PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

Metastatic melanoma is still a difficult-to-treat cancer type owing to its frequent resistance mechanisms to targeted and immunotherapy. Therefore, we aimed to unravel novel therapeutic strategies for melanoma patients. Preclinical and clinical studies show that melanoma patients may benefit from a treatment with poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this study, we focus on PARP1 as a potential biomarker to predict the response of melanoma cells to PARPi therapy. We found that melanoma cells with high basal PARP1 expression exhibit significantly increased cell death after PARPi treatment owing to higher PARP1 trapping compared with melanoma cells with low PARP1 expression. In addition, we could demonstrate that PARP1 expression levels are low in nonmalignant skin cells, and metastatic melanomas show considerably higher PARP1 levels compared with primary melanomas. Most strikingly, we found that high PARP1 levels correlate with worse overall survival of late stage metastasized melanoma patients. In conclusion, we show that PARP1 might act as a biomarker to predict the response to PARPi therapy, and that in particular the late stage metastasized melanoma patients are especially sensitive to PARPi therapy owing to elevated PARP1 expression. Our data suggest that the PARPi cytotoxicity primarily will affect the high PARP1 expressing melanoma cells, rather than the low PARP1 expressing nonmalignant skin cells resulting in only low side effects.

Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis.

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells.

HepaRG cells are increasingly accepted as model for human drug metabolism and other hepatic functions. We used lentiviral transduction of undifferentiated HepaRG cells to deliver Cas9 and two alternative sgRNAs targeted at NADPH:cytochrome P450 oxidoreductase (POR), the obligate electron donor for microsomal cytochromes P450 (CYP). Cas9-expressing HepaRGVC (vector control) cells were phenotypically similar to wild type HepaRG cells and could be differentiated into hepatocyte-like cells by DMSO. Genetic POR-knockout resulted in phenotypic POR knockdown of up to 90% at mRNA, protein, and activity levels. LC-MS/MS measurement of seven CYP-activities showed differential effects of POR-knockdown with CYP2C8 being least and CYP2C9 being most affected. Further studies on cytochrome b5 (CYB5), an alternative NADH-dependent electron donor indicated particularly strong support of CYP2C8-dependent amodiaquine N-deethylation by CYB5 and this was confirmed by genetic CYB5 single- and POR/CYB5 double-knockout. POR-knockdown also affected CYP expression on mRNA and protein level, with CYP1A2 being induced severalfold, while CYP2C9 was strongly downregulated. In summary our results show that POR/NADPH- and CYB5/NADH-electron transport systems influence human drug metabolizing CYPs differentially and differently than mouse Cyps. Our Cas9-expressing HepaRGVC cells should be suitable to study the influence of diverse genes on drug metabolism and other hepatic functions.