Kinetic Analysis of Prodrug Activation and ATP/UTP Substrate Preference of Nine Human Deoxycytidine Kinase Mutants

Deoxynucleoside analogues are prodrugs that canfunction as inhibitors of both viral and cellular DNA replication processes. They are important in anti-cancer therapy because they hinder DNA synthesis and cellular mitosis. Within the cell, deoxyribonucleotides are synthesized using the salvage pathways by converting the unphosphorylated nucleosides to their mono, di- and tri-phosphate forms using a phosphoryl donor: ATP or UTP. Human deoxycytidine kinase (dCK) is the first and rate-limiting enzyme in this process. The dCK protein uses nucleotide triphosphates to phosphorylate several clinically important nucleoside analogue prodrugs in addition to its natural substrates. The preferred physiological phosphoryl donor for dCK is UTP although it is less prevalent in the human body than ATP. Our objective is to improve the understanding of the phosphate-donor binding loop of dCK by kinetic analysis of a series of mutants of Asp241 and Phe242. These mutants were designed in an attempt to improve the activity of dCK with phosphate donors. Results show several mutants with improved kinetics and some with an ATP donor preference over UTP

Prognostic Significance of FLT3 and NPM1 Mutations in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most common and deadly hematopoietic malignancies. Currently, age, performance status (a patient’s general well being and activities), and cytogenetic status remain most predictive prognostic factors for AML. In addition, genomic mutations within nucleophosmin 1 (NPM1) and fms-like tyrosine kinase (FLT3) have been found to be clinically relevant prognostic factors for AML patients, especially those with normal cytogenetics. But it is unknown if the prognostic significance of FLT3 and NPM1 will apply to contemporary treatment strategies. Therefore, we examined the potential clinical significance of NPM1 and FLT3 mutations in a large cohort of adult de novo AML patients enrolled on South West Oncology Group (SWOG) trial S0106. In this randomized phase III trial, patients ages 18-60 with de novo AML were enrolled to compare the effects of adding Gemtuzumab Ozogamicin (GO) to standard induction therapy (Cytosine Arabinoside and Daunomycin, AD) vs. standard treatment alone. Samples from 198 of the 600 eligible patients were evaluated. Genomic mutation analyses for nucleotide insertions in exon 12 of the NPM1 gene and internal tandem duplications (ITDs) within exons 14-15 of FLT3 were performed using fragment analyses in diagnostic bone marrow (BM) and peripheral blood (PB) samples. Mutant/wild type allelic ratios were computed for all mutations. Analyses evaluating the prognostic significance of the mutation status of the two genes identified 3 mutational risk groups for overall survival (OS) and relapse-free survival (RFS). AML patients with NPM1 mutations and without FLT3-ITD mutations had a much more favorable prognosis than all other evaluated AML patients

Kinetic Analysis of Prodrug Activation and ATP/UTP Substrate Preference of Nine Human Deoxycytidine Kinase Mutants

Deoxynucleoside analogues are prodrugs that canfunction as inhibitors of both viral and cellular DNA replication processes. They are important in anti-cancer therapy because they hinder DNA synthesis and cellular mitosis. Within the cell, deoxyribonucleotides are synthesized using the salvage pathways by converting the unphosphorylated nucleosides to their mono, di- and tri-phosphate forms using a phosphoryl donor: ATP or UTP. Human deoxycytidine kinase (dCK) is the first and rate-limiting enzyme in this process. The dCK protein uses nucleotide triphosphates to phosphorylate several clinically important nucleoside analogue prodrugs in addition to its natural substrates. The preferred physiological phosphoryl donor for dCK is UTP although it is less prevalent in the human body than ATP. Our objective is to improve the understanding of the phosphate-donor binding loop of dCK by kinetic analysis of a series of mutants of Asp241 and Phe242. These mutants were designed in an attempt to improve the activity of dCK with phosphate donors. Results show several mutants with improved kinetics and some with an ATP donor preference over UTP