Purpose: To investigate the therapeutic role of a novel telomere-directed inhibitor, 6-thio-2′-deoxyguanosine (THIO) in gliomas both in vitro and in vivo. Experimental Design: A panel of human and mouse glioma cell lines was used to test therapeutic efficacy of THIO using cell viability assays, flow cytometric analyses, and immunofluorescence. Integrated analyses of RNA sequencing and reverse-phase protein array data revealed the potential antitumor mechanisms of THIO. Four patient-derived xenografts (PDX), two patient-derived organoids (PDO), and two xenografts of human glioma cell lines were used to further investigate the therapeutic efficacy of THIO. Results: THIO was effective in the majority of human and mouse glioma cell lines with no obvious toxicity against normal astrocytes. THIO as a monotherapy demonstrated efficacy in three glioma cell lines that had acquired resistance to temozolomide. In addition, THIO showed efficacy in four human glioma cell lines grown as neurospheres by inducing apoptotic cell death. Mechanistically, THIO induced telomeric DNA damage not only in glioma cell lines but also in PDX tumor specimens. Integrated computational analyses of transcriptomic and proteomic data indicated that THIO significantly inhibited cell invasion, stem cell, and proliferation pathways while triggering DNA damage and apoptosis. Importantly, THIO significantly decreased tumor proliferation in two PDO models and reduced the tumor size of a glioblastoma xenograft and a PDX model. Conclusions: The current study established the therapeutic role of THIO in primary and recurrent gliomas and revealed the acute induction of telomeric DNA damage as a primary antitumor mechanism of THIO in gliomas.

A select group of patients with hepatocellular carcinomas (HCC) benefit from surgical, radiologic, and systemic therapies that include a combination of anti-angiogenic and immune-checkpoint inhibitors. However, because HCC is generally asymptomatic in its early stages, this not only leads to late diagnosis, but also to therapy resistance. The nucleoside analogue 6-thio-dG (THIO) is a first-in-class telomerase-mediated telomere-targeting anticancer agent. In telomerase expressing cancer cells, THIO is converted into the corresponding 5′-triphosphate, which is efficiently incorporated into telomeres by telomerase, activating telomere damage responses and apoptotic pathways. Here, we show how THIO is effective in controlling tumor growth and, when combined with immune checkpoint inhibitors, is even more effective in a T-cell-dependent manner. We also show telomere stress induced by THIO increases both innate sensing and adaptive antitumor immunity in HCC. Importantly, the extracellular high-mobility group box 1 protein acts as a prototypical endogenous DAMP (Damage Associated Molecular Pattern) in eliciting adaptive immunity by THIO. These results provide a strong rationale for combining telomere-targeted therapy with immunotherapy.

Cell membrane transporters facilitate the passage of nucleobases and nucleosides for nucleotide synthesis and metabolism, and are important for the delivery of nucleoside analogues used in anticancer drug therapy. Here, we investigated if cell membrane transporters are involved in the cellular uptake of the nucleoside analogue DNA damage mediator 6-thio-2′-deoxyguanosine (6-thio-dG). A large panel of non–small cell lung cancer (NSCLC) cell lines (73 of 77) were sensitive to 6-thio-dG; only four NSCLC lines were resistant to 6-thio-dG. When analyzed by microarray and RNA sequencing, the resistant NSCLC cell lines clustered together, providing a molecular signature for patients that may not respond to 6-thio-dG. Significant downregulation of solute carrier family 43 A3 (SLC43A3), an equilibrative nucleobase transporter, was identified as a candidate in this molecular resistance signature. High levels of SLC43A3 mRNA predicted sensitivity to 6-thio-dG and therefore SLC43A3 could serve as a promising biomarker for 6-thio-dG sensitivity in patients with NSCLC. Significance: These findings identify a biomarker of resistance to the telomeric DNA damage mediator 6-thio-2′-deoxyguanosine.

The “Hayflick limit” is a “mitotic clock” and primary cells have a finite lifespan that correlates with telomere length. However, introduction of the telomerase catalytic protein component (TERT) is insufficient to immortalize most, but not all, human cell types under typical cell culture conditions. Originally, telomerase activity was only detected in cancer cells but is now recognized as being detectable in transit amplifying cells in tissues undergoing regeneration or in extreme conditions of wound repair. Here we report that in vitro low stress culture conditions allow normal human lung basal epithelial cells to grow for over 200 population doublings without engaging any telomere maintenance mechanism. This suggests that most reported instances of telomere-based replicative senescence are due to cell culture stress-induced premature senescence. One Sentence Summary: Human lung cells growing in reduced stress conditions can divide well beyond the Hayflick limit.

Telomerase is an attractive therapeutic target due to its expression in most cancer cells. This study focuses on harnessing the potential of telomerase to alter telomeres as a therapeutic modality. We designed and synthesized divalent dinucleotide prodrugs comprised of 6-thio-2′-deoxyguanosine (6-thio-dG; THIO) and 5-fluoro-2′-deoxyuridine (5-FdU) nucleosides. Although dinucleotides containing 5-FdU pharmacophores showed better activity in vitro versus compounds containing only THIO pharmacophores, we observed greater activity for THIO-containing compounds in vivo. The homopurine compounds MAIA-2022–12 and MAIA-2021–20, with two 6-thio-dG pharmacophores, linked by 3′, 5′- and 5′, 5′-phosphodiester bonds, respectively, demonstrated the greatest anticancer efficacy among the compounds tested and induced host immune-memory responses in vivo. The sequential combination of MAIA-2022–12 or MAIA-2021–20 with the immune anti-PD-1 or anti-PD-L1 checkpoint inhibitors demonstrated superior anticancer efficacy compared with the corresponding monotherapies. We conclude that MAIA-2022–12 and MAIA-2021–20 are promising candidates for future preclinical and potential clinical studies.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Nonetheless, deeper molecular understanding of NSCLC has resulted in novel therapeutic approaches, including targeted therapy and immunotherapy, which have improved patient prognosis and outcomes in recent years. Immune checkpoint inhibitors (ICIs), with or without chemotherapy, are now considered valuable components of treatment for NSCLC cases that do not have specific actionable genetic mutations. Patients with actionable genetic mutations are candidates for targeted therapies. The primary focus of this review is the rationale for using ICIs in the perioperative setting for patients with resectable NSCLC and in advanced disease settings. Furthermore, we compare the benefits of using ICIs with the challenges associated with their clinical implementation in resectable and advanced NSCLC. Finally, we emphasize the development of novel treatment strategies that potentially provide an optimal treatment choice for patients with advanced NSCLC.