Proliferating cells must enact a program of telomere lengthening to counteract the chromosome end replication problem. In most types of cancer cells, telomeres are maintained through the action of the ribonucleoprotein telomerase, but some cancer cells, particularly those of mesenchymal origin, utilize an alternative method of telomere repair and lengthening termed the alternative lengthening of telomeres (ALT) pathway. Since telomere maintenance is essential for tumor cell immortality, better understanding of the ALT mechanism could potentially reveal drug targets that could be used to develop novel therapies for tumors that use ALT. It has been previously observed that ALT tumors frequently carry mutations in ATRX, which partners with the protein DAXX in a chromatin remodeling complex, but how these mutations facilitate the ALT pathway is not well understood. Work in our lab identified an ALT-positive osteosarcoma cell line, identified here as OS1, in which DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. We find that knockdown of the DAXX-KIFC3 fusion neither impairs ALT nor cell proliferation, suggesting that the fusion represents a loss of function. Furthermore, inducible restoration of wild-type DAXX, reversibly abrogates ALT function in this cell line. One of the hallmarks of ALT is localization of telomeres and DNA recombination machinery to nuclear PML bodies, resulting in formation of ALT- associated PML Bodies, or APBs. Thus it may be considered that changes in PML body composition represent a key aspect of the ALT mechanism. We observe that in OS1 both DAXX and ATRX fail to localize to PML bodies. This finding is consistent with the fact that the DAXX-KIFC3 fusion results in loss of a C-terminal SUMO interaction motif that normally mediates PML body interaction. Leveraging our inducible system, using biochemical and imaging approaches, we are working to define the role of DAXX in maintaining PML body composition. Citation Format: Sarah F. Clatterbuck Soper, Soyeon A. Showman, Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sven Bilke, Paul S. Meltzer. A DAXX-KIFC3 fusion potentiates alternative lengthening of telomeres in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2017-3467
The unlimited proliferative capacity of cancer cells is closely linked to maintenance of their telomeres, which shorten with each cell division in normal cells. Cancer cells are able to maintain telomere length by multiple mechanisms, including activation of telomerase and the recombination based alternative lengthening of telomeres (ALT) pathway. ALT is prevalent in osteosarcoma, with approximately 50% of osteosarcoma cases using ALT for telomere maintenance. Mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 correlate with activation of the ALT pathway in several tumor systems. While loss of ATRX is a frequent event in osteosarcoma tumors, alterations of DAXX have not been reported. We characterized the telomere maintenance mechanisms utilized by 11 osteosarcoma cell lines. Of these, 45% (5/11) were ALT positive and 45% (5/11) were telomerase positive. One cell line possessed features of both telomere maintenance mechanisms. Among ALT positive osteosarcoma cell lines, we observed frequent loss of ATRX expression (4/5) and a previously unreported translocation resulting in disruption of DAXX. The translocation abolishes recruitment of DAXX to nuclear PML bodies and prevents normal DAXX function. By reintroducing full length DAXX, we were able to suppress telomere maintenance by ALT as evidenced by multiple assays including loss of C-circles and ALT-associated PML bodies, thus demonstrating that continued DAXX deficiency is necessary for maintenance of the ALT mechanism. Suppression of ALT by DAXX reintroduction did not result in compensatory activation of telomerase. This first demonstration of ALT suppression by DAXX supports a mechanistic connection between loss of the ATRX/DAXX chromatin remodeling complex and telomere maintenance by ALT. Understanding this relationship may uncover vulnerabilities specific to ALT tumors that could potentially lead to the development of targeted therapies for diverse cancers that depend on the ALT pathway. Citation Format: Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Ogan Abaan, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sean R. Davis, Sven Bilke, Paul S. Meltzer. Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2717.
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