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Mechanisms of resistance

Relapsed acute lymphoblastic leukemia-specific mutations in NT5C2 cluster into hotspots driving intersubunit stimulation

Abstract

Activating mutations in NT5C2, a gene encoding cytosolic purine 5′-nucleotidase (cN-II), confer chemoresistance in relapsed acute lymphoblastic leukemia. Here we show that all mutants became independent of allosteric effects of ATP and thus constitutively active. Structural mapping of mutations described in patients demonstrates that 90% of leukemia-specific allelles directly affect two regulatory hotspots within the cN-II molecule—the helix A region: residues 355–365, and the intersubunit interface: helix B (232–242) and flexible interhelical loop L (400–418). Furthermore, analysis of hetero-oligomeric complexes combining wild-type (WT) and mutant subunits showed that the activation is transmitted from the mutated to the WT subunit. This intersubunit interaction forms structural basis of hyperactive NT5C2 in drug-resistant leukemia in which heterozygous NT5C2 mutation gave rise to hetero-tetramer mutant and WT proteins. This enabled us to define criteria to aid the prediction of NT5C2 drug resistance mutations in leukemia.

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Acknowledgements

This work was supported by grant 15-06582S from the Czech Science Foundation and in part by the Ministry of Education of the Czech Republic (program “NPU I” LO1304 and program “InterBioMed” LO1302) and by European Regional Development Fund Project No. CZ.02.1.01/0.0/0.0/16_019/0000729. Institutional support was provided by projects RVO 61388963 and 68378050 of the Academy of Sciences of the Czech Republic. MZ and JT were funded by grant 15-30626A from Czech Health Research Council and grant Primus/MED/28 from Charles University. DBA was supported by a C. J. Martin Research Fellowship from the National Health and Medical Research Council of Australia (APP1072476) and the Jack Brockhoff Foundation (JBF 4186, 2016). We acknowledge beamline access at MX14.1 and MX14.3 of the BESSY (HZB Berlin, Germany) to collect crystal diffraction data. The authors would like to thank Irena Sieglova, MSc and Anna Soldanova, BSc for technical help during protein preparations and Katsiaryna Tratsiak, MSc for crystallization trials.

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Correspondence to Aleš Hnízda.

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Hnízda, A., Fábry, M., Moriyama, T. et al. Relapsed acute lymphoblastic leukemia-specific mutations in NT5C2 cluster into hotspots driving intersubunit stimulation. Leukemia 32, 1393–1403 (2018). https://doi.org/10.1038/s41375-018-0073-5

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