Abstract 3949: SMARCAL1 is a novel synthetic lethal target in ALT+ osteosarcoma and neuroblastoma

Abstract

Abstract Osteosarcoma (OS) is an aggressive pediatric solid tumor that is difficult to treat with established therapies. We performed CRISPR/Cas9 screening in 13 OS cell lines as part of the Dependency Map project. Mining this data to identify genes whose knockout (KO) leads to selective anti-viability in OS, we observed the helicase SMARCAL1 as a selective dependency in OS (p<0.0001; effect size = -0.275). Given the genomic instability of OS and SMARCAL1’s related, established role in replication stress, we first validated our screens by CRISPR KO in OS lines in vitro. This demonstrated selective antiviability of SMARCAL1 KO in dependent OS lines. To orthogonally validate SMARCAL1 dependency, we deployed a dTAG system, utilizing a hetero-bifunctional small molecule targeting the tagged protein for degradation by an E3 ubiquitin ligase. We observed antiviability of dependent lines after SMARCAL1 degradation. In parallel, in order to ascertain importance of this gene in vivo, we performed a barcoded CRISPR pooled screen against pre-selected OS dependencies in an OS cell line xenograft in NSG mice. After sequencing of tumor gDNA, we identified SMARCAL1 as a top hit, suggesting relevance in vivo.We next investigated biomarkers of SMARCAL1 dependency. A role for SMARCAL1 has been described in alternative lengthening of telomeres (ALT). We asked whether ALT+ OS cell lines are enriched for SMARCAL1 dependency. We found a correlation between ALT+ OS and SMARCAL1 dependence in our CRISPR screens. We then asked if this extends to other ALT+ tumors. Neuroblastoma (NB), the most common extracranial pediatric solid tumor, is enriched for ALT. When we looked specifically at NB models, we observed that SMARCAL1 was the most enriched dependency in ALT+ NB compared with other NB lines (p<0.0001; effect size = -0.785). We then performed SMARCAL1 KO in 2 ALT+ ATRX-altered NB lines and one ALT+ ATRX-WT NB line. We found that SMARCAL1 KO led to antiviability in the ATRX-altered ALT+ cell lines, while the ALT+ ATRX-WT cell line was unaffected. This suggests that dependency on SMARCAL1 is specific to ATRX alteration, rather than ALT alone.In order to confirm synthetic lethality between ATRX and SMARCAL1, we next utilized a human OS line not included in the DepMap screen, which has biallelic inactivation of SMARCAL1. We infected this line with our SMARCAL1 dTAG, leading to constitutive overexpression of SMARCAL1, and then performed ATRX KO. We observed that SMARCAL1 overexpression rescued the antiviability effect of ATRX KO. This could be reversed by degradation of exogenous SMARCAL1, suggesting that ATRX and SMARCAL1 can compensate for one another. Taken together, these findings demonstrate that SMARCAL1 is a selective dependency engendered by the loss of WT ATRX in OS and NB and that SMARCAL1 is playing a critical role in mediating ALT. Studies are ongoing to determine the mechanism of SMARCAL1’s activity and potential for targeting this enzyme for therapeutic benefit. Citation Format: Monika Wierdl, Jerrel L. Catlett, Nicole Ocasio-Martinez, Jon D. Johnson, Amy Herman, Neekesh V. Dharia, Gabriela Alexe, E. Alejandro Sweet-Cordero, Emily Bernstein, Kimberly Stegmaier, Lillian M. Guenther. SMARCAL1 is a novel synthetic lethal target in ALT+ osteosarcoma and neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3949.