Abstract 5821: The role of SMARCAL1 loss-of-function mutations in glioblastoma telomere maintenance
Authors: Liu Heng , Xu Cheng , Chen Lee H. , Diplas Bill H. , Ling Jinjie , Greer Paula K. , Roskoski Martin , Keir Stephen T. , Ashley David M. , Yan Hai , Waitkus Matthew S. 
Published: September 2020

Abstract
Malignant tumor cells overcome the restriction of progressive telomere shortening by employing telomere maintenance mechanisms (TMMs) to achieve replicative immortality. Over 80% of human cancers maintain telomere length using the enzyme telomerase, which uses an RNA template subunit. However, other cancers maintain their telomeres in a telomerase-independent manner, also known as Alternative Lengthening of Telomeres (ALT). Recently, we identified loss-of-function mutations in the SMARCAL1 gene as novel genetic alterations associated with the ALT phenotype in a subset of GBM that is wildtype for IDH1 and TERT promoter mutations. SMARCAL1 encodes an annealing helicase that localizes to sites of DNA damage and/or replication stress and resolves stalled replication fork structures to facilitate fork progression within difficult-to-replicate DNA sequences, such as telomeres. However, limited models are available to study the underlying mechanisms associated with SMARCAL1 deficiency and ALT induction, and tumorigenic patient-derived xenograft (PDX) models of SMARCAL1 deficient GBM have yet to be established. In this study, we sought to generate an ALT-positive, SMARCAL1-deficient PDX by subcutaneously transplanting the D06MG cell line, which harbors a native SMARCAL1 loss-of-function mutation, into immune-compromised mice. Transplanted cells formed palpable subcutaneous tumors in nude mice within approximately 6 months. Tissue from these tumors was harvested, disaggregated, and intracranially transplanted into nude mice. Immune compromised mice bearing intracranial xenografts showed neurological symptoms due to tumor growth within three months. Cell lines were derived from PDX tissue for further characterization of telomere-related endpoints. PDX-derived D06MG cell lines maintained ALT status, as evidence by a lack of telomerase activity, presence of APBs (ALT-associated promyelocytic leukemia (PML) bodies), and extrachromosomal C-circles. PDX-derived D06MG cell lines were characterized relative to the parental cell line using SKY (spectral karyotyping) and RNA-seq analyses. SKY analysis revealed that, during the in vivo selection, a unique homogeneous subpopulation emerged, which remained genetically stable even after intracranial injection. The majority of PDX-derived cells are diploid and share similar chromosome aberrations. RNA-sequencing and pathway enrichment analyses revealed that several signal transduction pathways were upregulated in the D06MG PDX lines, while cytosolic DNA sensing pathways were partially suppressed. In summary, we have generated and characterized a PDX model of SMARCAL1-deficient GBM that maintains telomeres via the ALT pathway.
Citation Format: Heng Liu, Cheng Xu, Lee H. Chen, Bill H. Diplas, Jinjie Ling, Paula K. Greer, Martin Roskoski, Stephen T. Keir, David M. Ashley, Hai Yan, Matthew S. Waitkus. The role of SMARCAL1 loss-of-function mutations in glioblastoma telomere maintenance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5821.