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Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia.

ABSTRACT: Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experience relapsed ALL (R367Q, R238W and L375F).Enzyme kinetics measurements revealed that the mutants are consitutively active without need for allosteric activators. This shows that hyperactivity is not caused by a direct catalytic effect but rather by misregulation of cN-II. X-ray crystallography combined with mass spectrometry-based techniques demonstrated that this misregulation is driven by structural modulation of the oligomeric interface within the cN-II homotetrameric assembly. These specific conformational changes are shared between the studied variants, despite the relatively random spatial distribution of the mutations.These findings define a common molecular mechanism for cN-II hyperactivity, which provides a solid basis for targeted therapy of leukemia. Our study highlights the cN-II oligomerization interface as an attractive pharmacological target.

SUBMITTER: Hnizda A

PROVIDER: S-EPMC5070119 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia.

Hnízda Aleš A Škerlová Jana J Fábry Milan M Pachl Petr P Šinalová Martina M Vrzal Lukáš L Man Petr P Novák Petr P Řezáčová Pavlína P Veverka Václav V

BMC biology 20161019 1

<h4>Background</h4>Relapsed acute lymphoblastic leukemia (ALL) is one of the main causes of mortality in childhood malignancies. Previous genetic studies demonstrated that chemoresistant ALL is driven by activating mutations in NT5C2, the gene encoding cytosolic 5´-nucleotidase (cN-II). However, molecular mechanisms underlying this hyperactivation are still unknown. Here, we present kinetic and structural properties of cN-II variants that represent 75 % of mutated alleles in patients who experie ...[more]

PMID: 27756303

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Project description:Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical as the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.

Project description:Opinion statementRelapse is still a common scenario in acute myeloid leukemia (AML) treatment and occurs in 40-50% of younger and the great majority of elderly patients. The prognosis in relapsed AML patients is generally poor but depends largely on the timing of relapse (early versus late) and the possibility of allogeneic hematopoietic stem cell transplantation (HSCT). At the time of relapse, we again perform a mutational screening and cytogenetic analysis in all AML patients as clonal evolution of disease is frequent. Clinical trials should be first priority in all relapsed patients. In fit patients without prior transplant, we aim to perform HSCT after salvage therapy. In AML patients relapsing after HSCT and good performance status, intensive therapy can be considered with subsequent cellular therapy such as donor lymphocyte infusion (DLI) or a second HSCT. However, less than 20% of these patients are alive after 5 years. For those patients that are unfit, the therapeutic aim is to prolong life with acceptable quality of life. Here, hypomethylating agents (HMA), low-dose AraC (LDAC), and solely cytoreductive therapy with hydroxurea are options depending on first-line therapy. For those patients that have not been treated with venetoclax in first line, the combination therapy of venetoclax with demethylating agents achieves encouraging response rates. Venetoclax is currently also studied in combination with intensive salvage therapy. Importantly, for patients with isocitrate dehydrogenase (IDH) 1/2-mutated AML, ivosidenib, an IDH1 inhibitor, and enasidenib, an IDH2 inhibitor, present well-tolerated options in the setting of refractory or relapsed (r/r) disease even in elderly and heavily pre-treated patients with response rates of 30-40%. Both substances have been approved by the U.S. Food and Drug Administration (FDA) for r/r AML patients with IDH1/2 mutations (but not yet by the European Medicines Agency (EMA)). For patients with FMS-like tyrosine kinase 3 (FLT3) mutations, treatment with the selective FLT3 inhibitor gilteritinib is well tolerated and leads to improved outcome compared with standard salvage therapy. The approval has been granted by the FDA and the EMA. Generally, we would recommend targeted therapy for IDH1/2- and FLT3-mutated AML if available. In order to improve outcome in relapsed AML, it will be important to intelligently combine novel substances with each other as well as chemotherapy in prospective clinical trials. The development of therapies with bispecific antibodies or chimeric antigen receptor T cells (CAR-T) are still in early development.

Dataset Information

Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia.

Publications

Oligomeric interface modulation causes misregulation of purine 5´-nucleotidase in relapsed leukemia.

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