Citations

Triple-negative breast cancer (TNBC) represents the most malignant subtype of breast cancer. The clinical application of PARP inhibitors (PARPi) is limited by the low frequency of BRCA1/2 mutations in TNBC. Here, we identified that MTAP deletion sensitized genotoxic agents in our clinical cohort of metastatic TNBC. Further study demonstrated that MTAP deficiency or inhibition rendered TNBC susceptibility to chemotherapeutic agents, particularly PARPi. Mechanistically, targeting MTAP that synergized with PARPi by disrupting the METTL16-MAT2A axis involved in methionine metabolism and depleting in vivo s-adenosylmethionine (SAM) levels. Exhausted SAM in turn impaired PARPi-induced DNA damage repair through attenuation of MRE11 recruitment and end resection by diminishing MRE11 methylation. Notably, brain metastatic TNBC markedly benefited from a lower dose of PARPi and MTAP deficiency/inhibition synergy due to the inherently limited methionine environment in the brain. Collectively, our findings revealed a feed-forward loop between methionine metabolism and DNA repair through SAM, highlighting a therapeutic strategy of PARPi combined with MTAP deficiency/inhibition for TNBC.

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.

Abstract Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were ‘unmasked’ only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

AbstractAlternative polyadenylation (APA) is a common genetic regulatory mechanism that generates distinct 3′ ends for RNA transcripts. Changes in APA have been associated with multiple biological processes and disease phenotypes. However, the role of hormones and their drug analogs in APA remains largely unknown. In this study, we investigated transcriptome‐wide the impact of glucocorticoids on APA in 30 human B‐lymphoblastoid cell lines. We found that glucocorticoids could regulate APA for a subset of genes, possibly by changing the expression of 142 RNA‐binding proteins, some with known APA‐regulating properties. Interestingly, genes with glucocorticoid‐mediated APA were enriched in viral translation‐related pathways, while genes with glucocorticoid‐mediated expression were enriched in interferon and interleukin pathways, suggesting that glucocorticoid‐mediated APA might result in functional consequences distinct from gene expression. For example, glucocorticoids, a pharmacotherapy for severe COVID‐19, were found to change the APA but not the expression of LY6E, an important antiviral inhibitor in coronavirus diseases. Glucocorticoid‐mediated APA was also cell‐type‐specific, suggesting an action of glucocorticoids that may be unique to immune regulation. We also observed evidence for genotype‐dependent glucocorticoid‐mediated APA (referred to as pharmacogenomic‐alterative polyadenylation quantitative trait loci), providing potential functional mechanisms for a series of common genetic variants that had previously been associated with immune disorders, but without a clear mechanism. In summary, this study reports a series of observations regarding the impact of glucocorticoids on APA, raising the possibility that this mechanism might have implications for both disease pathophysiology and drug therapy.

Abstract The human angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) proteins play key roles in the cellular internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus responsible for the coronavirus disease of 2019 (COVID-19) pandemic. We set out to functionally characterize the ACE2 and TMPRSS2 protein abundance for variant alleles encoding these proteins that contained non-synonymous single-nucleotide polymorphisms (nsSNPs) in their open reading frames (ORFs). Specifically, a high-throughput assay, deep mutational scanning (DMS), was employed to test the functional implications of nsSNPs, which are variants of uncertain significance in these two genes. Specifically, we used a ‘landing pad’ system designed to quantify the protein expression for 433 nsSNPs that have been observed in the ACE2 and TMPRSS2 ORFs and found that 8 of 127 ACE2, 19 of 157 TMPRSS2 isoform 1 and 13 of 149 TMPRSS2 isoform 2 variant proteins displayed less than ~25% of the wild-type protein expression, whereas 4 ACE2 variants displayed 25% or greater increases in protein expression. As a result, we concluded that nsSNPs in genes encoding ACE2 and TMPRSS2 might potentially influence SARS-CoV-2 infectivity. These results can now be applied to DNA sequence data for patients infected with SARS-CoV-2 to determine the possible impact of patient-based DNA sequence variation on the clinical course of SARS-CoV-2 infection.

Background and Purpose Acamprosate is an anti‐craving drug used for the pharmacotherapy of alcohol use disorder (AUD). However, only some patients achieve optimal therapeutic outcomes. This study was designed to explore differences in metabolomic profiles between patients who maintained sobriety and those who relapsed, to determine whether those differences provide insight into variation in acamprosate treatment response phenotypes. Experimental Approach We previously conducted an acamprosate trial involving 442 AUD patients, and 267 of these subjects presented themselves for a 3‐month follow‐up. The primary outcome was abstinence. Clinical information, genomic data and metabolomics data were collected. Baseline plasma samples were assayed using targeted metabolomics. Key Results Baseline plasma arginine, threonine, α‐aminoadipic acid and ethanolamine concentrations were associated with acamprosate treatment outcomes and baseline craving intensity, a measure that has been associated with acamprosate treatment response. We next applied a pharmacometabolomics‐informed genome‐wide association study (GWAS) strategy to identify genetic variants that might contribute to variations in plasma metabolomic profiles that were associated with craving and/or acamprosate treatment outcome. Gene expression data for induced pluripotent stem cell‐derived forebrain astrocytes showed that a series of genes identified during the metabolomics‐informed GWAS were ethanol responsive. Furthermore, a large number of those genes could be regulated by acamprosate. Finally, we identified a series of single nucleotide polymorphisms that were associated with acamprosate treatment outcomes. Conclusion and Implications These results serve as an important step towards advancing our understanding of disease pathophysiology and drug action responsible for variation in acamprosate response and alcohol craving in AUD patients.

Major depressive disorder (MDD), the number one psychiatric disease and a major cause of medical disability worldwide, is a heterogeneous disease. Efforts to identify biomarkers for antidepressant therapy response by the use of genome‐wide association studies (GWAS) have generally yielded disappointing results, e.g. only a few markers have been replicated across studies. By applying a “pharmacometabolomics‐informed pharmacogenomic” research strategy, we previously identified a genome‐wide significant single‐nucleotide polymorphism (SNP) signal in the glutamate‐rich 3 ( ERICH3 ) gene that was associated with plasma serotonin concentrations which were themselves associated with selective serotonin reuptake inhibitor (SSRI) response in MDD patients enrolled in the Mayo Clinic PGRN‐AMPS SSRI trial. The same ERICH3 SNPs were associated with SSRI response in other GWAS, including the STAR*D and ISPC studies. However, the function of ERICH3 and the molecular mechanism(s) by which it might be associated with plasma serotonin concentrations and with SSRI response remain unknown. Functional characterization of ERICH3 and its encoded protein might make it possible to identify novel mechanisms of antidepressant treatment response. In the present study, ERICH3 mRNA and protein were studied in cell lines from human peripheral blood and brain. ERICH3 cDNA constructs and specific antibodies were generated to make it possible to study its function. Two ERICH3 mRNA splice variants and protein isoforms, one “full‐length” and the other encoding a C‐terminal protein, were identified in a variety of human cell lines and in human brain tissue. ERICH3 mRNA expression and immunofluorescent staining suggested both cytosolic and vesicular localizations for ERICH3 as well as co‐localization with serotonin and dopamine in both mouse and human neurons. Co‐immunoprecipitation studies identified several ERICH3 interacting proteins, including PIK3C2A, CLTC and AP2A2, proteins known to play a role in vesicular trafficking, as well as ALK and CUX1, both of which display genetic polymorphisms that have been associated with SSRI response in GWAS studies of antidepressant treatment response in MDD patients. This series of experiments suggests that ERICH3 may play a role in vesicular trafficking and, as a result, may influence neurotransmitter actions in the brain that are associated with antidepressant treatment response. Support or Funding Information This work was supported by the NIH grants U19 GM61388, R01 GM28157 and T32 GM008685. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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 .

Abstract Background: With few targeted therapies available for triple-negative breast cancer (TNBC), there is a critical need to identify biomarkers that can predict chemotherapy-response. Copy number amplifications (CNAmps) have been implicated in tumorigenesis, progression, and treatment response. Previously, we performed a prospective neoadjuvant chemotherapy study (BEAUTY) and identified a 5 Mb CNAmp in TNBC patients associated with pathological complete response rates to taxane- and anthracycline-based chemotherapy. Here we further interrogated this amplicon and its role in chemotherapy response. Methods: Using siRNA screening of genes identified in the 5 Mb CNAmp, followed by cytotoxicity assays, we identified RDH10 significantly associated with chemo-sensitivity in TNBC cell lines. Functional studies were performed using RDH10 knockdown or overexpression followed by cytotoxicity, cell proliferation, luciferase reporter assays, HPLC or western blot. Results: RDH10 is a rate-limiting step involved in the synthesis of all-trans retinoic acid (ATRA) - the active metabolite of retinoid metabolism that is frequently aberrant in cancer. Previous studies demonstrated ATRA to be a potent inhibitor of PIN1, a key regulator of several oncogenic signaling pathways often amplified in TNBC. We discovered that genetic manipulation of RDH10 affected ATRA intracellular concentrations, contributing to PIN1 degradation and taxane-response. Furthermore, co-treatment of TNBC cells with ATRA significantly increased taxane-sensitivity. Conclusion: RDH10 amplification increases endogenous levels of ATRA and degrades PIN1 in TNBC. ATRA-mediated degradation of PIN1 sensitizes TNBC to chemotherapy, suggesting that RDH10 may be a novel biomarker of taxane response and the addition of ATRA to standard chemotherapy may improve chemo-sensitivity.

Abstract Triggering receptor expressed on myeloid cell 2 (TREM2) is a surface receptor that, in the central nervous system, is exclusively expressed on microglia. TREM2 variants have been linked to increased risk for neurodegenerative diseases, but the functional effects of microglial TREM2 remain largely unknown. To this end, we investigated TAR-DNA binding protein 43 kDa (TDP-43)-related neurodegenerative disease via viral-mediated expression of human TDP-43 protein (hTDP-43) in mice or inducible expression of hTDP43 with defective nuclear localization signals in transgenic mice. We found that TREM2 deficiency impaired microglia phagocytic clearance of pathological TDP-43, and enhanced neuronal damage and motor function impairments. Mass cytometry analysis revealed that hTDP-43 induced a TREM2-dependent subpopulation of microglia with high CD11c expression and higher phagocytic ability. Using mass spectrometry, we further demonstrated an interaction between TDP-43 and TREM2, in vitro and in vivo, in hTDP-43-expressing transgenic mouse brains. We computationally identified the region within hTDP-43 that interacts with TREM2 and observed the potential interaction in ALS patient tissues. Our data reveal the novel interaction between TREM2 and TDP-43, highlighting that TDP-43 is a potential ligand for microglial TREM2 and the interaction mediates neuroprotection of microglial TREM2 in TDP-43-related neurodegeneration.