RRM1 and RRM2 pharmacogenetics: association with phenotypes in HapMap cell lines and acute myeloid leukemia patients

Abstract

Background: Ribonucleotide reductase catalyzes an essential step in the cellular production of deoxyribonucleotide triphosphates and has been associated with clinical outcome in cancer patients receiving nucleoside analog-based chemotherapy.

Materials & methods: In the current study, we sequenced the genes RRM1 and RRM2 in genomic DNA from HapMap cell lines with European (Utah residents with northern and western European ancestry [CEU]; n = 90) or African (Yoruba people in Ibadan, Nigeria [YRI]; n = 90) ancestry.

Results: We identified 44 genetic variants including eight coding SNPs in RRM1 and 15 SNPs including one coding SNP in RRM2. RRM1 and RRM2 mRNA expression levels were significantly correlated with each other in both CEU and YRI lymphoblast cell lines, and in leukemic blasts from acute myeloid leukemia (AML) patients (AML97, n = 89; AML02, n = 187). Additionally, RRM1 expression was higher among patient features indicative of a high relapse hazard. We evaluated SNPs within the RRM1 and RRM2 genes in the HapMap lymphoblast cell lines from CEU and YRI panels for association with expression and cytarabine chemosensitivity. SNPs of potential significance were further evaluated in AML patients. RRM1 SNPs rs1042919 (which occurs in linkage disequilbrium with multiple other SNPs) and promoter SNP rs1561876 were associated with intracellular 1-β-D-arabinofuranosyl-CTP levels, response after remission induction therapy, risk of relapse and overall survival in AML patients receiving cytarabine and cladribine.

Conclusion: These results suggest that SNPs within ribonucleotide reductase might be helpful predictive markers of response to nucleoside analogs and should be further validated in larger cohorts.

Figure 1. SNPs in RRM1

(A) Snapshot from the UCSC genome browser [NCBI/hg18 (2006)] for the RRM1 locus. The regions sequenced in HapMap panels are shown by small boxes. (B) Linkage disequilibrium plot of RRM1 gene in European (CEU) and African (YRI) ancestry samples generated in Haploview using genotype data from the present study and from HapMap for both CEU and YRI samples from 10 kb upstream to 5 kb downstream. (C) SNPs that are linked, r² > 0.8 (and picked by the tagger program), are categorized in the same groups. SNPs without rs numbers are indicated by number corresponding to Table 1. CEU: Utah residents with northern and western European ancestry; MAF: Minimum allele frequency; YRI: Yoruba people in Ibadan, Nigeria.

Figure 2. SNPs in RRM2

(A) Snapshot from the UCSC genome browser [NCBI/hg18 (2006)] for the RRM2 locus. The regions sequenced in HapMap panels are shown by small boxes. (B) Linkage disequilibrium plot of RRM2 gene in European (CEU) and African (YRI) ancestry samples generated in Haploview using genotype data from the present study and from HapMap for both CEU and YRI samples 20 kb upstream to 5 kb downstream. (C) SNPs that are linked, r² ≥ 0.75 in CEU and r² ≥ 0.8 in YRI (and picked by the tagger program), are categorized in the same groups. SNPs located upstream of the translation start site (ATG) are denoted by an asterisk. SNPs without rs numbers are indicated by number corresponding to Table 1. CEU: Utah residents with northern and western European ancestry; MAF: Minimum allele frequency; YRI: Yoruba people in Ibadan, Nigeria.

Figure 3. Comparison of RRM1 and RRM2 mRNA expression in CEU and YRI cell lines

RRM1 and RRM2 mRNA expression extracted from GSE7761 in Epstein–Barr virus-transformed lymphoblast cell lines derived from subjects with European (CEU) and African (YRI) ancestries. CEU: Utah residents with northern and western European ancestry; YRI: Yoruba people in Ibadan, Nigeria.

Figure 4. Association of SNPs with mRNA expression and cytarabine cytotoxicity in HapMap lymphoblast cell lines

Box plots for the association of representative RRM1 SNPs with their expression and cytarabine area under the survival curve in YRI samples. Plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. The outliers are defined as data points that fall outside of the first and third quartiles by more than 1.5-times the interquartile range. Circles falling outside the whiskers represent outliers. YRI: Yoruba people in Ibadan, Nigeria.

Figure 5. Association of RRM2 SNP with cytarabine cytotoxicity in HapMap lymphoblast cell lines

Box plots for the association of representative RRM2 SNPs with cytarabine area under the survival curve in CEU lymphoblast cell lines. Plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. The outliers are defined as data points that fall outside of the first and third quartiles by more than 1.5-times the interquartile range. Circles falling outside the whiskers represent outliers. CEU: Utah residents with northern and western European ancestry.

Figure 6. Comparison of RRM1 and RRM2 mRNA expression in diagnostic leukemic blasts from acute myeloid leukemia patients

(A) RRM1 and RRM2 expression was extracted from Affymetrix U133A microarray data and correlation of mRNA expression levels between RRM1 and RRM2 was evaluated. (B) Box plot showing association of RRM1 expression with risk group. Plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. The outliers are defined as data points that fall outside of the first and third quartiles by more than 1.5-times the interquartile range. Circles falling outside the whiskers represent outliers.

Figure 7. Association of RRM1 and RRM2 SNPs with mRNA expression in diagnostic AML02 leukemic blasts

Box plots for the association of representative RRM1 SNPs rs11030918, rs1042927 and rs12806698 with its expression and RRM2 SNP rs1138729 with its mRNA expression levels in diagnostic leukemic blasts from acute myeloid leukemia patients. Plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. The outliers are defined as data points that fall outside of the first and third quartiles by more than 1.5-times the interquartile range. Circles falling outside the whiskers represent outliers.

Figure 8. Association of RRM1 SNP rs1561876 with clinical outcome in AML97 patients

(A) Box plot representing association of rs1561876 with intracellular levels of ara-CTP (nmol/2 × 10⁷ cells) determined at day 1. Plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. The outliers are defined as data points that fall outside of the first and third quartiles by more than 1.5-times the interquartile range. The circle falling outside the whiskers represents an outlier. (B) Association of rs1561876 with response after induction 1 in acute myeloid leukemia (AML) patients. Percentages of patients in each category are indicated in parenthesis. (C) Survival curve for association of rs1561876 with event-free survival in all AML patients. (D) Survival curve for association of rs1561876 with risk of relapse in AML patients. Ara-CTP: 1-β-D-arabinofuranosyl-CTP; CR: Complete response; NR: No response; PR: Partial response.

Figure 9. Association of RRM1 SNP rs1042919 with clinical outcome in Caucasian patients enrolled in AML97

(A) Box plot representing association of rs1042919 with intracellular levels of ara-CTP (nmol/2 × 10⁷ cells) determined at day 1 in Caucasian acute myeloid leukemia (AML) patients. Plots show medians as a line between boxes representing the first and third quartiles; the whiskers represent the range after excluding the outliers. (B) Survival curve for association of rs1042919 with event-free survival in white AML patients. (C) Survival curve for association of rs1042919 with risk of relapse in white AML patients. Ara-CTP: 1-β-D-arabinofuranosyl-CTP.