Evaluation of Nudt15 -Guided Thiopurine Dose Individualization In Vivo Using CRISPR/Cas9-Edited Mouse Model

Prolonged exposure to thiopurines (e.g., 6-mercaptopurine [MP]) is an essential component of curative therapy of acute lymphoblastic leukemia (ALL) in children and adults. However, this class of drugs are also associated with frequent dose-limiting hematopoietic and liver toxicities, at least in part explained by genetic polymorphisms in metabolizing enzymes such as TPMT . In fact, pre-emptive TPMT- genotype guided thiopurine dose reduction has been implemented in clinical practice and shown to effectively mitigate toxicity without compromising therapeutic efficacy. More recently, we and others identified NUDT15 as a novel genetic determinant of thiopurine toxicity ( Nature Genetics , 2014 and 2016), particularly in Asian and Hispanic populations. While results from correlative studies in patients point to potential effectiveness of NUDT15 -genotype based thiopurine individualization, the exact dosing algorithm remains unknown and there have been no laboratory models to test such strategies. To this end, we established a Nudt15 knockout mouse model on FVB/N background by CRISPR-Cas9 genome editing, with which we systematically evaluated NUDT15 -genotype directed thiopurine dose reduction and its effects on hematopoietic toxicity in vivo .

Nudt15^+/+ and Nudt15^-/- mice received daily intra-peritoneal MP injection at the dosage of 1, 5 or 20 mg/kg. Consistently across dosage levels, Nudt15^-/- mice experienced more severe and precipitous myelosuppression (especially reduction in neutrophil count), more rapid weight loss, and earlier toxic death, compared to wildtype mice (P < 0.001). For example at the MP dosage of 20 mg/kg/day, the median time to toxic death was 12 days in Nudt15^-/- mice whereas wildtype mice survived a median of 25 days (P = 6.9 × 10^-15). Myelosuppression (white blood cell count < 2.0 × 10⁶ /ml) occurred as early as 9 vs 22 days following the start of MP treatment in Nudt15^-/- vs wildtype mice, respectively (P < 0.01). Severe bone marrow hypocellularity at the time of death was confirmed by necropsy, and was particularly pronounced in deficient mice. Notably, MP treatment induced damage to the mucosal epithelial cells of the digestive tract (e.g., esophagus and small intestine) in Nudt15^-/- mice, whereas there were no mucosal lesions in wildtype animals. In Nudt15^-/- mice, toxicities were significantly reduced with decreasing MP dosage. In fact, at MP dosage of 1mg/kg/day, Nudt15^-/- mice showed a toxicity profile (bone marrow suppression, weight loss, and toxic death) that was comparable to that of Nudt15 wildtype mice receiving 20 mg/kg/day of MP, suggesting that a 95% dose reduction might be needed to completely mitigate MP toxicity in individuals with homozygous NUDT15 deficiency.

To assess the impacts of Nudt15 genotypes on thiopurine activation, we also characterized the acute accumulation of MP metabolite (i.e., DNA incorporated thioguanine, DNA-TG) following daily MP injection at increasing dosages (1, 5, 20 and 40 mg/kg/day). Although the level of DNA-TG in hematopoietic cells was positively correlated with MP dosage in both genotype groups, this active metabolite accumulated much more rapidly in Nudt15^-/- mice with increasing MP dosages than in wildtype mice (p < 0.001). Interestingly, DNA-TG level in Nudt15^-/- mice exposed to 1 mg/kg/day of MP was comparable to that in Nudt15^+/+ mice dosed at 20 mg/kg/day, consistent with our observation that a 20mg-to-1mg dosage reduction effectively eliminated the excessive MP toxicity associated with Nudt15 deficiency. Finally, we also examined intra-patient variations of leukocyte DNA-TG levels during MP dose titration in 46 children with ALL enrolled on the Japanese Pediatric Leukemia/Lymphoma Study Group ALL B-12 trial. The increase in DNA-TG when MP dose was titrated up was significantly higher in patients homozygous for NUDT15 risk alleles than those with heterozygous genotype and lowest in wildtype patients (P = 0.05), in a fashion similar to results from the Nudt15^-/- mouse model. Taken together, these data suggested that DNA-TG may be directly related to hematopoietic toxicity of MP and this metabolite may be used to guide MP dosage titration to avoid excessive toxicity.

In conclusion, we established a Nudt15 knockout mouse model, with which we comprehensively evaluated NUDT15 -genotype guided thiopurine dose individualization in vivo .