The cardiomyocyte "redox rheostat": Redox signalling via the AMPK-mTOR axis and regulation of gene and protein expression balancing survival and death.

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death

TRIM27 (tripartite motif containing 27)

Review on TRIM27 (tripartite motif containing 27), with data on DNA, on the protein encoded, and where the gene is implicated

Role of the Tripartite Motif Protein 27 in Cancer Development

Background The tripartite motif family protein 27 (TRIM27) is a transcriptional repressor that interacts with, and attenuates senescence induction by, the retinoblastoma-associated protein (RB1). High expression of TRIM27 was noted in several human cancer types including breast and endometrial cancer, where elevated TRIM27 expression predicts poor prognosis. Here, we investigated the role of TRIM27 expression in cancer development. Methods We assessed TRIM27 expression in human cancer using cancer profiling arrays containing paired tumor and normal cRNA (n=261) as well as in murine skin cancer induced by 7, 12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA). We generated mice with disrupted expression of murine TRIM27 (Trim27(-/-)) and assessed their susceptibility to DMBA/TPA-induced skin tumor development compared with isogenic littermates (n=26 mice per group). We assessed the effect of Trim27 loss on senescence propensity in mouse embryonic fibroblasts (MEFs) by quantifying cell proliferation alongside senescence markers (senescence-associated beta-galactosidase [SA-beta-gal] activity and hypertrophic cell morphology). The contribution of RB1 on senescence and cancer susceptibility (n>20 mice per group) in Trim27(-/-) backgrounds was also assessed. Data were analyzed using the Student’s t, chi(2), or log-rank test as indicated. All statistical tests were two-sided. Results TRIM27 transcript levels are statistically significantly increased in common human cancers, including colon and lung, vs normal tissues (TRIM27 expression relative to ubiquitin: cancers vs normal tissues, mean=0.59, 95% confidence interval [CI]=0.55 to 0.63 vs mean=0.46, 95% CI=0.43 to 0.49, P<.001) as well as in chemically induced mouse skin cancer compared with matched normal tissue (Trim27 expression relative to Gapdh control: tumor vs normal skin, mean=4.2, 95% CI=3.97 to 4.43 vs mean=0.96, 95% CI=0.69 to 1.2, P<.001). Trim27(-/-) mice (n=14) were resistant to chemically induced skin cancer development (eight [57.2%] of 14 mice were tumor free) compared with Trim27(+/+) wild-type littermates (n=13) (one [7.7%] of 13 mice was tumor free). Trim27(-/-) MEFs show enhanced senescence propensity in response to replicative (percentage of SA-beta-gal-positive cells: Trim27(+/+) MEFs vs Trim27(-/-) MEFs, mean=14.2%, 95% CI=11.1% to 17.4% vs mean=53.3%, 95% CI=48.7% to 57.9%, P<.001) or oncogenic stress (percentage of SA-beta-gal-positive cells: Trim27(+/+) MEFs + Ras vs Trim27(-/-) MEFs + Ras, mean=24.0%, 95% CI=19.9% to 28.1% vs mean=37.3%, 95% CI=32.2% to 42.4%, P<.05) compared with Trim27(+/+) MEFs. These responses were alleviated following inactivation of murine RB1 (Rb1). Furthermore, Trim27(-/-) mice are not protected from cancers arising as a consequence of Rb1 deletion (median survival: Trim27(-/-)Rb(+/-) vs Trim27(-/-) Rb+/-, 14 vs 13 months; difference=1.0 month, 95% CI=0.5 to 1.6 months, P=.14). Conclusion TRIM27 expression is a modifier of disease incidence and progression relevant to the development of common human cancers and is a potential target for intervention in cancer. J Natl Cancer Inst 2012;104:9412-95

Therapeutic vulnerability to PARP1,2 inhibition in RB1-mutant osteosarcoma

Loss-of-function mutations in the RB1 tumour suppressor are key drivers in cancer, including osteosarcoma. RB1 loss-of-function compromises genome-maintenance and hence could yield vulnerability to therapeutics targeting such processes. Here we demonstrate selective hypersensitivity to clinically-approved inhibitors of Poly-ADP-Polymerase1,2 inhibitors (PARPi) in RB1-defective cancer cells, including an extended panel of osteosarcoma-derived lines. PARPi treatment results in extensive cell death in RB1-defective backgrounds and prolongs survival of mice carrying human RB1-defective osteosarcoma grafts. PARPi sensitivity is not associated with canonical homologous recombination defect (HRd) signatures that predict PARPi sensitivity in cancers with BRCA1,2 loss, but is accompanied by rapid activation of DNA replication checkpoint signalling, and active DNA replication is a prerequisite for sensitivity. Importantly, sensitivity in backgrounds with natural or engineered RB1 loss surpasses that seen in BRCA-mutated backgrounds where PARPi have established clinical benefit. Our work provides evidence that PARPi sensitivity extends beyond cancers identifiable by HRd and advocates PARP1,2 inhibition as a personalised strategy for RB1-mutated osteosarcoma and other cancers

Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

Selective Elimination of Osteosarcoma Cell Lines with Short Telomeres by Ataxia Telangiectasia and Rad3-Related Inhibitors

To avoid replicative senescence or telomere-induced apoptosis, cancers employ telomere maintenance mechanisms (TMMs) involving either the upregulation of telomerase or the acquisition of recombination-based alternative telomere lengthening (ALT). The choice of TMM may differentially influence cancer evolution and be exploitable in targeted therapies. Here, we examine TMMs in a panel of 17 osteosarcoma-derived cell lines, defining three separate groups according to TMM and the length of telomeres maintained. Eight were ALT-positive, including the previously uncharacterized lines, KPD and LM7. While ALT-positive lines all showed excessive telomere length, ALT-negative cell lines fell into two groups according to their telomere length: HOS-MNNG, OHSN, SJSA-1, HAL, 143b, and HOS displayed subnormally short telomere length, while MG-63, MHM, and HuO-3N1 displayed long telomeres. Hence, we further subcategorized ALT-negative TMM into long-telomere (LT) and short-telomere (ST) maintenance groups. Importantly, subnormally short telomeres were significantly associated with hypersensitivity to three different therapeutics targeting the protein kinase ataxia telangiectasia and Rad3-related (ATR) (AZD-6738/Ceralasertib, VE-822/Berzoserib, and BAY-1895344) compared to long telomeres maintained via ALT or telomerase. Within 24 h of ATR inhibition, cells with short but not long telomeres displayed chromosome bridges and underwent cell death, indicating a selective dependency on ATR for chromosome stability. Collectively, our work provides a resource to identify links between the mode of telomere maintenance and drug sensitivity in osteosarcoma and indicates that telomere length predicts ATR inhibitor sensitivity in cancer

Selective Elimination of Osteosarcoma Cell Lines with Short Telomeres by ATR Inhibitors

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.0c00125. Origin and characteristics of the osteosarcoma cell lines, telomere length measurement from TRF, IC50 drug response in osteosarcoma cell lines, oligonucleotides used for this study, characterization of telomere status in osteosarcoma cell lines, APB assay, ATRi concentration–survival curves in osteosarcoma lines, relationship between sensitivity to methotrexate and telomere length, comparison of ATRi sensitivities between ALT-negative and positive lines, selective death of osteosarcoma with short telomeres exposed to ATR inhibitor BAY-1895344 (PDF).To avoid replicative senescence or telomere-induced apoptosis, cancers employ telomere maintenance mechanisms (TMMs) involving either the upregulation of telomerase or the acquisition of recombination-based alternative telomere lengthening (ALT). The choice of TMM may differentially influence cancer evolution and be exploitable in targeted therapies. Here, we examine TMMs in a panel of 17 osteosarcoma-derived cell lines, defining three separate groups according to TMM and the length of telomeres maintained. Eight were ALT-positive, including the previously uncharacterized lines, KPD and LM7. While ALT-positive lines all showed excessive telomere length, ALT-negative cell lines fell into two groups according to their telomere length: HOS-MNNG, OHSN, SJSA-1, HAL, 143b, and HOS displayed subnormally short telomere length, while MG-63, MHM, and HuO-3N1 displayed long telomeres. Hence, we further subcategorized ALT-negative TMM into long-telomere (LT) and short-telomere (ST) maintenance groups. Importantly, subnormally short telomeres were significantly associated with hypersensitivity to three different therapeutics targeting the protein kinase ataxia telangiectasia and Rad3-related (ATR) (AZD-6738/Ceralasertib, VE-822/Berzoserib, and BAY-1895344) compared to long telomeres maintained via ALT or telomerase. Within 24 h of ATR inhibition, cells with short but not long telomeres displayed chromosome bridges and underwent cell death, indicating a selective dependency on ATR for chromosome stability. Collectively, our work provides a resource to identify links between the mode of telomere maintenance and drug sensitivity in osteosarcoma and indicates that telomere length predicts ATR inhibitor sensitivity in cancer.KT and LCC were supported by Cancer Research UK (C36439/A12097) and European research council (281722-HRMCB). GZ and CAM were supported by Children with Cancer UK (17-244). CM was supported through a Cancer Research UK studentship (C416/A23233)

Signalling leading to CCT020312-dependent EIF2A phophorylation.

<p><b>A) Schematic showing EIF2A kinases and their regulation.</b> Pathway agonists thapsigargin, poly (I∶C) and NaAS2O3 and their locus of action are indicated. <b>B) Effect of EIF2AK3/PERK ablation on EIF2A phosphorylation by CCT020312.</b> U-2OS human osteosarcoma cells were transfected with either of two different EIF2AK3/PERK siRNA oligonucleotides (PERK-1, PERK-2) or an irrelevant control (NT) for 72 hours. Cells were treated with 10 µM CCT020312 (CCT) or 2 µM thapsigargin (Tg) for the indicated times. Lysates were analysed by immunoblot as indicated. <b>C) Quantitation of PERK mRNA expression in siPERK transfected cells.</b> PERK mRNA was quantified in siRNA tranfected cells using SYBR Green based quantitative PCR. The comparative cycle threshold method was used to determine the fold change in PERK mRNA relative to cells tranfected with irrelevant oligonucleotide. GAPDH was quantified in parallel and used to normalise between samples. Bars represent the mean fold change in triplicate technical replicates. <b>D) Ablation of EIF2AK3/PERK prevents CCT020312-mediated cyclin D loss and accumulation of underphosphorylated pRB.</b> HCT116 human colon cancer cells were transfected with EIF2AK3/PERK siRNA oligo ‘2’ or non-targeting siRNA (NT). After 72 hours cells were treated with 10 µM CCT020312, 2 µM thapsigargin (Tg) or DMSO for times indicated. Cell lysates were analysed by immunoblotting as indicated.</p

Mechanism of action predictions using cDNA microarray-based cluster analysis.

<p><b>A) Cluster analysis.</b> Data from CCT020312 and 22 other molecularly targeted agents, 296 samples in total, established using either the CRUK Human Whole Genome-wide Array v1.0.0 or the CRUKDMF_22K_v1.0.0 array were used. Genes that significantly varied with treatment (ANOVA p<0.05 with Bonferoni-correction) were used for hierarchical clustering. Clustering was performed using the Euclidian distance as a similarity measure. <b>B) Cluster deconvolution.</b> Conditions that cluster with (Cluster I) or adjacent to (Cluster II) CCT020312 are shown.</p