ATRX loss induces multiple hallmarks of the alternative lengthening of telomeres (ALT) phenotype in human glioma cell lines in a cell line-specific manner

Abstract

Cancers must maintain their telomeres at lengths sufficient for cell survival. In several cancer subtypes, a recombination-like mechanism termed alternative lengthening of telomeres (ALT), is frequently used for telomere length maintenance. Cancers utilizing ALT often have lost functional ATRX, a chromatin remodeling protein, through mutation or deletion, thereby strongly implicating ATRX as an ALT suppressor. Herein, we have generated functional ATRX knockouts in four telomerase-positive, ALT-negative human glioma cell lines: MOG-G-UVW, SF188, U-251 and UW479. After loss of ATRX, two of the four cell lines (U-251 and UW479) show multiple characteristics of ALT-positive cells, including ultrabright telomeric DNA foci, ALT-associated PML bodies, and c-circles. However, telomerase activity and overall telomere length heterogeneity are unaffected after ATRX loss, regardless of cellular context. The two cell lines that showed ALT hallmarks after complete ATRX loss also did so upon ATRX depletion via shRNA-mediated knockdown. These results suggest that other genomic or epigenetic events, in addition to ATRX loss, are necessary for the induction of ALT in human cancer.

MYCN amplification and ATRX mutations are incompatible in neuroblastoma

“…These data were extended to five other ATRX-mutant cell lines including two glioma cell lines that had isogenic ATRX-wild-type controls ( Supplementary Fig. 4) 23 . A marker of DNA-replicative stress (pRPA32) was upregulated in the absence of ATRX and in the presence of doxycycline.…”

Current understanding of extrachromosomal circular DNA in cancer pathogenesis and therapeutic resistance

“…The production of t-circles/c-circles using homologous recombination can be obviously inhibited by RAD52 deletion [81,82]. ATRX loss leads to multiple phenotypic features of ALT in two high-grade glioma cell lines, U-251 and UW479, including c-circle formation [83]. Knockdown of the Ku70/80 heterodimer by shRNAs reduces the levels of t-circles and activates the p53 pathway, ultimately resulting in significantly decreased cell growth in SaOS2 osteosarcoma cells [37].…”

Alternative Lengthening of Telomeres in Pediatric Cancer: Mechanisms to Therapies

“…Recent work has improved our understanding of the underlying mechanism of ALT, however, despite this, it is still not clear as to the exact process by which ALT development occurs. Due to the near universal loss of the SWI/SNF protein ATRX in ALT cancer, and the ability of ATRX to suppress markers of ALT in a DAXX dependent manner, it would appear that loss of ATRX is a key factor in the development of ALT (27)(28)(29). Indeed, one recent study showed that, in certain cell lines, markers of ALT could be triggered upon loss of ATRX alone (28).…”

“…Due to the near universal loss of the SWI/SNF protein ATRX in ALT cancer, and the ability of ATRX to suppress markers of ALT in a DAXX dependent manner, it would appear that loss of ATRX is a key factor in the development of ALT (27)(28)(29). Indeed, one recent study showed that, in certain cell lines, markers of ALT could be triggered upon loss of ATRX alone (28). This observation is in contrast to the majority of cases where ATRX loss alone is insufficient to trigger ALT and raises interesting questions as to the other underlying factors present in these cells.…”