Molecular biomarkers associated with targeted disruption of alternative lengthening of telomeres in atrx-deficient glioma models

Abstract Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism of telomere maintenance that occurs in approximately 10-15 percent of all cancers. ALT enables the replicative immortality of cancer cells and is characterized by elevated levels of DNA replication stress and telomere-localized DNA double-strand breaks (DSBs). In gliomas, ALT frequently co-occurs with inactivating mutations in ATRX, which is thought to promote ALT induction by disrupting ATRX-mediated histone H3.3 deposition at telomeres, thus leading to telomere synthesis by homology-directed repair mechanisms. To identify potential therapeutic vulnerabilities specific to ALT-positive tumors, we identified candidate ALT-positive cancer cell lines in the Cancer Dependency Map (DepMap) dataset and validated the presence or absence of ALT phenotypic markers, including ALT-associated PML bodies (APBs) and nuclear foci of single-stranded telomeric DNA (ssTeloC). Comparing ALT-positive cells to all other cancer cell lines in the DepMap dataset, we identified the replication fork remodeling enzymes SMARCAL1 and FANCM as gene dependencies with the highest degree of ALT-specificity. To validate SMARCAL1 as an ALT-specific dependency, we used inducible RNAi to deplete SMARCAL1 in ALT-positive glioma cells. SMARCAL1 depletion caused a marked increase in the abundance and intensity of ssTeloC nuclear foci, as well as increased γH2AX abundance in ALT+ glioma cells but not telomerase-positive cells. Inducible SMARCAL1 depletion did not coincide with detectable increases in apoptotic markers (e.g. caspase-3 cleavage). However, SMARCAL1 depletion caused a significant increase in the abundance of nuclear abnormalities indicative of mitotic catastrophe (e.g. micronuclei, nucleoplasmic bridges, and fragmented/multilobular nuclei). In an orthotopic model of ALT-positive glioma, doxycycline-inducible RNAi targeting SMARCAL1 extended the median survival of tumor-bearing animals to 155 days compared to 111 days in the non-targeting control condition. These data validate the therapeutic targetability of SMARCAL1 in ALT-positive gliomas and support the development of small molecule SMARCAL1 inhibitors for treatment of ALT-positive tumors.