Shirley Kow-Yin Kham

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10−5 and 0.0054, respectively; meta-analysis P = 4.45 × 10−8, allelic effect size = −11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

Thiopurine Methyltransferase polymorphisms in a multiracial Asian population and children with acute lymphoblastic leukemia

The purpose of this study was to determine the frequency of thiopurine methyltransferase (TPMT) polymorphisms in a multiracial Asian population and to assess its relevance in the management of childhood acute lymphoblastic leukemia (ALL). Six hundred unrelated cord blood samples from 200 Chinese, Malay, and Indian healthy newborns were collected at the National University Hospital, Singapore; an additional 100 children with ALL were analyzed for five of the commonly reported TPMT variant alleles using polymerase chain reaction/restriction fragment length polymorphism and allele-specific polymerase chain reaction-based assays. In the cord blood study, the TPMT *3C variant was detected in all three ethnic groups; Chinese, Malays, and Indians had allele frequencies of 3%, 2.3%, and 0.8%, respectively. The TPMT *3A variant was found only among the Indians at a low allele frequency of 0.5%. The TPMT *6 variant was found in one Malay sample. Among the children with ALL, two white and one Chinese were heterozygous for the TPMT *3A variant and showed intermediate sensitivity to 6-mercaptopurine during maintenance therapy. Three Chinese patients and one Malay patient were heterozygous for the TPMT *3C variant. Mercaptopurine sensitivity could be validated in only one out of four TPMT *3C heterozygous patients. The overall allele frequency of the TPMT variants in this multiracial population was 2.5%. The TPMT *3C was the most common variant allele;TPMT *3A and TPMT *6 were rare. These results support the feasibility of performing TPMT genotyping in all children diagnosed with acute leukemia to minimize toxicity from thiopurine chemotherapy.

TPMT*26 (208F→L), a novel mutation detected in a Chinese

AIMS Azathioprine, mercaptopurine and thioguanine are commonly used to treat autoimmune disorders, leukaemia and solid organ transplantation. However, azathiopurine and its metabolites can also cause adverse reactions such as myelosuppression. These manifestations may be attributed to polymorphisms or mutations in the thiopurine methyltransferase (TPMT) gene that might result in low TPMT enzyme activity. Our aim was to investigate if azathioprine-related myelosuppression is associated with TPMT polymorphism, which in turn affects its enzyme activity. METHODS A 61-year-old Chinese man with severe atopic eczema developed moderate myelosuppression with standard doses of azathioprine. His TPMT activity was measured using radiochemical assay. Genotyping of TPMT *3C, *3A and *6 were screened using polymerase chain reaction-restriction fragment length polymorphism. Novel mutation was detected by sequencing. Family studies of his three other siblings were performed. RESULTS After 4 weeks of azathioprine treatment, the patients white blood cells and absolute neutrophil count dropped by 40-45%. He was then taken off azathioprine, and blood counts returned to normal. TPMT activity test showed intermediate levels of 9.1 nmol h(-1) ml(-1) peripheral red blood cells (pRBC). Resequencing of the TPMT gene revealed a missense mutation Phe-->Leu at 208 aa position in exon 9 (ss105107120). Two of his three siblings were heterozygous for 208F-->L, which accounts for the decreased enzyme activity (brother 8.9 nmol h(-1) ml(-1) pRBC, sister 8.8 nmol h(-1) ml(-1) pRBC). The remaining sibling had wild-type allele with normal enzyme activity. Screening of 100 normal healthy Chinese subjects did not reveal any individual with this mutation. CONCLUSION We report a novel mutation TPMT*26 (208F-->L) associated with a decrease in TPMT enzyme activity.

Whole-transcriptome sequencing identifies a distinct subtype of acute lymphoblastic leukemia with predominant genomic abnormalities of EP300 and CREBBP

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n = 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384 gene with histone acetyltransferases EP300 and CREBBP ZNF384-rearranged ALL showed significant up-regulation of CLCF1 and BTLA expression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384 and CREBBP-ZNF384 fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300- and CREBBP-ZNF384 fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300 and CREBBP may cause epigenetic deregulation with potential for therapeutic targeting.

Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Childrens Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations.

FLT3 mutation and expression did not adversely affect clinical outcome of childhood acute leukaemia—a study of 531 Southeast Asian children by the Ma-Spore study group

FMS‐like tyrosine kinase 3 (FLT3) is critical for normal haematopoiesis and have been reported to be expressed in the majority of acute myeloid and lymphoid malignancies. We correlated the impact of FLT3 mutations and its expression with age, WHO 2008 classification and treatment outcome in 531 childhood acute leukaemias. Of 150 acute myeloid leukaemia (AMLs), 18 (12%) harboured FLT3‐ITD while nine (6%) had FLT3‐TKD. FLT3‐ITD and ‐TKD were rare in acute megakaryoblastic leukaemia (AMKL; FLT3‐ITD 0/26, FLT3‐TKD 1/26) and children below 3 years (n = 2/48). Acute promyelocytic leukaemia (APL) with t(15;17);PML‐RARα (n = 7/18; 39%) harboured the highest frequency of FLT3 mutations, followed by myelomonocytic (n = 4/18; 22%) and AML with t(8;21);RUNX1‐RUNX1T1 (n = 2/21; 9%). FLT3 expression levels were also lowest in AMKL, both in Downs and non‐Downs (p = 0.002) followed by patients <3 years (p = 0.001). The rarity of FLT3 mutations and expression levels in AMKL were independent of age. Conversely, only 2% of childhood acute lymphoblastic leukaemia (ALL) harboured FLT3 mutations (ITD = 1/381; TKD = 6/381). FLT3 was highly expressed in hyperdiploid ALL (p < 0.001). Of the 121 AMLs with clinical history, there were no significant differences in 4‐year event‐free survival (EFS) (46% vs. 38%; p = 0.46) and overall‐survival (OS) (55% vs. 43%; p = 0.30) between FLT3‐wildtype and ITD+ patients. Similarly, FLT3 expression levels did not influence survival in AML in both the good risk and non‐good risk subgroups. FLT3 does not appear to be involved in the pathogenesis of AMKL, both in Downs and non‐Downs. Therapeutic targets using FLT3 inhibitors may not be useful in AMKL and in young children with AML. Copyright

Whole-genome noncoding sequence analysis in T-cell acute lymphoblastic leukemia identifies oncogene enhancer mutations

Publishers Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Our understanding of acute lymphoblastic leukemia (ALL) has expanded tremendously in the past few years because of large-scale genomic studies.[1][2],[2][3] ALL is characterized by a relatively

Host genetic variants of ABCB1 and IL15 influence treatment outcome in paediatric acute lymphoblastic leukaemia

Background:Host germline variations and their potential prognostic importance is an emerging area of interest in paediatric ALL.Methods:We investigated the associations between 20 germline variations and various clinical end points in 463 children with ALL.Results:After adjusting for known prognostic factors, variants in two genes were found to be independently associated with poorer EFS: ABCB1 T/T at either 2677 (rs2032582) or 3435 (rs1045642) position (P=0.003) and IL15 67276493G/G (rs17015014; P=0.022). These variants showed a strong additive effect affecting outcome (P<0.001), whereby patients with both risk genotypes had the worst EFS (P=0.001), even after adjusting for MRD levels at the end of remission induction. The adverse effect of ABCB1 T/T genotypes was most pronounced in patients with favourable cytogenetics (P=0.011) while the IL15 67276493G/G genotype mainly affected patients without common chromosomal abnormalities (P=0.022). In both cytogenetic subgroups, increasing number of such risk genotypes still predicted worsening outcome (P<0.001 and=0.009, respectively).Conclusion:These results point to the prognostic importance of host genetic variants, although the specific mechanisms remain unclarified. Inclusion of ABCB1 and IL15 variants may help improve risk assignment strategies in paediatric ALL.

Preclinical evaluation of NUDT15-guided thiopurine therapy and its effects on toxicity and antileukemic efficacy

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.