The genetic architecture of methotrexate toxicity is similar in Drosophila melanogaster and humans

Abstract

The severity of the toxic side effects of chemotherapy varies among patients, and much of this variation is likely genetically based. Here, we use the model system Drosophila melanogaster to genetically dissect the toxicity of methotrexate (MTX), a drug used primarily to treat childhood acute lymphoblastic leukemia and rheumatoid arthritis. We use the Drosophila Synthetic Population Resource, a panel of recombinant inbred lines derived from a multiparent advanced intercross, and quantify MTX toxicity as a reduction in female fecundity. We identify three quantitative trait loci (QTL) affecting MTX toxicity; two colocalize with the fly orthologs of human genes believed to mediate MTX toxicity and one is a novel MTX toxicity gene with a human ortholog. A fourth suggestive QTL spans a centromere. Local single-marker association scans of candidate gene exons fail to implicate amino acid variants as the causative single-nucleotide polymorphisms, and we therefore hypothesize the causative variation is regulatory. In addition, the effects at our mapped QTL do not conform to a simple biallelic pattern, suggesting multiple causative factors underlie the QTL mapping results. Consistent with this observation, no single single-nucleotide polymorphism located in or near a candidate gene can explain the QTL mapping signal. Overall, our results validate D. melanogaster as a model for uncovering the genetic basis of chemotoxicity and suggest the genetic basis of MTX toxicity is due to a handful of genes each harboring multiple segregating regulatory factors.

Keywords: Drosophila synthetic population resource; chemotoxicity; methotrexate; pharmacogenomics; quantitative trait loci.

Figure 1

Experiment Design. (A) Each pair of RILs was crossed to produce the F1 hybrid. The hybrids were raised to adulthood, brother-sister mated, and placed into condo wells (three males and three females per well). Each condo contained four RIL crosses exposed to MTX simultaneously. After a 3-d exposure, each condo was recovered for 1 d, and the flies were allowed to lay eggs for 2 d after the recovery stage, after which the adult flies were cleared out of the condo. The eggs developed into adults for 14 d after the condos were cleared, frozen at −20°, and transferred onto adhesive sheets for counting. (B) The entire assay was split into eight blocks, with start dates on consecutive weeks. Each block took 8 wk to complete. F1 hybrids from populations A1×B2 were assayed over the first 3 wk, and any crosses that failed to complete the entire cycle were repeated in weeks 6 and 7. F1 hybrids from populations A2×B1 were assayed in weeks 4 and 5, and the crosses that failed in those two weeks were completed in week 8. (C) Density plot of the mean number of offspring produced by the three replicate females from each RIL. Different colors correspond to different weeks (blocks) of the experiment.

Figure 2

MTX cellular pathway. MTX enters cancer cells via the reduced folate carrier, and the efflux across the cell membrane is mediated by various ABC transporters. Inside the cell, MTX is converted to active methotrexate polyglutamates by folylpolyglutamate synthetase (FPGS), which adds glutamate residues to MTX. The primary action of MTX is inhibition of the enzyme dihydrofolate reductase (DHFR), which converts dihydrofolate to tetrahydrofolate. The effect of MTX depends on the function and expression of several other enzymes in the folate pathway, including methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and thymidylate synthetase. Degradation of MTXPGs to MTX depends on the activity of the lysosomal enzyme GGH, which catalyzes the removal of polyglutamates (Mikkelsen et al. 2011). AMP deaminase (AMPD1) converts adenosine monophosphate (AMP) to inosine monophosphate (IMP). Adenylosuccinate synthase (ADSS) and adenylosuccinate lyase (ADSL) counteract the action of AMPD1, convert IMP back to AMP in a two-step process. Adenylate kinase catalyzes the formation of two molecules of ADP from AMP and ATP; ADP is the substrate for oxidative phosphorylation that forms ATP. GSTs are not known to directly interact with methotrexate but are main mediators of detoxification by conjugation of xenobiotics with glutathione.

Figure 3

(A) Means and SEs of the number of offspring produced by each F1 female from each pA-pB cross. Different colors correspond to different weeks (blocks) of the experiment after removing weeks with poor dosing. (B) Density plot of the mean number of offspring produced by the three replicate females from each RIL. Different colors correspond to different weeks (blocks) of the experiment.

Figure 4

(A) MTX toxicity genome scan. The major chromosome arms are delineated by different background shading (white/yellow). The black line is the scan of the observed data. To give an example of the results obtainable by chance alone, the grey line is a scan of a single permutation of the observed data. Horizontal blue dotted lines indicate thresholds for various false positive rates (number of expected peaks per genome scan) obtained via permutations. Vertical dashed red lines indicate the location of fly orthologs of previously identified candidate genes for methotrexate toxicity. (B-C) Standardized founder haplotype means and SE at (B) QTL A and (C) QTL C. The number of each confidently assigned (probability > 0.95) founder genotype is listed above each plot. Only means with at least five observations are plotted.