Genome-Wide Association Analyses Identify Susceptibility Loci For Vincristine-Induced Peripheral Neuropathy In Children With Acute Lymphoblastic Leukemia

[Graphic][1] Introduction Vincristine, a vinca alkaloid, is one of the most widely used and effective medications for acute lymphoblastic leukemia (ALL). Vincristine exerts its cytotoxic effects by interfering with microtubule formation and mitotic spindle dynamics, leading to mitotic arrest and cell death. However its use often causes neuropathy characterized by abdominal pain, sensory and motor dysfunctions. To date, various candidate gene studies have failed to identify consistent genetic variants associated with an increased risk of vincristine-induced neuropathy. Methods Vincristine-induced neuropathy was assessed in children enrolled on SJCRH protocol study XIIIB for the treatment of newly diagnosed ALL and a subset of children with relapsed ALL enrolled on a Children’s Oncology Group protocol (COG AALL0433). Neuropathy was graded using CTCAE v1.0 (XIIIB) or a modified (“Balis”) pediatric scale (AALL0433). We performed a genome wide analysis of germline SNPs using the Affymetrix 500K or 6.0 array and imputed SNPs. Weighted logistic regression was used to test the association between genotypes and vincristine neuropathy in patients experiencing multiple episodes of grade 2-4 vincristine-induced neuropathy during their treatment. We also performed a meta-analysis that estimated p values across the two cohorts using Fisher’s method. Results Genome -wide SNP analysis and vincristine-induced neuropathy were assessed in 321 patients (222 on SJCRH protocol and 99 on COG protocol). The meta-analysis identified several loci that were significant in both patient cohorts in the same direction related to the risk of multiple episodes of neuropathy, with Rs924607 reaching genome-wide significance (p<10-8). After adjusting for genetically defined race and for vincristine dose, Rs924607 (localized in chromosome 5, near the CEP72 gene) remained significant (p = 4.68 x10-8). The cumulative incidence of developing multiple episodes of Vincristine-induced neuropathy differed among the three genotypes (p<0.0001) for this SNP, and this SNP was also associated with low expression of the CEP72 mRNA in the HapMap cells (CEU); the rs924607 risk allele (T) was associated with lower expression of CEP72 compared to the C allele. CEP72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation, making it plausible that this SNP alters the pharmacologic effects of vincristine. We subsequently confirmed that this SNP was associated with lower expression of CEP72 in a luciferase reporter construct containing 2612 bp of the CEP72 promoter region, including the Rs924607 SNP (comparing constructs containing either the C allele or the T allele of Rs924607). The T allele of this SNP creates a DNA binding sequence for the NKX family of transcription repressors. Molecular dynamics and binding free energy calculations indicated that NKX-6.3 bound with a higher affinity (the binding free energy of NKX-6.3 bound to T allele is 7.67 kcal/mol lower than bound to C allele, which is equivalent to approximately 400000 fold decrease of kd) to the T allele than the C allele, consistent with our finding of lower expression of CEP72 in the presence of the T allele. When expressed in SHSY5Y or NALM6 cells, the construct containing the T allele had significantly lower expression than the construct containing the C allele, and knockdown of NKX-6.3 rescued the low expression of the construct with the T allele. When CEP72 was knocked down in human ALL cells, there was increased accumulation of the cells at G2M after vincristine treatment, consistent with augmented vincristine effects. Thus, these findings have revealed a new and plausible inherited SNP in the CEP72 promoter region that is linked to the risk of vincristine-induced neuropathy in children with ALL. Conclusions Our genome-wide analysis has revealed a novel genomic region that is associated with increased risk of vincristine-induced neuropathy, providing new insights into this treatment complication and a means to identify patients at highest risk. Disclosures: No relevant conflicts of interest to declare. [1]: /embed/inline-graphic-2.gif