Genotype of susceptibility-linked and relapse-linked SNPs of ARID5B in BCP-ALL cell lines
We first analyzed ARID5B genotypes in 72 BCP-ALL cell lines established from Japanese patients . Our cell line bank contained 13 BCR/ABL1-positive, 13 TCF3/PBX1-positive, 12 MLL (KMT2A)-rearranged, 5 ETV6/RUNX1-positive, and 3 TCF3/HLF-positive cell lines, but no hyperdiploid cell lines. Thus, the majority of our cell lines had been established from BCP-ALL with high or intermediate risk karyotypes. We determined genotypes of two representative susceptibility-linked SNPs  (rs7923074 and rs10821936, Fig. 1) and three representative relapse-linked SNPs  (rs4948488, rs2893881, and rs6479778, Fig. 1) in each cell line after direct sequencing of each genomic PCR product. Allele frequencies of each SNP in BCP-ALL cell lines were in Hardy–Weinberg equilibrium. Due to linkage disequilibrium, genotypes of rs7923074 and rs10821936 were identical in 71 of 72 cell lines. Genotypes of rs2893881 and rs6479778 were also identical in 71 cell lines. In HapMap project database (Table 2), we compared the allele frequency of each SNP between our cell lines and the Japanese population, but no significant differences were observed in the genotypes of both the susceptibility-linked SNPs and the relapse-linked SNPs of ARID5B.
No association of susceptibility or relapse-linked SNPs of ARID5B with ARID5B expression
Since both the susceptibility-linked SNPs and the relapse-linked SNPs of ARID5B are located in intronic regions, we next performed expression quantitative trait locus (eQTL) analysis. We quantified ARID5B gene expression level in each cell line by real-time RT-PCR using ACTB gene expression as an internal control. However, in eQTL analysis of 72 BCP-ALL cell lines, neither genotypes of the susceptibility-linked rs7923074 and rs10821936 nor those of the relapse-linked rs4948488, rs2893881, and rs6479778 were significantly associated with ARID5B expression level (Fig. 2). These observations demonstrated that genotypes of both susceptibility-linked SNPs and the relapse-linked SNPs of ARID5B were not clearly associated with ARID5B expression levels in the BCP-ALL cell line.
Association of relapse-linked SNPs of ARID5B with drug sensitivity
We next verified whether genotype of the relapse-linked SNPs of ARID5B in BCP-ALL cell lines is associated with their sensitivities to chemotherapeutic agents. We performed an alamarBlue assay to determine IC50 values (concentration that needs to kill 50% of the cells) of nine representative agents used for ALL chemotherapy including Pred, Dex, VCR, DNR, L-Asp, AraC, MTX, 6MP, and CY (Maf). Of note, IC50 values of VCR (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of non-risk allele (T) (Fig. 3a). In addition to VCR, sensitivities to CY (Maf) (Fig. 3b) and AraC (Fig. 3c) tended to be associated with the genotype of the relapse-linked rs4948488. Similar associations were observed in genotypes of rs2893881 and rs6479778 (Fig. 3a-c). Among the nine agents, IC50 values of six agents (Dex, Pred, DNR, L-Asp, MTX, and 6MP) were not significantly associated with genotypes of the relapse-linked rs4948488, rs2893881, and rs6479778 (Supplemental Fig. 1a-f).
We further analyzed association of the susceptibility-linked rs7923074 and rs10821936 with drug sensitivities. In contrast to the genotypes of the relapse-linked SNPs, no significant associations were observed in genotypes of rs7923074 and rs10821936 with sensitivities to VCR, CY and AraC (Fig. 3a-c) and the other six agents (Supplemental Fig. 1a-f). These observations suggest that the risk allele of relapse-linked SNPs, but not susceptibility-linked SNPs, may be associated with a higher relapse rate in pediatric BCP-ALL patients due to reduced sensitivities to VCR, CY and AraC.
Association of ARID5B gene expression with drug sensitivity
We finally verified whether gene expression level of ARID5B is associated with drug sensitivities of BCP-ALL cell lines. To address this issue, we simply divided our 72 BCP-ALL cell lines into two groups—36 cell lines with higher gene expression levels than the median value and the other 36 cell lines with lower gene expression levels than the median value—and compared the IC50 values of each drug. Of note, the IC50 values of MTX in 36 cell lines with lower ARID5B expression (median IC50: 37.1 ng/ml) was significantly higher (p = 0.023 in Mann–Whitney U test) than those in the other 36 cell lines with lower expression (16.9 ng/ml) (Fig. 4a). In contrast, although the sensitivities to VCR, CY, and AraC were associated with genotypes in the relapse-linked SNPs of ARID5B, no significant differences were observed in the IC50 values of VCR, CY, and AraC between the two groups (Fig. 4b-d). Furthermore, although genotypes in the susceptibility-linked SNPs of ARID5B were associated with sensitivities to Pred and Dex, no significant differences were observed in the IC50 values of Pred and Dex between the two groups (Supplement Fig. 2a, b). In the IC50 values of the remaining three agents (DNR, L-Asp, and 6MP), there were no statistically significant differences between the two groups (Supplement Fig. 2c-e). These observations suggest that lower ARID5B expression may be a genetic marker for MTX resistance in BCP-ALL.