Cancer-associated SMARCAL1 loss-of-function mutations promote alternative lengthening of telomeres and tumorigenesis in telomerase-negative glioblastoma cells

Abstract

Background: Telomere maintenance mechanisms are required to enable the replicative immortality of malignant cells. While most cancers activate the enzyme telomerase, a subset of cancers uses telomerase-independent mechanisms termed alternative lengthening of telomeres (ALT). ALT occurs via homology-directed-repair mechanisms and is frequently associated with ATRX mutations. We previously showed that a subset of adult glioblastoma (GBM) patients with ATRX-expressing ALT-positive tumors harbored loss-of-function mutations in the SMARCAL1 gene, which encodes an annealing helicase involved in replication fork remodeling and the resolution of replication stress. However, the causative relationship between SMARCAL1 deficiency, tumorigenesis, and de novo telomere synthesis is not understood.

Methods: We used a patient-derived ALT-positive GBM cell line with native SMARCAL1 deficiency to investigate the role of SMARCAL1 in ALT-mediated de novo telomere synthesis, replication stress, and gliomagenesis in vivo.

Results: Inducible rescue of SMARCAL1 expression suppresses ALT indicators and inhibits de novo telomere synthesis in GBM and osteosarcoma cells, suggesting that SMARCAL1 deficiency plays a functional role in ALT induction in cancers that natively lack SMARCAL1 function. SMARCAL1-deficient ALT-positive cells can be serially propagated in vivo in the absence of detectable telomerase activity, demonstrating that the SMARCAL1-deficient ALT phenotype maintains telomeres in a manner that promotes tumorigenesis.

Conclusions: SMARCAL1 deficiency is permissive to ALT and promotes gliomagenesis. Inducible rescue of SMARCAL1 in ALT-positive cell lines permits the dynamic modulation of ALT activity, which will be valuable for future studies aimed at understanding the mechanisms of ALT and identifying novel anticancer therapeutics that target the ALT phenotype.

Keywords: ATRX; Adult gliomas; SMARCAL1; alternative lengthening of telomeres; gliomagenesis; telomere maintenance.

Figure 1.

D06MG tumor cells maintain alternative lengthening of telomeres features after in vivo propagation. (A) Western blot detection of ATRX, DAXX, and SMARCAL1 proteins. U2OS (osteosarcoma), HeLa (endocervical adenocarcinoma) and NY (osteosarcoma) cells were used for positive and negative controls for ATRX and SMARCAL1 detection. (B) IF-FISH analysis of D06MG-NSC cells for detection of telomeric foci and nuclear PML foci. (C) H&E staining of formalin-fixed paraffin-embedded orthotopic D06MG tumor tissue using 10x and 40x objectives. (D) TeloTAGG assay for the detection of telomerase activity between cell lines. HeLa and U2OS were included as positive and negative controls for telomerase activity, respectively. Three independent replicates are shown for each condition and the error bars represent the mean +/- standard deviation. (E) Western blot for the detection of SMARCAL1 protein from D06MG cell lines, as well as positive controls HeLA and U2OS. (F) C-circle assay for the detection of extrachromosomal circular telomeric repeats. Three independent replicates are shown for each condition and the error bars represent the mean +/− standard deviation. (G) Confocal imaging using a 63x objective of IF-FISH co-staining for telomeric DNA using a TelC-AlexaFlour-647 probe and an anti-PML antibody.

Figure 2.

D06MG xenografts are diploid and genomically stable. (A) Spectral karyotyping (SKY) analyses of D06MG-MSC primary cell line cultures prior to in vivo propagation. A subset of triploid and tetraploid metaphase spreads was identified, with the majority of cells being diploid. (B) SKY analysis of D06MG cells derived from subcutaneous tumors and orthotopic tumors. (C) Graph showing the total number of chromosomes present in each of the 20 metaphase spreads analyzed per condition.

Figure 3.

Enrichment of alternative lengthening of telomeres (ALT)-associated gene expression and associated PML bodies (APBs) in D06MG-SubQ and D06MG-IC lines. (A) Volcano plot of genes encoding proteins localized to ALT telomeres plotted by log2 fold change and adjusted P-value when comparing mRNA expression of D06MG-IC cells relative to D06MG-NSC. The red dashed box indicates the expanded portion of the plot visualized to the right. Gene identifiers are labeled next to the individual data points. (B) IF-FISH staining of D06MG cell lines cells with antibodies to PML and BLM, costained with a TelC-647 telomere probe and counterstained with DAPI. Dashed lines represent the location of nuclei and white arrows note the presence of BLM-positive APBs in selected cells. (C) Quantitation of the proportion of nuclei that contain an APB in each cell line. (D) Quantitation of the proportion of nuclei with APBs that exhibit colocalization with nuclear BLM foci.

Figure 4.

Inducible SMARCAL1 rescue suppresses alternative lengthening of telomeres indicators and in vivo tumorigenesis. (A) Western blot analyses measuring doxycycline-inducible expression of SMARCAL1^WT or SMARCAL1R764Q in D06MG-IC. (B) C-circle analysis of D06MG-IC without or with doxycycline-induced expression (5 days) of SMARCAL1^WT or SMARCAL1^R764Q. Differences between conditions were assessed using a student’s t-test. (C) D06MG-IC cells with inducible SMARCAL1^WT (left panel) were orthotopically implanted into mice. 5 days after implantation, mice were switched to a control or doxycycline-containing chow diet, and their survival was determined according to loss of body weight and/or the onset of neurological symptoms. The same experimental framework was used for orthotopic tumors expressing doxycycline-inducible SMARCAL1^R764Q as a control to account for possible effects of doxycycline chow (right panel). Survival differences between groups were analyzed via the log-rank test.

Figure 5.

SMARCAL1 activity suppresses telomeric DNA double-strand breaks and de novo telomere synthesis in D06MG cells. (A) D06MG-IC cells without or with doxycycline-inducible SMARCAL1 rescue were analyzed for associated PML bodies (APBs) and γH2AX abundance. Representative images are shown. Cells were cultured in a chamber slide and SMARCAL1 expression was induced with doxycycline (1 ug/ml for 5 days). Cells were fixed and processed for IF-FISH with a TelC probe, anti-γH2AX antibody, and anti-PML antibody. Bar graph depicts the proportion of nuclei that exhibit APBs with co-staining of γH2AX. (B) D06MG-IC cells without or with doxycycline-induced SMARCAL1 expression were pulse-labeled with EdU (2 hours) before processing for IF-FISH detection of APBs and EdU foci. EdU staining at APBs was used to detect nascent telomeric DNA synthesis. (C) Experiment performed as in (B) with NY osteosarcoma cells.

Figure 6.

Alternative pathways of alternative lengthening of telomeres (ALT) induction in human cancers. Cancer-associated loss-of-function genetic alterations occurring in ATRX/DAXX (top) or SMARCAL1 (bottom) can independently lead to ALT induction in cancer cells. ATRX/DAXX deficiency compromises the canonical H3.3 chaperone function of these proteins, leading to increased G-quadruplex formation and replication stress at telomeres. SMARCAL1 deficiency leads to a loss of replication fork reversal and stabilization, thus leading to degeneration into double-strand breaks, which in turn facilitate break-induced replication of telomeres. Illustration was created with Biorender.com.