Abstract PR004: SMARCAL1 is a selective dependency and a novel synthetic lethal target in ATRX/DAXX mutant ALT+ osteosarcoma and neuroblastoma
Authors: Wierdl Monika , Catlett Jerrel L. , Ocasio-Martinez Nicole , Johnson John D. , Herman Amy , Dharia Neekesh V. , Alexe Gabriela , Sweet-Cordero E. Alejandro , Bernstein Emily , Stegmaier Kimberly , Guenther Lillian M. 
Published: January 2024

Abstract
Osteosarcoma (OS) is an aggressive pediatric solid tumor that is difficult to treat with established therapies. We mined the DepMap data in 13 OS cell line this data to identify enriched dependencies whose knockout (KO) leads to selective anti-viability in OS. We observed that SMARCAL1 as a selective dependency in OS (p&lt;0.0001; effect size = -0.275). We reasoned that SMARCAL1 is important given the genomic instability of OS and SMARCAL1’s known role in replication stress. We first validated screen findings by CRISPR KO in OS lines in vitro. This demonstrated selective antiviability effect of SMARCAL1 KO in dependent OS lines. To orthogonally validate SMARCAL1 dependency, we deployed a dTAG system that utilizes a hetero-bifunctional small molecule targeting the tagged protein for degradation by an E3 ubiquitin ligase. We observed significant antiviability of dependent OS after SMARCAL1 degradation. In parallel, in order to ascertain SMARCAL1’s relevance as a target in OS in vivo, we performed a pooled barcoded CRISPR screen (n=1,626 guides) 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 its importance in OS in vivo. We next investigated biomarkers of SMARCAL1 dependency. A role for SMARCAL1 has been described in alternative lengthening of telomeres (ALT). With this knowledge, we asked whether ALT+ OS lines are enriched for SMARCAL1 dependence. 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 ALT-enriched. When we looked specifically at NB, we observed that SMARCAL1 was the most enriched dependency in ALT+ NB compared with other NB lines (p&lt;0.0001; effect size = -0.785). When then performed KO of SMARCAL1 in 2 ALT+ ATRX altered NB lines and one ALT+ ATRX wildtype (WT) NB line. We found that SMARCAL1 KO led to antiviability in ATRX altered ALT+ cell lines, while the ALT+, ATRX WT cell line was unaffected. This suggests that dependency on SMARCAL1 is specific to ATRX/DAXX alteration, rather than only ALT. In order to confirm synthetic lethality between ATRX and SMARCAL1, we next utilized a human OS line which has biallelic inactivation of SMARCAL1. We infected this line with our SMARCAL1 dTAG, leading to constitutive overexpression of tagged SMARCAL1, and then performed ATRX CRISPR KO. We observed that SMARCAL1 overexpression rescued the antiviability effect of ATRX KO, and that 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 that is engendered by the loss of WT ATRX/DAXX 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, John D. Johnson Jr, Amy Herman, Neekesh V. Dharia, Gabriela Alexe, E. Alejandro Sweet-Cordero, Emily Bernstein, Kimberly Stegmaier, Lillian M. Guenther. SMARCAL1 is a selective dependency and a novel synthetic lethal target in ATRX/DAXX mutant ALT+ osteosarcoma and neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR004.