SAMHD1 single nucleotide polymorphisms impact outcome in children with newly diagnosed acute myeloid leukemia.

Cytarabine arabinoside (Ara-C) has been the cornerstone of acute myeloid leukemia (AML) chemotherapy for decades. After cellular uptake, it is phosphorylated into its active triphosphate form (Ara-CTP), which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in the Ara-C metabolic pathway has been shown to affect intracellular abundance of Ara-CTP and, thus, its therapeutic benefit. Recently, SAMHD1 (SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1) has emerged to play a role in Ara-CTP inactivation, development of drug resistance, and, consequently, clinical response in AML. Despite this, the impact of genetic variations in SAMHD1 on outcome in AML has not been investigated in depth. In this study, we evaluated 25 single nucleotide polymorphisms (SNPs) within the SAMHD1 gene for association with clinical outcome in 400 pediatric patients with newly diagnosed AML from 2 clinical trials, AML02 and AML08. Three SNPs, rs1291128, rs1291141, and rs7265241 located in the 3' region of SAMHD1 were significantly associated with at least 1 clinical outcome: minimal residual disease after induction I, event-free survival (EFS), or overall survival (OS) in the 2 cohorts. In an independent cohort of patients from the COG-AAML1031 trial (n = 854), rs7265241 A>G remained significantly associated with EFS and OS. In multivariable analysis, all the SNPs remained independent predictors of clinical outcome. These results highlight the relevance of the SAMHD1 pharmacogenomics in context of response to Ara-C in AML and warrants the need for further validation in expanded patient cohorts.

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia: Results from the HEAT-AML trial.

BACKGROUND: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol.

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).

Pharmacogenetics of deoxycytidine kinase: identification and characterization of novel genetic variants.

Deoxycytidine kinase (DCK) is a rate-limiting enzyme in the activation of nucleoside analogs such as cytarabine (ara-C), gemcitabine, clofarabine, and others. The present study was undertaken to identify and to determine the functional consequences of genetic variants in DCK. We sequenced 1.5 kilobases of the DCK proximal promoter and all seven coding exons in International HapMap Project panels (n = 90 each) with European (Centre d' Etude du Polymorphisme Humain; CEPH) or African (Yoruba people in Ibadan, Nigeria; YRI) ancestry. Sixty-four genetic polymorphisms, including three nonsynonymous coding changes (I24V, A119G, and P122S) were identified. Compared with DCK-wild-type (WT) protein, the activity of the recombinant DCK24Val, DCK119Gly, and DCK122Ser proteins was 85 +/- 5, 66 +/- 3, and 43 +/- 4%, respectively. DCK119Gly and DCK122Ser mutants had lower Km (p < 0.01) and Vmax (p < 0.001) compared with DCK-WT protein. Lymphoblast cell lines from subjects heterozygous for the coding changes had significantly lower DCK activity compared with homozygous WT subjects. Ethnic differences were observed, with African ancestry subjects demonstrating significantly higher DCK mRNA expression compared with subjects with European ancestry. In both CEPH and YRI subjects, the C allele of a 3'-untranslated region single-nucleotide polymorphism (SNP) (35708 C>T) was significantly associated with lower DCK mRNA expression. This SNP was strongly linked with other intronic SNPs, forming a major haplotype block in both ethnic groups. In an exploratory analysis, the 35708C allele was also associated with lower blast ara-C-5'-triphosphate (ara-CTP) levels in acute myeloid leukemia patients receiving ara-C as continuous infusion. These results suggest that genetic variation in DCK influences its activity and expression and may predict the variability observed in intracellular levels of the ara-C active metabolite ara-CTP.

Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia.

The importance of the cellular pharmacokinetics of cytarabine triphosphate (ara-CTP) with regard to therapeutic efficacy is well established. In vitro and in vivo monitoring of pharmacokinetic parameters of leukemic blast cells were initiated in order to contribute to the pharmacological basis of optimal ara-C treatment strategies. Peripheral or bone marrow blast cells from 66 leukemic patients [51 acute myelogenous leukemia (ALL), 15 acute lymphoblastic leukemia (AML) were separated and incubated with ara-C for 1 hour and in ara-C-free medium for another 3 hours, and the intracellular formation and retention of ara-CTP was measured. In eight children who received continuous ara-C infusion for induction treatment, the ara-CTP concentration in circulating blast cells was monitored in vivo. The in vitro values observed in this assay corresponded to the cellular levels monitored in vivo. The ara-CTP retention differed clearly among the individual groups, as classified by immunophenotype at the time of the initial diagnosis: non-T-ALL 67+/-25% (x+/-SD, n=33), T-ALL 37+/-15% (n=8), and AML 34+/-18% (n=14). The difference in ara-CTP retention between non-T-All and AML (P<0.05) as well as T-ALL (P<0.05) was significant. There was a tendency toward lower ara-CTP retention in relapsed as compared with newly diagnosed ALL, but the difference was not significant. The maximal accumulation of ara-CTP (after 1 hour incubation) was comparable in AML, T-ALL, non-T-ALL, and blast cells from children in relapse. The observed similarity of cellular accumulation in all groups and the significantly more rapid decrease in T-ALL and AML provide the pharmacokinetic rationale supporting the prolonged infusion duration for ara-C in these subgroups as an alternative to the intensification by high-dose ara-C schedules with short-term infusion.

Effect of liposomally trapped antitumour drugs on a drug-resistant mouse lymphoma in vivo.

A TLX-5 mouse lymphoma which was resistant to 1-ß-D-arabinofuranosyl cytosine (AraC) was used in vivo to study the possibility of using liposomes as drug-delivery vehicles in order to overcome drug resistance.The effects of free drugs (AraC, AraCTP and methotrexate) and the liposomally associated drugs on the survival time of tumour-bearing mice were determined.As a more sensitive measure of cell survival, (125)IUdR was incorporated into the DNA of the ascites TLX-5 cells before i.p. injection. Cell survival and the cytotoxic effects of the drugs on the tumour cells were determined by using a double-headed gamma counter to measure the retention of the (125)I label.Both AraC and AraCTP, either as the free drugs or liposomally associated, had no effects on the tumour. Due to the lack of response of tumour cells to these drugs, further studies were initiated with free and liposomally associated methotrexate (MTX), a drug to which the cells were known to be sensitive. It was found that the liposomally associated MTX, at a 5-10-fold lower dose than the free drug, was (a) more effective in prolonging the survival of tumour-bearing mice and (b) as effective as the free drug in killing tumour cells (as measured by the (125)I retention).In vivo MTX was more effective in the liposomally associated form, whereas liposomally entrapped AraC and AraCTP were ineffective. It is proposed that in vivo liposomally associated drugs may be acting not by actively localizing in the tumour cells, but by the liposomes providing a slow-release drug depot, improving the pharmacokinetic properties of MTX.

Studies on biologically active nucleosides and nucleotides. 5. Synthesis and antitumor activity of some 2,2'-anhydro-1-(3',5'-di-O-acyl-beta-D-arabinofuranosyl)cytosine salts and 2,2'-anhydro-1-(3'-O-acyl-beta-D-arabinofuranoxyl)cytosine 5'-phosphates.

A series of 3',5'-diesters of a 2,2'-anhydro-1-(beta-D-arabinofuranosyl)cytosine salt bearing functional substituents on the ester side chains (4--16) have been synthesized. The synthesis of these diesters involved the reaction between cytidine and the corresponding acid anhydride or acid chloride in the presence of boron trifluoride etherate. Similar reaction of bis(cytidine 5'-)suberate (21) with pivaloyl chloride gave bis[2,2'-anhydro-1-(3'-O-pivaloyl-beta-D-arabinofuranosyl)cytosine 5'-]suberate dihydrochloride (22). The reaction could also be extended to a one-step synthesis of 3'-esters of 2,2'-anhydro-1-(beta-D-arabinofuranosyl)cytosine 5'-phosphate (24) from 5'-cytidylic acid. These compounds have been examined for antitumor activity against L1210 leukemia in BDF1 mice. The diesters with a long-chain carboxylic acid (4c, 7c, 12, and 24d) showed high activity when administered intraperitoneally. The compound 24d exhibited moderate activity when administered orally.