Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies
Utthara Nayar, … , Kenneth M. Kaye, Ethel Cesarman
Utthara Nayar, … , Kenneth M. Kaye, Ethel Cesarman
Published May 15, 2017
Citation Information: J Clin Invest. 2017;127(6):2066-2080. https://doi.org/10.1172/JCI83936.
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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 5

ADK levels determine sensitivity to 6-ETI.

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ADK levels determine sensitivity to 6-ETI. (A) A panel of KSHV+ and KSHV...
(A) A panel of KSHV+ and KSHV– lymphoma cell lines were plated at viability assay conditions (i.e., 1 × 105 cells/ml in RPMI with 20% FBS), and whole cell extracts obtained at 24 hours were examined for ADK levels by Western blotting. GAPDH was used as a loading control. Results shown are representative of more than 3 independent experiments. (B) Successful lentiviral shRNA-mediated knockdown of ADK in the pancreatic adenocarcinoma cell line PA-Tu-8988T was examined by Western blot using an ADK antibody. A representative blot of 3 independent experiments is shown. 6-ETI viability assays at 24 hours were performed on the knockdown cell line compared with control. LC50 graphed is the average of 5 independent experiments (mean ± SEM). Statistical analysis was performed using Mann-Whitney unpaired test (P = 0.0079, **P ≤ 0.05). (C) ADK normally phosphorylates adenosine to convert it into adenosine monophosphate (AMP) and similarly adds a phosphate from ATP into 6-ETI to convert it into p6-ETI. (D) PEL cell lines BC3 and BC2 and DLBCL cell line IBL1 were treated with increasing concentrations of 6-ETI and phospho–6-ETI for 48 hours. Results plotted are the average of 4 independent experiments (mean ± SEM). Statistical analysis was performed using Mann-Whitney unpaired test (P = 0.0317, *P ≤ 0.05).