Abstract
Post-translational modifications (PTMs) profoundly impact protein function and are involved in almost all cell biological processes. Various PTMs, including ADP-ribosylation, have also been linked to different pathologies such as cancer. The discovery of the synthetic lethal interaction between PARP1 inhibitors and BRCA-deficient tumors has led to the development of ADP-ribosylation inhibitors as a novel class of anticancer drugs. This has highlighted the potential of ADP-ribosyltransferases as targets for cancer therapy. Recently, it has been reported that PARP7 promotes tumor growth in a cell-autonomous manner and suppresses the anti-tumor immune response. However, the molecular mechanism underlying the effects of PARP7-mediated ADP-ribosylation in cancer cells have not yet been fully understood. Therefore, it remained crucial to identify PARP7 targets as potential biomarkers for patient stratification and to comprehensively understand the mode of action of PARP7 inhibition during tumor growth. Here, we identified PARP7 as a nuclear mono-ADP-ribosyltransferase that is specific to cysteine residues and modifies targets that are crucial for transcription regulation, including the AP-1 transcription factor FRA1. Inhibition of PARP7 or mutation of the FRA1 ADP-ribosylation site C97 resulted in increased FRA1 degradation by the proteasome via an enhanced interaction with PSMC3, a 19S proteasomal subunit. The reduction of FRA1 protein levels led to the IRF1 and IRF3-dependent expression of cytokine and pro-apoptotic genes, ultimately resulting in CASP8-mediated apoptosis. Among the nucleic acid-sensing pathways only RIG1-like receptor (RLR)-singling was responsible for activating IRF1/IRF3, while the cGAS/STING pathway did not contribute to apoptosis. Furthermore, elevated levels of PARP7 expression indicated susceptibility to PARP7 inhibition in FRA1-positive lung and breast cancer cells. Collectively, our findings emphasize the clinical potential of PARP7 inhibitors for FRA1-driven cancers. In addition to FRA1, we identified approximately 80 high-confidence targets of PARP7 with an unknown function of the PARP7-mediated ADP-ribosylation. To determine which of the identified ADP-ribosylation sites make a physiologically relevant contribution to the function of the modified proteins, we used thermal protein profiling (TPP) to analyze the biophysical state of proteins following the treatment with RBN-2397. RBN-2397 strongly increased the melting point of FRA1 and several RNA-binding proteins, including splicing proteins. This suggests that PARP7 may promote cancer cell viability by controlling mRNA splicing, in addition to regulating FRA1 complex formation with the proteasome. In conclusion, our study provides evidence that PARP7 activity is crucial for maintaining cancer cell progression by stabilizing FRA1 and potentially by regulating mRNA splicing. These findings may help to stratify cancer patients and identify novel combinatorial approaches for utilizing PARP7 inhibition in cancer treatment.
Manetsch, Patrick