NPM1 phosphorylation-mediated telomere maintenance via stabilization of POLD3 in ALT-positive osteosarcoma: unraveling mechanisms and therapeutic opportunities

Maintaining telomere integrity is essential for cellular survival, and reactivation of telomerase or alternative lengthening of telomeres (ALT) represents a hallmark of cancer, ensuring replicative immortality. Osteosarcoma (OS), a malignancy in which many tumors rely on ALT for telomere maintenance, lacks effective therapeutic strategies targeting this pathway. This study aimed to identify and characterize novel molecular regulators of ALT activity and explore their potential as therapeutic targets in OS. <b>Methods:</b> Immunohistochemistry was performed to evaluate the expression of phosphorylated NPM1 (pT199-NPM1) in OS tissues. Functional experiments including NPM1 knockdown and rescue assays were conducted to assess the impact of NPM1 on break-induced telomere replication (BITR) and cell viability in ALT-positive cells. Mechanistic studies involving phosphorylation analysis, ubiquitination assays, and co-immunoprecipitation were used to determine how ATR-mediated phosphorylation of NPM1 regulates POLD3 stability and its interaction with the CST complex. Pharmacological screening was performed to identify compounds that inhibit ALT activity, followed by <i>in vitro</i> proliferation assays and <i>in vivo</i> mouse xenograft experiments to evaluate therapeutic efficacy and synergy with doxorubicin. <b>Results:</b> We identified pT199-NPM1 as a novel, highly expressed protein factor in ALT-positive OS tissues. NPM1 depletion impaired break-induced telomere replication and significantly reduced the viability of ALT-positive cells. ATR signaling phosphorylated NPM1 at Thr199, which stabilized POLD3 by preventing its ubiquitin-mediated degradation. Recruitment and function of pT199-NPM1 at telomeric damage sites required STN1, defining a CST/pT199-NPM1/POLD3 regulatory axis essential for ALT activity. Clinically, elevated Thr199 phosphorylation correlated with poor survival in OS patients, while expression of a phosphorylation-deficient T199A mutant failed to sustain ALT telomere maintenance. Pharmacological screening identified EPZ-6438, an EZH2 inhibitor, as a potent ALT suppressor that reduced NPM1 transcription, inhibited homologous recombination-mediated telomere synthesis, and suppressed OS cell proliferation. In mouse xenografts, EPZ-6438 enhanced OS cell sensitivity to doxorubicin, suggesting therapeutic synergy. <b>Conclusions:</b> This study uncovers a novel CST/pT199-NPM1/POLD3 regulatory module that is critical for ALT telomere maintenance in OS. Targeting NPM1 or its downstream effectors effectively suppresses ALT activity and enhances chemotherapy response. These findings provide new mechanistic insights into telomere regulation in ALT-positive tumors and highlight the therapeutic potential of NPM1-centered pathways in OS.