IF dual AAV-mediated chromatin restoration — intratumoral AAV9-DAXX (week 0) followed by AAV9-ATRX (week 1), both at ~1×10¹¹ vg/tumor, administered without any concurrent G-quadruplex (G4) stabilizer — is delivered to Saos-2 ALT-positive osteosarcoma xenografts in female athymic nude mice (6–8 weeks), and then supplemented at week 3 with intratumoral AAV9-RNaseH1 overexpression to resolve residual TERRA R-loops at telomeres,

THEN ≥70% reduction in APB foci (immuno-FISH, PML/telomere colocalization) and ≥70% reduction in C-circle signal (quantitative rolling-circle amplification C-circle assay) will be observed by week 6 compared to vehicle-AAV controls, with secondary reductions in telomere sister-chromatid exchange (T-SCE) frequency and telomere length heterogeneity,

BECAUSE of the following mechanistic chain:

1. Loss of ATRX is causally sufficient to activate ALT, as demonstrated by C-circle production, APB formation, fragile telomeres, and elevated T-SCEs in ATRX-null sarcoma models (ATRX loss drives ALT hallmarks in primary CAST/EiJ sarcoma)[https://doi.org/10.1101/2023.11.06.565874]. Restoring ATRX expression re-establishes G4 resolution at TERRA-rich telomeric repeats and re-deposits H3.3 into heterochromatin, directly suppressing ALT (ATRX restoration suppresses C-circles and APBs)[https://doi.org/10.1038/ncomms8538].

2. DAXX, ATRX's obligate chromatin-remodeling partner, independently and rapidly suppresses ALT in the osteosarcoma line G292 when stably re-expressed; this requires intact C-terminal SUMO-interaction motif–mediated PML body localization and acts through a distinct mechanism from ATRX alone, likely by enforcing PML-nuclear body sequestration of ALT recombination machinery (DAXX re-expression rapidly suppresses ALT in osteosarcoma)[https://doi.org/10.1038/s41598-019-41058-8]. Sequential DAXX-then-ATRX delivery reconstitutes the native DAXX/ATRX histone chaperone complex at PML bodies before full G4 resolution occurs, avoiding a window of unopposed G4 stress.

3. [CRITICAL MECHANISTIC INVERSION] G4 stabilizers including RHPS4 — and by mechanistic class, pyridostatin (PDS) — do not suppress ALT in ALT-positive osteosarcoma; instead they paradoxically fuel ALT by inducing telomeric replication stress that is converted to APB enhancement, T-SCE elevation, and C-circle accumulation, while reducing RAD51 and CHK1 by only ~30% (G4 stabilization fuels the ALT pathway in ALT-positive osteosarcoma)[https://doi.org/10.3390/genes11030304]. Furthermore, G4 stabilization directly impairs ATRX-dependent ALT suppression because ATRX must bind and unwind G4-forming G-rich repeats to enforce heterochromatin (G4 stabilization impairs ATRX-dependent ALT suppression)[https://doi.org/10.1038/ncomms8538]. Therefore, PDS co-treatment is antagonistic to ATRX restoration and must be omitted; the original combination premise requires fundamental revision.

4. Once DAXX/ATRX restoration resolves the chromatin remodeling deficit, res...

SENS category: OncoSENS

Key references: • doi.org/10.1101/2023.11.06.565874]. • doi.org/10.1038/ncomms8538]. • doi.org/10.1038/s41598-019-41058-8]. • doi.org/10.3390/genes11030304]. • doi.org/10.1093/narcan/zcab031].