Loss of PARP7 Increases Type I Interferon Signaling in EO771 Breast Cancer Cells and Prevents Mammary Tumor Growth by Increasing Antitumor Immunity.

PARP7 is a member of the ADP-ribosyltransferase diphtheria toxin-like (ARTD) family and acts as a repressor of type I interferon (IFN) signaling. PARP7 inhibition causes tumor regression by enhancing antitumor immunity, which is dependent on the stimulator of interferon genes (STING) pathway, TANK-binding kinase 1 (TBK1) activity, and cytotoxic CD8+ T cells. To better understand PARP7's role in cancer, we generated and characterized PARP7 knockout (Parp7KO) EO771 mouse mammary cancer cells in vitro and in a preclinical syngeneic tumor model using catalytic mutant Parp7H532A mice. Loss of PARP7 expression or inhibition of its activity increased type I IFN signaling, as well as the levels of interferon-stimulated gene factor 3 (ISGF3) and specifically unphosphorylated-ISGF3 regulated target genes. This was partly because PARP7's modification of the RelA subunit of nuclear factor κ-B (NF-κB). PARP7 loss had no effect on tumor growth in immunodeficient mice. In contrast, injection of wildtype cells into Parp7H532A mice resulted in smaller tumors compared with cells injected into Parp7+/+ mice. Parp7H532A mice injected with Parp7KO cells failed to develop tumors and those that developed regressed. Our data highlight the importance of PARP7 in the immune cells and further support targeting PARP7 for anticancer therapy.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

Analysis of the effects of aryl hydrocarbon receptor expression on cancer cell invasion via three-dimensional microfluidic invasion assays.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds to xenobiotics and activates expression of response elements to metabolize these compounds. The AHR pathway has been associated with a long list of diseases including cancer; however, it is debated whether AHR is tumorigenic or tumour-inhibiting. In particular, there are contradictory reports in the literature regarding the effects of AHR expression level on metastatic breast cancer. Here we used a 3D invasion assay called cell invasion in digital microfluidic microgel systems (CIMMS) to study the effect of AHR expression on invasion. In this study, MDA-MB-231 cells with stable knockout of AHR (AHRko) showed enhanced invasive characteristics and reduced proliferation, and cells with transient overexpression of AHR showed reduced invasiveness. Overexpression of AHR with a mutation in the DNA binding domain showed no difference in invasiveness compared to control, which suggests that the changes in invasiveness are related to the expression of AHR. CIMMS also allowed for extraction of sub-populations of invaded cells for RNA sequencing experiments. A comparison of the transcriptomes of invaded subpopulations of wild-type and AHRko cells identified 1809 genes that were differentially expressed, with enriched pathways including cell cycle, proliferation, survival, immunoproteasome activation, and activation of matrix metalloproteases. In sum, the data reported here for MDA-MB-231 cells suggests some new interpretations of the discrepancy in the literature on the role of AHR in breast cancer. We propose that the unique combination of functional discrimination with transcriptome profiling provided by CIMMS will be valuable for a wide range of mechanistic invasion-biology studies in the future.