A Genome-Wide Approach Identifies Variations in the Aspartate Metabolism Pathway That Are Associated with Asparaginase Sensitivity

Asparaginase is an important drug for acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in asparaginase sensitivity, we employed a genome-wide association approach using the HapMap lymphoblastoid cell lines from 87 individuals of European ancestry (CEU) and diagnostic ALL blasts from 42 newly diagnosed, genomically-determined white patients. In vitro sensitivity was based on IC₅₀ values measured following 48 hour exposures to native E. coli asparaginase (0.003–100 IU/ml) in CEU cell lines and 96 hour exposures (0.003–10 IU/ml) in patient samples using the methylthiazol tetrazolium assay. For CEU cell lines, single nucleotide polymorphism (SNP) genotypes were downloaded from the International HapMap database (www.hapmap.org) and gene expression data (Affymetrix GeneChip Human Exon 1.0 ST Array) were downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE7761. For patients with ALL, we used the 500K SNP arrays to interrogate germline DNA and Affymetrix U133A GeneChip Array to assess gene expression in ALL blasts. We tested whether 2,390,203 SNP genotypes were associated with asparaginase IC₅₀ using a linear mixed effect model in CEU cell lines, setting a p value threshold of p < 0.001 for individual SNPs and p < 0.05 at the gene level. This approach yielded 329 SNPs representing 94 genes. Combining these germline SNPs with those representing genes whose expression was also associated with IC₅₀ at the p < 0.05 level (1,706 genes), there were 6 SNPs representing 5 genes, two of which (rs8135371 and rs17001863, both in the ADSL gene) contributed to asparaginase sensitivity (p = 6.9 × 10⁻⁴ and 9.1 × 10^{− 4}, respectively) through their effects on ADSL gene expression. The top ranked KEGG pathway overrepresented by the 94 top-ranked genes (329 SNPs) was that of aspartate metabolism, which may be directly linked to the mechanism of action of asparaginase. The two most highly ranked genes (ADSL and DARS) in this pathway encompassed 7 SNPs (rs8135371, rs17001863, rs3768998, rs2278683, rs11893318, rs2322725, and rs7587285), all with p < .001. Using multiple linear regression analysis, 32% of the variability in asparaginase IC₅₀ among the CEU cell lines could be accounted for by these 7 SNPs (p = 5.9 × 10⁻⁷). To examine the overall contribution of the aspartate metabolism pathway to asparaginase IC₅₀, we compared all SNPs (935 in cell lines, 717 in patients) representing the aspartate pathway with those SNPs representing other pathways, using a random forest model. We found that the SNP genotypes in the aspartate pathway explained significantly more variation in asparaginase IC₅₀ in cell lines (11.4%, p = 6.9 × 10⁻⁴) and in ALL patient samples (11.2%, p = 0.02) than other pathways. The expression of ADSL differed among ALL subtypes, with more sensitive subtypes (hyperdiploid and TEL-AML1 ALL) having lower ADSL expression than more resistant subtypes (T-ALL) (p = 1.1 × 10⁻⁵ and 2.9 × 10⁻⁹, respectively). Genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited and acquired genomic interindividual variation in a plausible candidate pathway contribute to asparaginase sensitivity.