Zhang, Lingxin

Abstract Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited therapeutic strategy and unfavorable prognosis, especially for patients bearing brain metastasis. Here, we demonstrate that MTAP gene deletion or MTAP inhibition by MTAP inhibitor, MTDIA, sensitizes breast cancer cells to poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi). Mechanistically, PARPi suppresses the expression of methionine adenosyltransferase 2A (MAT2A) by inducing METTL16 auto-inhibition mediated intron retention. Given that MAT2A and MTAP are key enzymes in methionine metabolism, the dual inhibition of MAT2A and MTAP disrupts methionine metabolism and leads to reduced s-adenosylmethionine. Dietary methionine restriction further boosts the combined anti-tumor efficacy of MTAP depletion/inhibition and PARPi. Most importantly, in brain metastatic TNBC, due to the natural low methionine environment in the brain, the combination of MTAP depletion/inhibition and PARPi confers significant therapeutic efficacy. Therefore, our findings provide a promising strategy for inhibiting primary or brain metastatic TNBC.

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. Because of the very large number of missense variants in ORFs, it is difficult to link the genotypes of these variants to their phenotypes using “one at a time” expression systems. Therefore, the functional characterization of variants of unknown significance in clinically important pharmacogenes remains a major challenge in pharmacogenomics. Deep mutational scanning (DMS) is a high‐throughput technique that make it possible to analyze hundreds of different missense variants in a parallel and scalable fashion. Engineered “landing pad” HEK 293T cells were used as a platform to integrate pooled variant libraries, resulting in one variant per cell for further assessment. We adapted this DMS system to analyze the function of missense variants in the ORFs of CYP2C9 and CYP2C19 . The ORFs of CYP2C9 and CYP2C19 were fused to green fluorescent protein, and individual variants with different fluorescence intensities were used to indicate different levels of protein expression. Pooled variant libraries were generated by nicking mutagenesis, and we then used recombinase to integrate the libraries into landing pad cells, one per cell. Multiplexed functional selection performed with Fluorescence Activated Cell Sorting separated cells into different bins by fluorescence readout at the single–cell level. Amplicon sequencing of DNA collected in each bin, followed by computational analysis of the frequency of variants appearing in each bin, was used to determine levels of protein expression. To identify potentially severely damaging variants for these two important pharmacogenes, we studied 243 missense variants in the CYP2C9 and CYP2C19 ORFs that had been identified by the Exome Aggregation Consortium Project and by the Mayo Clinic RIGHT 1K Study. We found that 13 of 115 CYP2C9 and 25 of 128 CYP2C19 missense variants displayed less than approximately 25% of the wildtype protein expression, a level that may have clinical relevance for drug metabolism. Variant calling by the DMS method for the severely damaging variants was compared with the results of individual variant assay by western blot and with the predictions of computational algorithms such as Polyphen and SIFT. Our results suggest that DMS is an efficient method for the high‐throughput identification of damaging ORF variants that might have potential clinical application in pharmacogenomics. Support or Funding Information Funded by NIH grant U19 GM61388 (The Pharmacogenomics Research Network), R01 GM28157, R01 GM125633, T32 GM08685 and by the Mayo Clinic Center for Individualized Medicine This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .