Importance With cure rates of childhood acute lymphoblastic leukemia (ALL) exceeding 85%, there is compelling need to mitigate treatment toxicities that can compromise quality of life. Peripheral neuropathy is the major dose-limiting toxicity of the microtubule inhibitor vincristine, an anticancer agent given to every child with ALL. Objective Identify genetic germline variants associated with the occurrence or severity of vincristine-induced peripheral neuropathy in children with ALL. Design, Setting and Participants All patients had been enrolled in one of two prospective clinical trials for childhood ALL that included treatment with 36–39 doses of vincristine. Genome-wide single nucleotide polymorphism (SNP) analysis and vincristine-induced peripheral neuropathy were assessed in all patients from whom DNA was available (n=321 patients); 222 patients (median age at 6.0 years, range 0.1–18.8 years) enrolled between 1994–1998 on the St. Jude Children’s Research Hospital protocol Total XIIIB (St. Jude cohort) with toxicity followed through January 2001, and 99 patients (median age 11.4 years, range 3.0–23.8 years) enrolled between 2007–2010 on the Children’s Oncology Group protocol AALL0433 (COG cohort) with toxicity followed through May 2011. Human leukemia cells and induced pluripotent stem cell neurons were used to assess the effects of lower CEP72 expression on vincristine sensitivity. Exposures Treatment with vincristine at a dosage of 1.5 or 2.0 mg/m2 as a component of protocol directed chemotherapy for childhood ALL. Main Outcomes and Measures Vincristine-induced peripheral neuropathy was assessed at each clinic visit using the National Cancer Institute Common Terminology Criteria for Adverse Events and prospectively graded as mild (grade 1), moderate (grade 2), serious/disabling (grade 3), or life-threatening (grade 4). Results Grade 2–4 vincristine-induced neuropathy during continuation therapy occurred in 28.8% of patients (n=64 of 222) in the St. Jude cohort and in 22.2% of patients (n=22 of 99) in the COG cohort. A SNP in the promoter region of the CEP72 gene, which encodes a centrosomal protein involved in microtubule formation, had a significant association with vincristine neuropathy (meta p =6.3 × 10−9). This SNP had a minor allele frequency of 37% (235/642), with 50 of 321 patients (16%, 95% CI 11.6%–19.5%) homozygous for the risk allele (TT at rs924607). Among patients with the high-risk CEP72 genotype (TT at rs924607), 28 of 50 patients (56%, 95% CI 41.2–70.0) developed at least one episode of grade 2–4 neuropathy, a higher rate than in patients with the CEP72 CC or CT genotype (58 of 271 patients; 21.4%, 95% CI 16.9–26.7); p=2.4×10−6. The severity (grade) of neuropathy was greater (2.4-fold by Poisson regression (p<0.0001), 2.7-fold based on mean grade of neuropathy (1.23 [95% CI 0.74 – 1.72] versus 0.45 [95% CI 0.3 – 0.6]; t test p=0.004)) in patients homozygous for the CEP72 risk allele (TT genotype), compared to patients with the CC or CT genotype. The CEP72 promoter SNP was show...

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and leukemia cell resistant to glucocorticoids confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the sensitivity to prednisolone of primary leukemia cells from 444 newly diagnosed ALL patients, revealing significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases.

NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3-5 exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3 terminus of DNA. The activity is dependent upon the presence of Mg 2؉ , is most pronounced with single-stranded substrates or mismatched bases at the 3 terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2-deoxyadenosine analogue, into the 3-terminal position. The 3-5 exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3-5 exonuclease activity resulting from a Lys 12 to glutamine substitution within the protein. 3-5 Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1.nm23-H1 was first classified as a metastasis suppressor gene on the basis of its reduced expression in several metastatic melanoma cell lines relative to nonmetastatic counterparts (1). Subsequently, low expression of the NM23-H1 protein has been linked to increased metastatic potential in human breast carcinoma (2), hepatoma (3), and gastric carcinoma (4). NM23-H1 overexpression has been shown experimentally to inhibit the metastatic phenotype and/or promote differentiation in melanoma (5), breast carcinoma (6), and transformed neural cell lines (7,8). The human nm23-H1 gene is one of eight related NM23 family members identified to date (reviewed in Ref. 9). Each exhibits nucleoside-diphosphate kinase (NDPK) 1 activity, catalyzing the transfer of ␥-phosphate between nucleoside triphosphate and nucleoside diphosphate via a "ping-pong" mechanism (10). NDPK activity does not appear to be relevant to metastasis suppression, however, as catalytically inactive mutants retain metastasis suppressor activity (11,12). In addition to NDPK, a number of other biological activities have been reported for NM23 proteins, some of which have been proposed to underlie metastasis suppression. Two spontaneous mutations in NM23-H1, a serine 120 to glycine (S120G) substitution seen frequently in aggressive neuroblastomas (13), and a proline 96 to serine mutation that corresponds to the killer of prune mutation (Kpn; P96S) in the Drosophila homologue of NM23 (awd), both result in loss of antimetastatic activity (14). Interestingly, both mutations also abrogate a histidine-dependent protein kinase activity of NM23-H1 (15). This activity has ...

Treatment-related toxicity can be life-threatening and is the primary cause of interruption or discontinuation of chemotherapy for acute lymphoblastic leukemia (ALL), leading to an increased risk of relapse. Mercaptopurine is an essential component of continuation therapy in all ALL treatment protocols worldwide. Genetic polymorphisms in thiopurine S-methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity; however, some patients with wild-type TPMT develop toxicity during mercaptopurine treatment for reasons that are not well understood. To identify additional genetic determinants of mercaptopurine toxicity, a genome-wide analysis was performed in a panel of human HapMap cell lines to identify trans-acting genes whose expression and/or single-nucleotide polymorphisms (SNPs) are related to TPMT activity, then validated in patients with ALL. The highest ranking gene with both mRNA expression and SNPs associated with TPMT activity in HapMap cell lines was protein kinase C and casein kinase substrate in neurons 2 (PACSIN2). The association of a PACSIN2 SNP (rs2413739) with TPMT activity was confirmed in patients and knock-down of PACSIN2 mRNA in human leukemia cells (NALM6) resulted in significantly lower TPMT activity. Moreover, this PACSIN2 SNP was significantly associated with the incidence of severe gastrointestinal (GI) toxicity during consolidation therapy containing mercaptopurine, and remained significant in a multivariate analysis including TPMT and SLCO1B1 as covariates, consistent with its influence on TPMT activity. The association with GI toxicity was also validated in a separate cohort of pediatric patients with ALL. These data indicate that polymorphism in PACSIN2 significantly modulates TPMT activity and influences the risk of GI toxicity associated with mercaptopurine therapy.

The metastasis suppressor NM23-H1 possesses 3 enzymatic activities in vitro, a nucleoside diphosphate kinase (NDPK), a protein histidine kinase and a more recently characterized 3 0 -5 0 exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3 0 -5 0 exonuclease to metastasis suppressor function has not been addressed in detail. To this end, site-directed mutagenesis and biochemical analyses of bacterially expressed mutant NM23-H1 proteins have identified mutations that disrupt the 3 0 -5 0 exonuclease alone (Glu 5 to Ala, or E 5 A), the NDPK and histidine kinase activities tandemly (Y 52 A, H 118 F) or all 3 activities simultaneously (K 12 Q). Although forced expression of NM23-H1 potently suppressed spontaneous lung metastasis of subcutaneous tumor explants derived from the human melanoma cell line 1205LU, no significant metastasis suppressor activity was obtained with the exonuclease-deficient variants E 5 A and K 12 Q. The H 118 F mutant, which lacked both the NDPK and histidine kinase while retaining the 3 0 -5 0 exonuclease, also exhibited compromised suppressor activity. In contrast, each mutant retained the ability to suppress motility and invasive characteristics of 1205LU cells in culture, indicating that the NM23-H1 molecule possesses an additional activity(s) mediating these suppressor functions. These studies provide the first demonstration that the 3 0 -5 0 exonuclease activity of NM23-H1 is necessary for metastasis suppressor function and further indicate cooperativity of the 3 enzymatic activities of the molecule on suppression of the metastatic process.Metastasis suppressors are a class of genes defined by their ability to selectively inhibit the metastatic process with little or no impact on primary tumor growth. 1 The first metastasis suppressor gene to be identified was nm23-M1, which was discovered by virtue of its low expression in K-1735-derived melanoma cell lines with elevated metastatic potential. 2 Subsequent studies have confirmed that the human homolog nm23-H1 exhibits potent metastasis suppressor activity in breast carcinoma and melanoma cell lines and is underexpressed in multiple forms of metastatic cancer. 3 Although the mechanisms underlying metastasis suppressor activity of NM23-H1 are not fully understood, the protein exhibits 3 different enzymatic activities in vitro that represent potential antimetastatic functions. First to be described was its nucleoside diphosphate kinase (NDPK) activity, which maintains balance in intracellular nucleotide pools by catalyzing transfer of c-phosphate between nucleoside triphosphates and diphosphates. 4 Although a role for the NDPK in metastasis suppression has been challenged, 5-7 the concept has yet to be addressed directly with in vivo models of metastatic growth. NM23-H1 is also reported to exhibit a protein histidine kinase activity that mediates its antimotility function, 8 possibly via serine phosphorylation of the kinase suppressor...