Loss of DAXX induces alternative lengthening of telomeres (ALT)-associated hallmarks in prostate cancer cells in a context-specific manner
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Background
The 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.
Methodology
Mutations 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.
Results
Four CWR22Rv1 DAXX mutant clones ( DAXX Mut 1 - 4 ) and five LAPC-4 clones ( DAXX Mut 1-5 ) were evaluated. In CWR22Rv1, DAXX Mut 1, DAXX Mut 2, and DAXX Mut 4 were true knockout clones with frameshift mutations in both copies, while CWR22Rv1 DAXX Mut 3 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 DAXX Mut 3 . In LAPC-4, DAXX Mut 1 was a true knockout, while DAXX Mut 2, DAXX Mut 3, DAXX Mut 4 , and DAXX Mut 5 clones had at least one in-frame mutation. Among these LAPC-4 clones, only DAXX Mut 1 had undetectable protein by western blotting. ALT-associated characteristics such as APBs, C-circles, and telomere length heterogeneity were observed only in CWR22Rv1 DAXX Mut 4 . All the clones maintained telomerase activity, regardless of whether ALT-associated hallmarks were observed.
Implications
The 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.
