATRX structure and function
Alpha Thalassemia/mental Retardation syndrome X-linked (ATRX) is a highly conserved nuclear protein. It is composed of three main regions: a C-terminal region containing a helicase/ATPase domain identifying ATRX as a part of SNF2, the ATP dependent SWI/SNF chromatin remodeling complex, a central DAXX-binding domain, and an N-terminal region containing a nuclear localization signal. These regions prime ATRX for chromatin remodeling and interactions. ATRX interacts with DAXX to form a complex that deposits the H3.3 histone variant onto chromatin rendering it transcriptionally inactive suggesting a role for ATRX in transcriptional regulation. Additionally, ATRX has been shown to play a role in homologous recombination DNA repair and replication stress, which are critical components of cell cycle regulation.1–3
ATRX was initially identified in patients with the intellectual disability now known as ATRX syndrome and is highly expressed in fetal brain implicating an important role in brain development.3 Mutations in ATRX have been linked to a variety of brain cancers but its precise role in gliomas is still unknown.4
ATRX is a critical marker for classifying gliomas
The World Heath Organization released a new classification system for central nervous system (CNS) tumors in 2016 that incorporated molecular and histological phenotypes, which has made a dramatic impact on diagnosis and management of CNS tumors. This system clarified the classification of diffuse low-grade gliomas and their high-grade variants. Four molecular parameters were established to classify diffuse low-grade gliomas based on the presence or absence of IDH mutation, 1p/19q chromosomes codeletion, TP53 mutation, and ATRX loss. Immunohistochemistry has been used to determine expression and mutation status of ATRX, providing an easy mechanism to evaluate ATRX in clinical samples that can then be used for the development of optimal therapeutic strategies.2
Developing an ATRX-deficient glioblastoma mouse model
Transgenic loss of ATRX in a mouse model is embryonic lethal, and post-natal conditional loss of ATRX alone impairs brain development but does not result in tumor formation. These challenges in mutating ATRX to producing an ATRX-mutant mouse model of glioma led to the use of the Sleeping Beauty transposase system to create a model of ATRX-deficient glioblastoma. This model system incorporated the knockdown sequence of p53 (shp53), expression of the oncogene NRAS, and the knockdown sequence of ATRX (shATRX) onto plasmids that were injected along with the Sleeping Beauty Transposase into the lateral ventricle of neonatal mice. The p53 and NRAS modifications were included because human glial tumors that have mutated ATRX frequently (30-80%) also have mutated p53 and dysregulated receptor tyrosine kinase-RAS-PI3 kinase pathways often resulting in NRAS upregulation. These mice had larger tumors at earlier time points and a decreased rate of survival compared to mice injected with only shp53/NRAS but not shATRX. Additionally, these tumors were found to be more sensitive to double-strand DNA-damaging treatments such as radiation, doxorubicin, irinotecan (SN-38), and topotecan. suggesting a possible mechanism for why glioma patients with ATRX mutation have increased survival compared to ATRX wild type patients.5
In the current study, authors wanted to advance the work they found with this new ATRX deficient glioblastoma mouse model by elucidating the mechanism driving radiation sensitivity and to determine whether ATRX loss in human models of glioma also contributes to changes in radiation sensitivity.6
ATRX loss in human glioma models correlates with mouse model
To evaluate function of ATRX in human gliomas, CRISPR was used to knockout ATRX in three human glioblastoma cell lines: U251, SF188, and UW479, and two primary pediatric high-grade glioma (HGG) cell lines: SJ-GBM2 (TP53 mutant, ATRX mutant, IDH WT) and KNS42 (TP53 mutant, ATRX WT, IDH WT) cell lines were examined. ATRX levels were confirmed to be reduced in all ATRX knockdown cell lines compared to control cells, and these cell lines were positive for c-circle assays, which measure alternative lengthening of telomeres and are a phenotypic attribute of human ATRX-deficient gliomas. Similar to the mouse model, ATRX-deficient cells had higher sensitivity to irradiation (measured by decreased proliferation after irradiation) compared to their appropriate wild type controls. Phospho-histone 3 staining was used to measure mitotic indexes because it specifically marks the four phases of mitosis and late G2.7 In this assay, ATRX-deficient cells returned to active replication twice as fast as wild type controls after irradiation. This indicates that ATRX plays a critical role in controlling G2/M checkpoint after irradiation and without ATRX cells continue to replicate with DNA damage creating checkpoint dysregulation.
Transcriptional analysis reveals ATRX regulates DNA repair and cell cycle regulation gene expression
The authors set out to elucidate the mechanism for this checkpoint dysregulation. ATRX-deficient glioblastoma neurospheres were generated from the mouse model and used for CHIP/seq and RNA/seq analysis. Integrating this new murine glioblastoma neurosphere data with existing murine neuronal progenitor cell data, ATRX was found to bind both DNA repair and cell cycle regulation genes. Five genes were identified that had significant ATRX binding and reduced expression in the absence of ATRX: Ccnd1, Ccne2, Cdk1, Chek1, and Wee1.
Single-cell RNA-sequencing was performed on U251 wild type and ATRX-deficient cells. Thirteen unique clusters were identified, with two clusters showing striking differences between the wild type and ATRX-knockout populations and two clusters having dramatic differences in cycling cells. CHEK1 (Checkpoint Kinase 1) was identified in multiple clusters as one of the leading-edge genes (top or bottom ranked gene set) of several mitotic/cell cycle regulation processes. Examination of previously published human glioma tumor datasets found that ATRX mutant tumors had increased proportions of cycling cells and cells expressing genes associated with both S and G2/M phases. Together, these findings indicate that ATRX-deficient glioma cells have dysregulation of cell-cycle regulatory genes.
Chk1 and upstream regulators ATM and ATR mediate cell cycle dysregulation in the absence of ATRX after irradiation
The transcriptomics studies pointed to cell-cycle checkpoint regulatory protein Checkpoint Kinase 1 (Chk1) as a potential regulator of irradiation responses in ATRX deficient cells. During the cell cycle there are two checkpoint pathways that respond to DNA damage, ATR-Chk1 and ATM-Chk2. While ATM-Chk2 typically recognizes double-strand breaks, ATR-Chk1 plays a role in across multiple types of DNA damage. These checkpoint pathways are critical for inhibiting or stalling cell cycle progression specifically during S phase, and at the G1/S and G2/M transitions.8
ATRX plays a role in the epigenetic control of CHEK1 expression, so this was then examined in both the glioblastoma mouse model and human cell lines. CHEK1 RNA and protein levels (unmodified and phosphorylated) were reduced in both mouse and human ATRX deficient cells and glioma models. Subsequently, Chek1 was overexpressed in ATRX deficient U251 cells, and this overexpression caused cell cycling to decrease to a rate similar to ATRX wild type U251 cells. Together, these data support a critical role for Chk1 in ATRX-mediated cell cycle dysregulation following irradiation.
The observation that ATRX expression affects cell cycle regulation genes like Chek1 indicates that in the absence of ATRX other upstream cell cycle regulators might be able to counter this cell cycle dysregulation. ATM and ATR, two master regulators of cell-cycle checkpoint activation that can impact Chk1 activation, were examined for their ability to reduce radiation-induced cell death. In irradiated ATRX deficient cells, ATM rather than ATR was activated. The addition of ATM inhibitors to these cells returned ATM and its substrates to pre-irradiation levels and decreased the proliferation of these cells.
ATM inhibitors improve outcomes for ATRX deficient tumors.
These studies indicate that ATM inhibitors could be used in combination with irradiation therapy to enhance survival and decrease tumor size. To examine this hypothesis, p53-/- tumors or p53-/-ATRX-/- tumors were implanted into C57BL/6 mice. The mice with ATRX deficient tumors had reduced survival (24 days) compared to the p53 only mutant mice (41 days). Radiation increased the survival of both groups of mice such that they had similar levels of survival (43 days). The addition of an ATM inhibitor dramatically increased the survival of mice with p53-/-ATRX-/- tumors (>100 days), while having a slight reduction in survival of the mice with p53-/- tumors (39 days). Additionally, the ATM inhibitor reduced tumor size in animals with p53-/-ATRX-/- tumors compared to radiation treatment alone.
ATRX is frequently mutated in cancer and is an important marker in classifying stage and grade of glioma. The studies performed in this paper provide a mechanism for ATRX-mutant sensitivity to irradiation therapy and provide additional therapeutic strategies for ATRX-deficient glioma.6 This paper also highlights how mechanistic insights can further provide strategies for precision medicine.
Fortis Products Featured in the Article:
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ATRX
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Regulates expression of cell cycle regulation genes.
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A301-045A
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IHC, IP, WB
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Hu
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Rabbit
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Polyclonal
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