Loss of DAXX induces alternative lengthening of telomeres (ALT)-associated hallmarks in prostate cancer cells in a context-specific manner

Mori, J. O.; Da, J.; Kim, J.; Rizzo, A.; Davis, C.; Lanza, C.; Brosnan-Cashman, J. A.; Meeker, A. K.; Heaphy, C. M.; Graham, M. K.

doi:10.1101/2025.08.06.668744

bioRxiv · 10.1101/2025.08.06.668744

Loss of DAXX induces alternative lengthening of telomeres (ALT)-associated hallmarks in prostate cancer cells in a context-specific manner

Mori, J. O.·Da, J.·Kim, J.·Rizzo, A.·Davis, C.·Lanza, C.·Brosnan-Cashman, J. A.·Meeker, A. K.·Heaphy, C. M.·Graham, M. K.

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Abstract

BackgroundThe histone chaperone complex, consisting of the death domain-associated protein (DAXX) and the alpha-thalassemia/mental retardation X-linked protein (ATRX), plays a pivotal role in maintaining chromatin through the deposition of the histone variant H3.3. Mutations leading to loss of ATRX or DAXX function are linked to the non-telomerase, alternative lengthening of telomeres (ALT) phenotype in certain cancers. Engineered ATRX mutations have previously been found to induce features of ALT in prostate cancer cell lines, notably in LAPC-4, but not in CWR22Rv1. This study determined the impact of DAXX mutations on ALT-associated characteristics in CWR22Rv1 and LAPC-4. MethodologyMutations were induced in CWR22Rv1 and LAPC-4 cells by targeting exon 2 of DAXX using the CRISPR-Cas9 genome editing strategy. The resulting mutant clones were then evaluated for ALT-associated characteristics, including the presence of ALT-associated PML bodies (APBs), C-circles, telomere length heterogeneity, and a lack of telomerase activity. ResultsFour CWR22Rv1 DAXX mutant clones (DAXXMut1-4) and five LAPC-4 clones (DAXXMut1-5) were evaluated. In CWR22Rv1, DAXXMut1, DAXXMut2, and DAXXMut4 were true knockout clones with frameshift mutations in both copies, while CWR22Rv1 DAXXMut3 had a frameshift mutation in one copy and an in-frame mutation in the other. Protein expression was undetectable in all the CWR22Rv1 clones, including CWR22Rv1 DAXXMut3. In LAPC-4, DAXXMut1 was a true knockout, while DAXXMut2, DAXXMut3, DAXXMut4, and DAXXMut5 clones had at least one in-frame mutation. Among these LAPC-4 clones, only DAXXMut1 had undetectable protein by western blotting. ALT-associated characteristics such as APBs, C-circles, and telomere length heterogeneity were observed only in CWR22Rv1 DAXXMut4. All the clones maintained telomerase activity, regardless of whether ALT-associated hallmarks were observed. ImplicationsThe CWR22Rv1 and LAPC-4 DAXX mutant clone models provide useful tools for future studies on telomere maintenance mechanisms and DAXX-related biology, particularly in prostate cancer.

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Mori, J. O., Da, J., Kim, J., Rizzo, A., Davis, C., Lanza, C., Brosnan-Cashman, J. A., Meeker, A. K., Heaphy, C. M., Graham, M. K.. 2025-08-09. Loss of DAXX induces alternative lengthening of telomeres (ALT)-associated hallmarks in prostate cancer cells in a context-specific manner. https://doi.org/10.1101/2025.08.06.668744

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