Massive parallel variant characterization identifiesNUDT15alleles associated with thiopurine toxicity

Abstract

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs.Recently, NUDT15 deficiency was identified as a novel genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction is quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single aminoacid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in 2,398 patients treated with thiopurines, with 100% sensitivity and specificity, in contrast with poor performance of bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,103 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogeneticsguided thiopurine treatment individualization.

Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity

“…In recent years NUDT15 has also been shown to be a major player in thiopurine metabolism. Inactivating mutations in NUDT15 are strongly associated with thiopurine toxicity (19), and thiopurine dosage adjustment is recommended based on NUDT15 genotyping (20,21). In addition to the role of NUDT15 in thiopurine hypersensitivity, other studies have suggested that thiopurine-based therapies in patients with WT NUDT15, can be improved by NUDT15 inhibition.…”

“…These variants in TPMT therefore lead to excessive accumulation of thioguanine in the genome, which ultimately causes the severe hematopoietic toxicity observed in patients undergoing thiopurine-based chemotherapy (15)(16)(17)(18)(19). For this reason, it is recommended to adjust the thiopurine dosage according to the results of preemptive TPMT genotyping (20,21).…”

Defining the Sensitivity Landscape of 74,389 EGFR Variants to Tyrosine Kinase Inhibitors

“…Despite the significance of this work, there are several important caveats: (1) We only used PC9 cells for cytotoxicity screening. The genetic background of this cell line might have had an unwanted influence on the functional interpretation of variants [12,19]. Use of multiple NSCLC cell lines for parallel functional screening can significantly reduce such interference; (2) The expression of the exogenous EGFR variants was higher than that of endogenous EGFR in PC9 cells, which may lead to an overestimation of the TKI resistance of specific variants; (3) The endogenous EGFR of PC9 cells was deleted by CRISPR/Cas9.…”

“…Usually, a mutant library is first constructed employing synthetic biology or genome editing methods [11,12], and functional impacts of variants are determined through parallel functional assays. To date, variants of a few clinically actionable genes, such as BRCA1, PPARG, TP53, PTEN, TPMT, NUDT15, SCN5A, CYP2C9, CYP2C19, CXCR4, CCR5, ADRB2, and MSH2 [12][13][14][15][16][17][18][19][20][21][22][23][24], have been extensively studied using the DMS method, highlighting its potential for assessing the TKI sensitivity of rare and potential EGFR mutations. In this study, based on the DMS method, we developed massively parallel cytotoxicity assays and systematically determined the sensitivity landscape of the variants to five EGFR-TKIs (Fig.…”

A Review of the Important Role of CYP2D6 in Pharmacogenomics

“…In this approach, a mutagenesis library of missense variants is constructed that represent all possible amino acid substitutions within a protein and their in vitro functionality is determined. This high-throughput functional screening approach has been successfully applied to NUDT15 and could be conceivably applied to CYP2D6 [111]. Analysis of large datasets is another approach to assign functional effects to these undetermined haplotypes, and is being increasingly carried out to interrogate CYP2D6 [112,113].…”