Loss of the Chromatin Remodeler, ATRX, Promotes Aggressive Features of Osteosarcoma and Activates NF-κB Signaling and Integrin Receptor Binding

Abstract

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Recent studies of OS have begun to recognize the relatively common mutations in and subsequent loss-of-function of the gene, ATRX, however the biological impact of the loss of this gene on tumor biology is not well-understood. Thus, exploring the role of ATRX expression loss in OS tumorigenesis may provide a better understanding of this disease as well as guide us to potential new and much needed targeted therapies based on expression of this gene. Here we investigated how loss of function of the chromatin remodeler, ATRX, impacts OS biology and contributes to aggressive disease phenotypes, and we identified the cellular signaling pathways driving these altered phenotypes. In order to explore the role of ATRX loss in mouse OS, we used a previously established Osterix-Cre driven genetically engineered mouse model of OS to examine tumor initiation in mice with loss of Rb and p53 compared to loss of Rb, p53, and ATRX. In this mouse model, ATRX loss correlated with increased tumor initiation relative to wildtype ATRX expression. To investigate how ATRX loss impacts human OS cellular phenotypes of aggression, we stably transduced the human 143B OS cell line with a non-silencing shRNA or one of two independent shRNA constructs for ATRX knockdown, and ATRX knockout 143B and MG63 cell lines were generated using CRISPR-Cas9. These cell lines were then manipulated both in vitro and in vivo to characterize the resulting phenotypic changes of tumor growth, migration, invasion and metastasis in human OS. ATRX knockdown or knockout in the 143B human OS cell line enhanced growth and local invasion of established xenograft tumors. These knockdown and knockout cells displayed significantly greater migration and invasion than the wildtype cells in both scratch wound and transwell migration and invasion assays. An orthotopic mouse model showed increased lung metastasis in mice injected with the 143B ATRX knockout cell line compared to wildtype. In order to further elucidate the cellular mechanisms that correspond with the phenotypic changes found with loss of ATRX expression, RNA-Seq and ATAC-Seq were performed on the 143B ATRX shRNA knockdown and non-silenced control cells. In the sequencing results, several NF-κB-associated pathways were most significantly upregulated (based on normalized enrichment scores) and several extracellular matrix (ECM) pathways were most significantly downregulated, supporting a role for ATRX in matrix remodeling and invasion. NF-κB ELISA assays were used to further validate the sequencing data, and a chromatin binding motif analysis identified a common overlap with ETS transcription factor motifs. Wildtype or CRISPR-Cas9 knockout MG63 cells were screened with 2,100 bioactive small molecule inhibitors to identify drugs for which ATRX loss of function led to increased drug efficacy, and there was significant sensitization of the ATRX knockout to an integrin inhibitor, SB273005. An in vivo study in an ATRX-null U2OS xenograft mouse model showed reduced tumor growth with this integrin inhibitor compared to vehicle-treated mice. Additionally, this integrin inhibitor reversed the increased phenotypes of aggression seen with ATRX loss in vitro, including migration and invasion, and this drug partially reversed the nuclear upregulation of the NF-κB transcription factors. Thus, our studies show that functional ATRX plays an important tumor suppression role in OS, and decreased ATRX expression is associated with more aggressive cellular phenotypes, including increased growth, migration, invasion and metastasis. Here we show a correlation between ATRX deficiency, these aggressive phenotypes, activation of NF-κB signaling, increased integrin αvβ3 expression, altered ECM remodeling, and ETS transcription factor binding. However, ATRX-deficient cells display substantially increased sensitivity to integrin signaling inhibition. The relationship of ATRX expression with integrin-binding, NF-κB activation, and ETS transcription factor binding has not been noted in previous studies, but may impact other known diseases with ATRX loss, including other cancers and ATR-X mental retardation syndrome. Future studies are needed to explore integrin inhibition as a potential new targeted therapy for ATRX-deficient OS.