Abstract
BACKGROUND: Heat shock protein 90 (HSP90) stabilizes numerous oncogenic client proteins, including factors required for telomere maintenance. Telomere dysfunction triggers chromosomal instability (CIN). To quantify telomere-directed activity and separate it from general mitotic effects, we utilized a human artificial chromosome (HAC) assay with isogenic lines carrying a linear, telomere-containing EGFP HAC or a circular, telomere-lacking EGFP HAC. METHODS: Four HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) were tested at each compound's cell-specific LC50 in HT1080 (linear and circular HACs) and HEK293 (linear HAC) cells, with GRN163L as a positive control. HAC loss was quantified by flow cytometry. Telomere length was measured by Southern blot and qPCR in parental HT1080 and HEK293 cells, and telomere signal intensity by FISH in HEK293. Telomere dysfunction (TIFs; γH2AX/TRF2) and micronuclei (MNi) were also scored. RESULTS: TAS-116 showed the strongest telomere-specific activity among HSP90 inhibitors, significantly increasing linear HAC loss in both HT1080 and HEK293 while circular HACs showed only minimal instability. TAS-116 shortened telomeres in both lines (qPCR: ~1.8-fold in HT1080; ~2-fold in HEK293) and, in HEK293, reduced FISH telomere signal to 59.66% of control. TAS-116 also increased telomere-associated damage (~ 1.8 TIFs per TIF-positive nucleus), DNA double-strand breaks, and micronuclei relative to the other HSP90 inhibitors. CONCLUSIONS: TAS-116 consistently disrupts telomere maintenance-driving linear HAC loss, telomere shortening, reduced FISH signal, elevated TIFs, and increased MNi-thereby validating the HAC-based framework for discriminating telomere-directed activity. These findings support the therapeutic promise of HSP90 inhibition against telomere maintenance in cancer.