Genetic Determinants of Susceptibility to Cytarabine Arabinoside.

Cytarabine arabinoside (ara-C) is an essential component of treatment for acute myeloid leukemia (AML) and resistance to this agent is a main reason for treatment failure. To identify genetic factors that are important in susceptibility to the cytotoxic effects of ara-C, we utilized an unbiased whole-genome approach using lymphoblastoid cell lines that are part of the International HapMap Project. We exposed 85 cell lines derived from Utah residents with ancestry from northern and western Europe (CEU) and 89 cell lines derived from individuals of Yoruba in Ibadan, Nigeria (YRI) to a range of concentrations of ara-C (1 – 80 μM) for 72 hours. We used a short-term proliferation assay, alamarBlue®, to measure cell growth inhibition at each concentration of ara-C. In addition, we used the Affymetrix Human Exon 1.0 ST array to determine gene expression of each cell line. We utilized publicly available HapMap (release #22) single nucleotide polymorphisms (SNPs) to perform genome wide association after filtering to remove Mendelian errors and SNPs with mean allele frequencies <5%, leaving approximately 2 million SNPs. We then performed a 3-step analysis to identify genetic signatures associated with gene expression important in susceptibility to ara-C cytotoxicity. The first step involved a Quantitative Transmission Disequilibrium Test (QTDT) association analysis between SNP genotype and percent survival at each concentration of ara-C. Using the SNPs identified in this initial step, we performed a second QTDT analysis between genotype and gene expression of approximately 17,000 transcripts from the exon array to identify SNPs that were associated with the expression of any genes. Finally, we performed a linear regression analysis between the expression of genes identified in this second step and percent survival to identify a subset of these genes whose expression is correlated with susceptibility to ara-C. Using a multivariable backward elimination approach we also determined how much variation in ara-C percent survival is explained by the SNPs identified in step 2. The mean (range) percent survival in the CEU compared to the YRI population was 53.3 (20.8 – 82.8) vs. 47.3 (25.2 – 73.7) at 5μM (p = .0018), 46.8 (18.7–73.2) vs. 39.9 (22.2 – 72.4) at 10μM (p = 0.0001), 40.3 (20.3 – 68.2)vs. 32.8 (17.5 – 53.9) at 40μM (p < 0.0001), and 37.3 (19.1 – 61.3) vs. 30.4 (15.8 – 50.0) (p < 0.0001) at 80μM ara-C. Among the 85 CEU cell lines we identified 177 SNPs for which genotype significantly associated with percent survival at 1μM, using an FDR cutoff of 0.05. Eleven of these 177 SNPs were significantly associated with the expression of a total of 24 genes and all were located trans to the associated gene. The expression of 23 of these genes significantly correlated with percent survival. Of the 11 SNPs that were associated with percent survival and gene expression, 4 SNPs explained 61.2% of the variation in susceptibility to ara-C cytotoxicity among the CEU cell lines. By using an cell-based model and a whole-genome approach that integrates genetic variation, expression variation and sensitivity to drug, we were able to identify a genetic signature for susceptibility to the cytotoxic effects of ara-C. We can now concentrate on the candidate genes identified from this analysis for further functional validation studies.