Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies
Utthara Nayar, Jouliana Sadek, Jonathan Reichel, Denise Hernandez-Hopkins, Gunkut Akar, Peter J. Barelli, Michelle A. Sahai, Hufeng Zhou, Jennifer Totonchy, David Jayabalan, Ruben Niesvizky, Ilaria Guasparri, Duane Hassane, Yifang Liu, Shizuko Sei, Robert H. Shoemaker, J. David Warren, Olivier Elemento, Kenneth M. Kaye, Ethel Cesarman
Utthara Nayar, Jouliana Sadek, Jonathan Reichel, Denise Hernandez-Hopkins, Gunkut Akar, Peter J. Barelli, Michelle A. Sahai, Hufeng Zhou, Jennifer Totonchy, David Jayabalan, Ruben Niesvizky, Ilaria Guasparri, Duane Hassane, Yifang Liu, Shizuko Sei, Robert H. Shoemaker, J. David Warren, Olivier Elemento, Kenneth M. Kaye, Ethel Cesarman
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Research Article Oncology

Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies

Abstract

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase–inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI–sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

Authors

Utthara Nayar, Jouliana Sadek, Jonathan Reichel, Denise Hernandez-Hopkins, Gunkut Akar, Peter J. Barelli, Michelle A. Sahai, Hufeng Zhou, Jennifer Totonchy, David Jayabalan, Ruben Niesvizky, Ilaria Guasparri, Duane Hassane, Yifang Liu, Shizuko Sei, Robert H. Shoemaker, J. David Warren, Olivier Elemento, Kenneth M. Kaye, Ethel Cesarman

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Figure 4

ADK is required for 6-ETI activation.

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ADK is required for 6-ETI activation. (A) Figure indicates locations of ...
(A) Figure indicates locations of independent recurrent mutations G239E and E243K on ADK, discovered by SNP calling on RNA-Seq data from 6-ETI–resistant clones. Schrödinger’s Maestro was used to model ADK binding to adenosine (ADO) (top panel), and postulated binding to 6-ETI (bottom panel). (B) BC3 cells treated at a range of concentrations of A-134974 or ABT-702 were analyzed by CellTiter-Glo assay for viability at 48 hours. Results are shown as the mean ± SEM of at least 3 independent experiments where LC50s were determined using online EC50 software. (C) 6-ETI viability dose curves were set up on BC3 cells pretreated with 1 μM ADK inhibitors A-134974 and ABT-702 or DMSO, followed by treatment with 6-ETI. The fold increases in 6-ETI LC50s are shown via bar graph, representing the mean ± SEM of at least 3 independent experiments. (D) ADK kinase assay to test 6-ETI phosphorylation was carried out by mixing of kinase assay components with 6-ETI, followed by acetonitrile quenching at various time points. Reaction mixture at each time point was injected into HPLC to detect the appearance of the phosphorylated peak. Chromatogram for the 6-ETI standard under the HPLC conditions indicates the appearance of a peak at retention time 1.76 minutes, indicated by a star. Chromatograms at t = 0 and t = 60 minutes both contain this peak, while t = 60 reveals the appearance of a secondary peak at 1.57 minutes (indicated by double star). (E) Mass spectrometry analysis was performed on the fraction of the t = 60 reaction mixture retrieved at 1.57 minutes of HPLC. The MS revealed a prominent peak at 391.3 (indicated by asterisk), corresponding exactly to the expected molecular weight of phospho–6-ETI.