Over-expression of the translesion synthesis polymerase (TLS pol) hpol κ in glioblastomas has been linked to a poor patient prognosis; however, the mechanism promoting higher expression in these tumors remains unknown. We determined that activation of the aryl hydrocarbon receptor (AhR) pathway in glioblastoma cells leads to increased hpol κ mRNA and protein levels. We blocked nuclear translocation and DNA binding by the AhR in glioblastoma cells using a small-molecule and observed decreased hpol κ expression. Pharmacological inhibition of tryptophan-2,3-dioxygenase (TDO), the enzyme largely responsible for activating the AhR in glioblastomas, led to a decrease in the endogenous AhR agonist kynurenine (Kyn) and a corresponding decrease in hpol κ protein levels. Importantly, we discovered that inhibiting TDO activity, AhR signaling, or suppressing hpol κ expression with RNA interference led to decreased chromosomal damage in glioblastoma cells. Epistasis assays further supported the idea that TDO activity, activation of AhR signaling and the resulting over-expression of hpol κ function primarily in the same pathway to increase endogenous DNA damage. These findings indicate that up-regulation of hpol κ through glioblastoma-specific TDO activity and activation of AhR signaling likely contributes to the high levels of replication stress and genomic instability observed in these tumors.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced—indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation—a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.

Metabolism of the essential amino acid L-tryptophan (TRP) is implicated in a number of neurological conditions including depression, neurodegenerative diseases, and cancer. The TRP catabolite kynurenine (KYN) has recently emerged as an important neuroactive factor in brain tumor pathogenesis, with additional studies implicating KYN in other types of cancer. Often highlighted as a modulator of the immune response and a contributor to immune escape for malignant tumors, it is well known that KYN has effects on the production of the co-enzyme nicotinamide adenine dinucleotide (NAD+), which can have a direct impact on DNA repair, replication, cell division, redox signaling, and mitochondrial function. Additional effects of KYN signaling are imparted through its role as an endogenous agonist for the aryl hydrocarbon receptor (AhR), and it is largely through activation of the AhR that KYN appears to mediate malignant progression in gliomas. We have recently reported on the ability of KYN signaling to modulate expression of human DNA polymerase kappa (hpol κ), a translesion enzyme involved in bypass of bulky DNA lesions and activation of the replication stress response. Given the impact of KYN on NAD+ production, AhR signaling, and translesion DNA synthesis, it follows that dysregulation of KYN signaling in cancer may promote malignancy through alterations in the level of endogenous DNA damage and replication stress. In the following perspectives article, we discuss the connections between KYN signaling, DNA damage tolerance, and genomic instability, as they relate to cancer.

Aberrant expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy and immune response in gliomas in part through kynurenine (KYN)-mediated activation of the aryl hydrocarbon receptor (AhR). In the current study, we investigated the hypothesis that TDO activation in gliomas has a broad impact upon genome maintenance -promoting tolerance of replication stress (RS) and repair of DNA damage. We report that inhibition of TDO activity attenuated recovery from hydroxyurea (HU)-induced RS and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU), as fork progress was impeded when TDO-deficient glioma cells were treated with BCNU. Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to treatment with BCNU, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced -indicative of increased fork collapse. Restoration of KYN levels protected against some replication-associated effects of BCNU. Inhibition of TDO activity had a strong anti-proliferative effect on glioma-derived cells -enhancing the cytotoxic effects of BCNU. Analysis of results obtained using quantitative proteomics revealed TDOdependent changes in several signaling pathways -including down-regulation of DNA repair factors and sirtuin signaling. Consistent with these observations, inhibition of TDO diminished SIRT7 recruitment to chromatin, which increased histone H3K18 acetylation -a key mark involved in 53BP1 recruitment to sites of DNA damage. Cells lacking TDO activity exhibited defective recruitment of 53BP1 to gH2AX foci, which corresponded with delayed repair of BCNU-induced DNA breaks. Addition of exogenous KYN increased the rate of break repair. The discovery that TDO activity modulates sensitivity to DNA damage by fueling SIRT7/53BP1 localization to chromatin and repair of BCNU-induced DNA damage highlights the potential for tumor-specific metabolic changes to influence genome stability and may have implications for glioma biology and treatment strategies.

A small library of (Z)-2-(benzo[d][1,3]dioxol-5-yl) and (Z)-2,3-dihydrobenzo [b][1,4] dioxin-6-yl analogs of 2- and 3-phenylacetonitriles has been synthesized and evaluated for their anti-cancer activities against a panel of 60 human cancer cell lines. The dihydrodioxin analog 3j and dioxol analogs 5e and 7e exhibited the most potent anti-cancer activity of all the analogs synthesized in this study, with GI50 values of <100 nM against almost all of the cell lines in the human cancer cell panel. Of these three, only compound 3j inhibited tubulin polymerization to any degree in vitro. The binding modes of 3j and the structurally related tubulin-inhibitor DMU-212 were determined by virtual docking studies with tubulin dimer. Compound 3j docked at the colchicine-binding site at the dimer interface of tubulin. The Full-Fitness (FF) score of 3j was observed to be substantially higher than DMU-212, which agrees well with the observed anti-cancer potency (GI50 values). The mechanism by which dioxol analogs 5e and 7e exert their cytotoxic effects remains unknown at this stage, but it is unlikely that they affect tubulin dynamics. Nevertheless, these findings suggest that both dioxol and dihydrodioxin analogs of phenylacrylonitrile may have potential for development as clinical candidates to treat a variety of human cancers.