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High expression of programmed death-ligand 1 (PD-L1) in cancer cells drives immune-independent, cell-intrinsic functions, leading to resistance to DNA-damaging therapies. We find that high expression of the ubiquitin E3 ligase FBXO22 sensitizes nonsmall cell lung cancer (NSCLC) cells to ionizing radiation (IR) and cisplatin, and that activation of FBXO22 by phosphorylation is necessary for this function. Importantly, FBXO22 activates PD-L1 ubiquitination and degradation, which in turn increases the sensitivity of NSCLC cells to DNA damage. Cyclin-dependent kinase 5 (CDK5), aberrantly active in cancer cells, plays a crucial role in increasing the expression of PD-L1 in medulloblastoma [R. D. Dorand et al., Science 353, 399–403 (2016)]. We show in NSCLC cells that inhibiting CDK5 or reducing its expression increases the level of FBXO22, decreases that of PD-L1, and increases the sensitivity of the cells to DNA damage. We conclude that FBXO22 is a substrate of CDK5, and that inhibiting CDK5 reduces PD-L1 indirectly by increasing FBXO22. Pairing inhibitors of CDK5 with immune checkpoint inhibitors may increase the efficacy of immune checkpoint blockade alone or in combination with DNA-damaging therapies.

Programmed death ligand 1 (PD-L1), an immune-checkpoint protein expressed on cancer cells, also functions independently of the immune system. We found that PD-L1 inhibits the killing of cancer cells in response to DNA damage in an immune-independent manner by suppressing their acute response to type I interferon (IFN; IFN-I). In addition, PD-L1 plays a critical role in sustaining high levels of constitutive expression in cancer cells of a subset of IFN-induced genes, the IFN-related DNA damage resistance signature (IRDS) which, paradoxically, protects cancer cells. The cyclic GMP-AMP synthase-stimulator of the IFN genes (cGAS-STING) pathway is constitutively activated in a subset of cancer cells in the presence of high levels of PD-L1, thus leading to a constitutive, low level of IFN-β expression, which in turn increases IRDS expression. The constitutive low level of IFN-β expression is critical for the survival of cancer cells addicted to self-produced IFN-β. Our study reveals immune-independent functions of PD-L1 that inhibit cytotoxic acute responses to IFN-I and promote protective IRDS expression by supporting protective chronic IFN-I responses, both of which enhance the resistance of cancer cells to DNA damage.

A single high dose of interferon-β (IFNβ) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNβ-induced proteins, and to constitutive resistance to DNA damage., IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage IFNβ induces the formation of a novel transcriptional complex, U-ISGF3, which contains un-phosphorylated STATs. U-ISGF3 regulates the expression of a subset of IFNβ-stimulated genes to promote resistance to virus infection and DNA damage.

A single high dose of IFN β activates powerful cellular responses in which many antiviral, pro-apoptotic, and anti-proliferative proteins are highly expressed. The expression of IFN-stimulated genes (ISGs) is mediated by ISGF3, consisting of IRF9 and tyrosine phosphorylated STATs 1 and 2. This initial response to IFN β is rapid and transient, so that the expression of most ISGs returns to basal levels within a day. However, the expression of some ISGs is prolonged for many days. We found that high levels of STAT1, STAT2, and IRF9 proteins that lack tyrosine phosphorylation are critical for the prolonged second phase response to IFN β . The expression of the STAT1, STAT2, and IRF9 proteins is increased by IFN β at late times (>24 h), and these proteins form a complex resembling ISGF3 (named unphosphorylated ISGF3, U-ISGF3), and bind to ISREs of target genes independently of tyrosine phosphorylation of STATs 1 and 2. U-ISGF3 induces only a subset of ISGs (20–30% of the total), which are mostly known to be antiviral genes and are also highly expressed in therapy-resistant cancer cells, but not cytotoxic ISGs. We observed that, in the absence of interferon treatment, high levels of U-ISGF3 significantly reduced the replication of many RNA viruses. High U-ISGF3 levels are correlated with resistance of cancer cells to DNA damage induced by radiation, etoposide, or doxorubicin. Knocking STAT1 and IRF9 down to decrease U-ISGF3 levels sensitized cancer cells to doxorubicin-induced DNA damage. Continuous exposure of cells to a low level of IFN β , often seen in cancers, leads to steady-state increases in the expression of only the U-ISGF3-dependent genes, with no sustained increases of IFN β -induced cytotoxic genes. Our study has elucidated that the U-ISGF3-dependent response to IFN β prolongs the antiviral effects, but leads to DNA damage resistance, which inhibits responses to cancer therapy.

Ionizing radiation and DNA damaging chemotherapeutic agents are used widely to treat many cancers, but the presence of cells that resist DNA damage often leads to failure to cure. One of the specific features of therapy-resistance is high expression of Interferon-Related DNA damage resistance Signature (IRDS) genes. The IRDS genes are a subset of (about 30 genes) Interferon Stimulated Genes (ISGs) including IFI27, OAS2, Mx1, and IFIT1. However, pro-apoptotic and anti-proliferative ISGs, such as IRF1, caspases, and FASL, are not included in this signature. In this study, we elucidated the molecular mechanism of how the expression of IRDS genes are increased, with no increase of other ISGs, in therapy-resistant cancer cells. When cells expressed high levels of IRF9 and STATs 1 and 2 in the absence of phosphorylation of Tyr-701-STAT1 and Tyr-690-STAT2, the expression of IRDS genes was increased, but that of other ISGs was not. The un-phosphorylated STAT1, STAT2, and IRF9 proteins formed a complex resembling Interferon Stimulated Gene Factor 3 (ISGF3), a major transcription factor in type I interferon signaling. While ISGF3, consisting of phosphorylated STATs 1 and 2 and IRF9, mediates the expression of whole set of ISGs, the unphosphorylated ISGF3 (U-ISGF3) induces only IRDS genes. ChIP assay revealed that U-ISGF3 selectively bound to the promoters of IRDS genes (IFI27, OAS2, and Mx1), but not to those of other ISGs (MyD88, IFI16, and IRF1). Continuous exposure of cells to a low level of interferonβ, which might be produced by cancer cells or surrounding immune cells, lead to steady-state increases in the expression of only U-ISGF3 and IRDS genes with no increase of phosphorylated ISGF3 and non-IRDS ISGs. We examined the correlation between the U-ISGF3 levels and DNA damage resistance in various small cell lung carcinoma (SCLC) cell lines. The expression of IRF9 and STATs 1 and 2 proteins varied in different SCLC cell lines, and high levels of U-ISGF3 correlated with resistance to DNA damage. H82, H1048, and H146 cells, which express low levels of U-ISGF3, were sensitive to DNA damage induced by radiation, etoposide, and doxorubicin, while H196, H1688, and H2195 cells expressing high levels of U-ISGF3 were resistant to DNA damage. In response to radiation, γH2A.X levels were significantly increased and remained high up to 48 hrs in H1048 cells, but the increased γH2A.X levels returned to lower levels in H196 cells, suggesting that U-ISGF3-induced gene products may increase DNA damage repair. When STAT1 was knocked down to reduce U-ISGF3 levels in H196 cells, γH2A.X levels increased by radiation remained high. Knocking IRF9 down also decreased the resistance to DNA damage. In conclusion, U-ISGF3, which is increased by a low dose of interferons, plays an important role in inducing DNA damage resistance, and its targeting sensitizes cancer cells to DNA damage induced by various cancer therapies. Citation Format: HyeonJoo Cheon, Elise G. Holvey-Bates, George R. Stark. Chronic exposure to a low dose of interferon-β increases levels of DNA damage resistance signature genes through unphosphorylated ISGF3. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2014-485