The immunomodulatory anticancer agent, RRx-001, induces an interferon response through epigenetic induction of viral mimicry

Abstract

Background: RRx-001, a dinitroazetidine derivative, is a novel anticancer agent currently in phase II clinical trials. It mediates immunomodulatory effects either directly through polarization of tumor associated macrophages or indirectly through vascular normalization and increased T-lymphocyte infiltration. With multiple additional mechanisms of action including upregulation of oxidative stress, depletion of GSH and NADPH, anti-angiogenesis and epigenetic modulation, RRx-001 is being studied as a radio- and chemo-sensitizer to resensitize tumors to prior therapy and to prime tumors to respond to radiation, chemotherapy and immunotherapy in combination therapy studies. Here, we identified another mechanism, viral mimicry, which refers to the "unsilencing" of epigenetically repressed viral genes present in the tumor that provokes an immune response and may contribute to the anticancer activity of RRx-001.

Results: RRx-001 inhibited the growth of colon cancer cells (HCT 116) and decreased levels of the DNA methyltransferases DNMT1 and DNMT3a in a time and dose-dependent manner. Treatment of HCT 116 cells with 0.5 μM RRx-001 for 24 h significantly increased transcripts of interferon (IFN)-responsive genes and this induction was sustained for up to 4 weeks after transient exposure to RRx-001. ELISA assays showed that RRx-001 increased secretion of type I and III IFNs by HCT 116 cells, and these IFNs were confirmed to be bioactive. Transcription of endogenous retrovirus ERV-Fc2 and LTRs from the ERV-L family (MLT2B4 and MLT1C49) was induced by RRx-001. The induction of ERV-Fc2-env was through demethylation of ERV-Fc2 LTR as determined by methylation-specific polymerase chain reaction and combined bisulfite restriction analysis. Immunofluorescence staining with J2 antibody confirmed induction of double-stranded RNA.

Conclusions: Transient exposure of HCT 116 cells to low-dose RRx-001 induced transcription of silenced retroviral genes present in the cancer cell DNA with subsequent synthesis of IFN in response to this "pseudo-pathogenic" stimulus, mimicking an antiviral defense. RRx-001-mediated IFN induction may have the potential to improve the efficacy of immunotherapies as well as radiotherapy, standard chemotherapies and molecularly targeted agents when used in combination. The striking safety profile of RRx-001 in comparison to other more toxic epigenetic and immunomodulatory agents such as azacitidine makes it a leading candidate for such clinical applications.

Keywords: Cancer; Epigenetics; Interferon response; Pseudo-infection; RRx-001; Viral mimicry.

Fig. 1

RRx-001 inhibited cell growth and DNMT1 and DNMT3a expression in HCT 116 cells. Exponentially growing cells were treated with 5-AZA, RRx-001 or 0.025% DMSO as a vehicle control for 1 to 3 days. Media containing RRx-001, 5-AZA, or DMSO were replaced daily over the 3-day period. a Concentration-survival curve of HCT 116 cells treated with RRx-001. SCC VII cells were used as a positive control. Data are mean ± SD from 3 independent experiments. b Protein levels of DNMT1 and DNMT3a in HCT 116 cells treated with RRx-001 as determined by Western blot. β-actin was used as an internal control for sample loading. 5-AZA was used as a positive control for inhibition of DNMT1 and DNMT3a expression. Bar graphs show the relative expression of DNMT1 and DNMT3a in treated cells compared to control cells

Fig. 2

Transient treatment with RRx-001 induced a sustained IFN response in HCT 116 cells. Expression of IFN-stimulated genes (ISGs) over the course of 4 weeks in response to 24 h of treatment with 0.5 μM 5-AZA (a) or RRx-001 (b). Transcript levels of ISGs (ISG15, IRF7, OASL, and DDX58) were determined by qPCR 7 days after treatment. Upregulation of gene expression observed in 5-AZA- treated cells was statistically significant at all time points. Gene expression changes observed in RRx-001-treated cells that were not significantly different from DMSO-treated cells were marked by # and those that were significantly downregulated by ##

Fig. 3

RRx-001 induced IFN response through upregulation of type I and III IFN expression and JAK/STAT pathway. Cells were transiently (24 h) treated with 0.5 μM RRx-001 or 0.5 μM 5-AZA and subsequently maintained in drug-free medium for an additional 7 days. RRx-001 induced a significant increase in type I IFN (IFN-β) (a) and type III IFN (IL-29/IL-28B) (b) secretion into culture medium by HCT 116 cells as measured by ELISA. Transcript levels of IL29/IL28A were also increased as determined by qPCR (c). ISGs (IFI27, IFi44, IFI44L, and IFI6) were upregulated by 5-AZA and RRx-001 but blocked by the JAK/STAT inhibitor ruxolitinib (rux) at 2 μM concentration (d). Expression of ISGs (IRF7, ISG15, DDX58, and OASL) was also upregulated in HCT 116 cells cultured in conditioned medium containing secreted IFNs induced by 5-AZA and RRx-001 as determined by qPCR (e)

Fig. 4

RRx-001 upregulated ERV transcription. Cells were transiently (24 h) treated with drugs and maintained for an additional 5 days in drug-free medium. a Immunofluorescence staining of dsRNA using J2 antibody (red) in DMSO-, 5-AZA-, or RRx-001-treated cells. DAPI was used to stain the nucleus (blue). White arrows point to cells that were negative for dsRNA. b Transcript levels of two ERVs were upregulated by 5-AZA and RRx-001 compared to DMSO as determined by qPCR

Fig. 5

RRx-001 induced ERV-Fc2-env expression through DNA demethylation. Cells were transiently (24 h) treated with drugs and maintained for an additional 5 days in drug-free medium. a Transcript levels of ERV-Fc2-env were upregulated by 5-AZA and RRx-001 compared to DMSO as determined by qPCR. b Methylation of ERV-Fc2 LTR was decreased by 5-AZA and RRx-001 compared to DMSO as determined by methylation-specific PCR. c Methylation of ERV-Fc2 LTR was decreased by 5-AZA and RRx-001 compared to DMSO as determined by COBRA. Bisulfite-treated DNA was amplified and digested with the AciI enzyme. Unmethylated DNA (U) was not digested and remained intact as a 199 bp band. Methylated DNA (M) was digested into two bands (155 and 44 bp). Signal intensity was quantified using ImageJ software (https://imagej.net)