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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.

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Supplementary Figure 1. (A-B) Successful labeling of CellTrace Violet for M059K and H4 cells. M059K and H4 cells were labeled with a cell membrane dye CellTrace Violet and verified the success of labeling by FACS. (C-D) Cell viability of M059K and H4 following THIO treatment. M059K and H4 cells were treated with control medium or THIO at 5µM and then determined the cell viability percentage via detecting PSVue643 and PI by FACS. (E-F) Western blot results of time-course and dose-titration experiments in M059K and H4. For the time-course experiment, cells were treated with THIO at 5µM and proteins were tested at 0, 24, 48 and 72 hours. For dose-titration experiments, cells were treated with serious doses of THIO for 72 hours. TMZ functions as control. Actin serves as a loading control. Supplementary Figure 2. (A) Crystal violet staining images for acquired TMZ-resistant (TR) cell lines after treatment. (B) The heatmap of quantification results of the crystal violet images of TR cells. % relative to control. (C) Senescence assay for DBTRG-05MG, M059K and H4 cells following THIO treatment at 1 µM for 7 days, and then fixed and stained using Senescence β-Galactosidase Staining Kit and taken images by microscope. (D-F) Quantification results of senescence assay for DBTRG-05MG, M059K and H4 cells. 7 fields were counted for each group. (G-J) Neurosphere growth curves for T98G, U87MG, 13.0302 and TS-603 treated with or without THIO at 5 µM.

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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.

Supplementary Figure 1. (A-B) Successful labeling of CellTrace Violet for M059K and H4 cells. M059K and H4 cells were labeled with a cell membrane dye CellTrace Violet and verified the success of labeling by FACS. (C-D) Cell viability of M059K and H4 following THIO treatment. M059K and H4 cells were treated with control medium or THIO at 5µM and then determined the cell viability percentage via detecting PSVue643 and PI by FACS. (E-F) Western blot results of time-course and dose-titration experiments in M059K and H4. For the time-course experiment, cells were treated with THIO at 5µM and proteins were tested at 0, 24, 48 and 72 hours. For dose-titration experiments, cells were treated with serious doses of THIO for 72 hours. TMZ functions as control. Actin serves as a loading control.

Supplementary Figure 3. (A) Relative initial telomere length of 17 glioma cell lines and NHA. (B) Correlation of IC50s of THIO and initial telomere length in 17 glioma cell lines. (C) Baseline relative expression of hTERT in 17 glioma cell lines and NHA. (D) RNAseq data of hTERT expression in 11 glioma cell lines obtained from CCLE. (E) Correlation of IC50s of THIO and baseline expression of hTERT in 17 glioma cell lines. (F) Correlation of IC50s of THIO and baseline expression of hTERT in 11 glioma cell lines from CCLE. (G) The heatmap of the enrichment scores of 5 telomere/telomerase-related gene signatures for 65 glioma cell lines from CCLE. (H-I) Relative telomere length of M059K and DBTRG-05MG cells following THIO treatment at 2.5 µM for 5 days. (J-K) Telomerase activity of M059K and DBTRG-05MG cells following THIO treatment at 5 µM for 72 hours. (L) Baseline telomerase activity of 10 glioma cell lines, including 5 cell lines that more sensitive to THIO and 5 cell lines that more resistant to THIO. (M) Correlation of baseline telomerase activity and cell viability (%) following THIO treatment. (N) Quantification of western blot of γH2AX expression in 8 glioma cell lines treated with THIO for 72 hours.

Supplementary Figure 5. (A-C) The heatmap of proteins changed by THIO in M059K, LN229 and U87MG cells from RPPA data. (D-F) Western blot was performed to confirm the change of these proteins in H4 (more sensitive to THIO) (D), U87MG (less sensitive to THIO) (E), and U251MG (more resistant to THIO) (F) after treatment with THIO at 5 µM. Actin serves as a loading control. (G) Heatmap of pathways regulated by THIO in M059K, LN229 and U87MG cells.

Supplementary Figure 4. (A-D) The heatmap of significantly expressed gene in M059K, H4, D2224MG and DBTRG-05MG cells treated with or without THIO for 72 hours. |Log2 FC|>2.5 and p

Supplementary Figure 6. (A) Creation of primary patient-derived glioblastoma organoids. (B) Quantification of tumor weight of U87MG xenograft model. (C) The curve of body weight of mice of U87MG xenograft model during THIO treatment. (D) Quantification of tumor weight of GBM PDX model. (E) The curve of body weight of mice of GBM PDX model during THIO treatment. (F) The curve of body weight of mice of U251MG xenograft model during THIO treatment.

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30 to 40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the “Hayflick limit”). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase (hTERT) or treatment with the telomerase-mediated telomere targeting agent 6-thio-2’deoxyguanosine (6-thio-dG) abrogates colony growth in these late passage cultures (>200PD) but not in early passage cultures (

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.

BackgroundTelomeres and telomerase in cancer cells are highly attractive targets for specific anti-tumor therapy, since telomerase is almost universally expressed in cancer cells, but not in the majority of normal counterparts.MethodsIn this presentation we summarize an unexpected, yet promising functional property of a modified nucleoside - 6-thio-2'-deoxyguanosine (6-thio-dG; THIO), as a potential anticancer agent with a unique mechanism of action. In vitro and in vivo, THIO is readily converted into the corresponding 5'-triphosphate, which is a substrate for mammalian telomerase. Incorporation of THIO into de novo synthesized cancer cell telomeres by telomerase leads to a fast induction of DNA damage responses and cancer cell senescence and apoptosis. Importantly, cancer cell death occurs in a telomere length-independent manner. THIO treatment leads to the generation of chromosomal bridges and, eventually, to the formation of cytosolic micronuclei structures, containing de novo modified telomeric DNA fragments. In addition to activation of innate immunity (i.e., cGAS pathway and NK cells), these in situ produced neo-adjuvants are exported extracellularly and then sensed by host-derived naïve dendritic cells, resulting in an enhanced cross-priming and tumor-specific T- cell (both CD4+ and CD8+) activation.ResultsTreatment with THIO overcomes resistance to checkpoint blockade (by aPD-1 or aPD-L1 agents) in advanced in vivo cancer models, leading to profound and persistent tumor regression with induction of cancer type specific long-term immune memory. Thus, in vivo cancer curative activity was observed in murine syngeneic models of colorectal (MC-38) and lung (LLC) cancers, when THIO administration was followed sequentially by atezolizumab. Combinations with other immune checkpoint inhibitors (cemiplimab; pembrolizumab) were also highly effectiveConclusionsIn summary, our findings demonstrate the importance of cancer cell telomeric DNA structural and functional integrity, as well as a therapeutically attractive opportunity to induce telomerase-mediated telomere replication stress. THIO modified telomeres increase innate sensing and adaptive antitumor immunity via “cancer cell self-produced” chemical modification of telomeres.AcknowledgementsSupported in part by CA070907 to JWS

Neurotrophins constitute a family of growth factors initially characterized as predominant mediators of nervous system development, neuronal survival, regeneration and plasticity. Their biological activity is promoted by the binding of two different types of receptors, leading to the generation of multiple and variegated signaling cascades in the target cells. Increasing evidence indicates that neurotrophins are also emerging as crucial regulators of metabolic processes in both neuronal and non-neuronal cells. In this context, it has been reported that neurotrophins affect redox balance, autophagy, glucose homeostasis and energy expenditure. Additionally, the trophic support provided by these secreted factors may involve the regulation of cholesterol metabolism. In this review, we examine the neurotrophins’ signaling pathways and their effects on metabolism by critically discussing the most up-to-date information. In particular, we gather experimental evidence demonstrating the impact of these growth factors on cholesterol metabolism.

Summary Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells. Here, we show that treatment with a telomere-targeting drug, 6-thio-2′-deoxyguanosine (6-thio-dG), leads to tumor regression through innate and adaptive immune-dependent responses in syngeneic and humanized mouse models of telomerase-expressing cancers. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/interferon I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8+ T cell activation. Moreover, 6-thio-dG overcomes resistance to checkpoint blockade in advanced cancer models. Our results unveil how telomere stress increases innate sensing and adaptive anti-tumor immunity and provide strong rationales for combining telomere-targeting therapy with immunotherapy.

Background:Statins represent a class of medications widely prescribed to efficiently treat dyslipidemia. These drugs inhibit 3-βhydroxy 3β-methylglutaryl Coenzyme A reductase (HMGR), the rate-limiting enzyme of mevalonate (MVA) pathway. Besides cholesterol, MVA pathway leads to the production of several other compounds, which are essential in the regulation of a plethora of biological activities, including in the central nervous system. For these reasons, statins are able to induce pleiotropic actions, and acquire increased interest as potential and novel modulators in brain processes, especially during pathological conditions. Objective: The purpose of this review is to summarize and examine the current knowledge about pharmacokinetic and pharmacodynamic properties of statins in the brain. In addition, effects of statin on brain diseases are discussed providing the most up-to-date information. Methods: Relevant scientific information was identified from PubMed database using the following keywords: statins and brain, central nervous system, neurological diseases, neurodegeneration, brain tumors, mood, stroke. Results: 315 scientific articles were selected and analyzed for the writing of this review article. Several papers highlighted that statin treatment is effective in preventing or ameliorating the symptomatology of a number of brain pathologies. However, other studies failed to demonstrate a neuroprotective effect.Conclusion:Even though considerable research studies suggest pivotal functional outcomes induced by statin therapy, additional investigation is required to better determine the pharmacological effectiveness of statins in the brain, and support their clinical use in the management of different neuropathologies.

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 thatin vitrolow 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 SummaryHuman lung cells growing in reduced stress conditions can divide well beyond the Hayflick limit.

Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.

Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells but not in normal cells. Here we show that treatment with a telomerase-mediated telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression in innate and adaptive immune-dependent manners in syngeneic and humanized mouse colon cancer models. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/IFN-I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8 T-cell activation. Moreover, 6-thio-dG overcomes resistances to checkpoint blockade in advanced mouse cancer models. Together, our results unveil how 6-thio-dG induced telomere stress increases innate sensing and adaptive anti-tumor immunity and provide a strong rationale for sequentially combining telomerase directed telomere-targeted therapy with immunotherapy. Citation Format: Anli Zhang, Ilgen Mender, Zhenhua Ren, Chuanhui Han, Yafang Deng, Silvia Siteni, Huiyu Li, Jerry W. Shay, Yang-Xin Fu. Telomere stress potentiates host STING-dependent anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 973.

ObjectiveCancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies.DesignTo discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents.ResultsThe drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368.ConclusionsLY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

AMP-activated protein kinase (AMPK) is considered the master metabolic regulator in all eukaryotes, as it maintains cellular energy homeostasis in a variety of tissues, including the brain. In humans, alterations in AMPK activity can lead to a wide spectrum of metabolic disorders. The relevance of this protein kinase in the pathogenesis of diabetes and metabolic syndrome is now well established. On the contrary, correlations between AMPK and brain physiopathology are still poorly characterized. The aim of this review is to summarize and discuss the current knowledge about the prospective involvement of AMPK in the onset and the progression of different neurological diseases.