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.

Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.

The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.Therapy resistance represents a major hurdle in the management of cancer patients. Several resistance mechanisms have been described (e.g., overexpression of efflux pumps, mutations in the DNA repair system, activation of the antiapoptotic machinery). Multicellular resistance, suggested to be important for the resistance of advanced solid tumors, 1 is due both to limited penetration of drugs into tumor parenchyma 2 and to the presence of quiescent cell populations in hypoxic regions. These populations are insensitive to clinically used drugs and are believed to repopulate tumors between therapy cycles ("regrowth resistance"). 3 Most experimental studies of resistance mechanisms and anticancer drug testing are performed with monolayer cell cultures, which is not an optimal experimental system and has limitations. Three-dimensional (3D) cell cultures, as the well-established multicellular spheroid (MCS) system, reflect the in vivo structure of solid tumors and more adequately reproduce the metabolic microenvironment of tumor tissue. The formation of a 3D structure by tumor cells is accompanied by changes in cells and their microenvironment: the cells are more tightly packed than in 2D and have differences in nutrient supply, oxygen tension, pH, proliferation, resistance to apoptosis. 4,5 MCS are more resistant to chemotherapy than cells grown in 2D. 6,7 To examine the mechanisms of drug resistance, we performed a gene expression profiling of a colon cancer cell line MCS. Among highly upregulated genes in MCS we found a subset of type I interferonstimulated genes (ISGs). A similar signature designated the interferon-related DNA damage signature (IRDS) was previously associated with resistance to radiotherapy. 8,9 The full set of ISGs is activated by the transcription factor complex ISGF3, consisting of tyrosine-phosphorylated STAT1 and STAT2, and the DNA-binding protein IRF9. However, ISGF3 lacking tyrosine phosphorylation of STAT1 and STAT2 (unphosphorylated ISGF3, U-ISGF3) can ...

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.

Type I interferons (IFN-I) protect us from viral infections. Signal transducer and activator of transcription 2 (STAT2) is a key component of interferon-stimulated gene factor 3 (ISGF3), which drives gene expression in response to IFN-I. Using electron microscopy, we found that, in naive cells, U-STAT2, lacking the activating tyrosine phosphorylation, forms a heterodimer with U-STAT1 in an inactive, anti-parallel conformation. A novel phosphorylation of STAT2 on T404 promotes IFN-I signaling by disrupting the U-STAT1-U-STAT2 dimer, facilitating the tyrosine phosphorylation of STATs 1 and 2 and enhancing the DNA-binding ability of ISGF3. IKK-ε, activated by virus infection, phosphorylates T404 directly. Mice with a T-A mutation at the corresponding residue (T403) are highly susceptible to virus infections. We conclude that T404 phosphorylation drives a critical conformational switch that, by boosting the response to IFN-I in infected cells, enables a swift and efficient antiviral defense.