To better understand the mechanisms by which ATRX contributes to osteosarcoma aggressiveness, we used both in vitro and in vivo methods to examine changes in tumor initiation, growth and proliferation, migration, invasion and metastasis that correspond with loss of ATRX expression. An Osterix-Cre driven genetically engineered mouse model of osteosarcoma was developed to examine tumor development in mice with loss of Rb and p53 compared to loss of Rb, p53, and ATRX.

Additionally, human 143B osteosarcoma cells were stably transduced with non-silencing shRNA or one of two independent shRNA constructs for ATRX knockdown, and an ATRX knockout 143B cell line was generated using CRISPR-Cas9. Control and ATRX knockdown/knockout cells were injected subcutaneously in SCID-beige mice, and tumor growth rates were compared. In an orthotopic model of osteosarcoma, SCID-beige mice were injected subperiosteally in the tibia with luciferase-labelled 143B wildtype or CRISPR knockout cells, and lung metastases were measured using IVIS imaging. In parallel, RNA-Seq was performed on the ATRX knockdown cell lines. CRISPR-Cas9 also was used to knock out ATRX in the human MG63 osteosarcoma cell line, and the wild-type or knockout MG63 cell lines were screened with 2,100 bioactive small molecule inhibitors to identify drugs for which ATRX loss of function led to increased drug efficacy. One drug of interest, an integrin inhibitor, was tested in vivo in an ATRX-null U2OS xenograft mouse model.

In a genetically engineered mouse model, ATRX loss correlated with increased tumor initiation relative to wildtype ATRX expression. ATRX shRNA knockdown and CRISPR knockout in the 143B human osteosarcoma cell line enhanced growth and local invasion of established xenograft tumors. The orthotopic mouse model showed increased lung metastasis in mice injected with the ATRX knockout cell line compared to wildtype. Whole transcriptomic profiling of the ATRX knockdown cell lines by RNA-Seq showed significant upregulation of the NF-κβ pathway, which has been shown to play a role in cancer cell proliferation, decreased apoptosis, and increased angiogenesis. Additionally, this sequencing showed significant downregulation of several extracellular matrix pathways, supporting a role for ATRX in matrix remodeling and invasion. Consistent with the RNA-Seq data, a drug screen of 2,100 bioactive small molecule inhibitors showed significant sensitization of the ATRX knockout to an integrin inhibitor. An in vivo study with this integrin inhibitor showed reduced tumor growth 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 partially reversed the upregulation of the NF-κβ transcription factors.

In conclusion, decreased ATRX expression in osteosarcoma is associated with more aggressive tumors exhibiting increased proliferation, growth, migration, invasion and metastasis, which are correlated with activation of NF-κβ signaling and altered extracellular matrix signaling. However, this increased aggression comes at a cost: ATRX-null cells display substantially increased sensitivity to integrin signaling inhibition. Future studies will explore integrin inhibition as a potential new targeted therapy for ATRX-null osteosarcomas.