Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical antimyeloma activity
Rodger E. Tiedemann, Xinliang Mao, Chang-Xin Shi, Yuan Xiao Zhu, Stephen E. Palmer, Michael Sebag, Ron Marler, Marta Chesi, Rafael Fonseca, P. Leif Bergsagel, Aaron D. Schimmer, A. Keith Stewart
Rodger E. Tiedemann, Xinliang Mao, Chang-Xin Shi, Yuan Xiao Zhu, Stephen E. Palmer, Michael Sebag, Ron Marler, Marta Chesi, Rafael Fonseca, P. Leif Bergsagel, Aaron D. Schimmer, A. Keith Stewart
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Research Article Oncology

Identification of kinetin riboside as a repressor of CCND1 and CCND2 with preclinical antimyeloma activity

Abstract

Knockout and transgenic studies in mice demonstrate that normal somatic tissues redundantly express 3 cyclin D proteins, whereas tumor cells seem dependent on a single overexpressed cyclin D. Thus, selective suppression of the individual cyclin D deregulated in a tumor represents a biologically valid approach to targeted cancer therapy. In multiple myeloma, overexpression of 1 of the cyclin D proteins is a ubiquitous feature, unifying at least 7 different initiating genetic events. We demonstrate here that RNAi of genes encoding cyclin D1 and cyclin D2 (CCND1 and CCND2, respectively) inhibits proliferation and is progressively cytotoxic in human myeloma cells. By screening a chemical library using a cell-based assay for inhibition of CCND2 trans-activation, we identified the plant cytokinin kinetin riboside as an inhibitor of CCND2 trans-activation. Kinetin riboside induced marked suppression of CCND2 transcription and rapidly suppressed cyclin D1 and D2 protein expression in primary myeloma cells and tumor lines, causing cell-cycle arrest, tumor cell–selective apoptosis, and inhibition of myeloma growth in xenografted mice. Mechanistically, kinetin riboside upregulated expression of transcription repressor isoforms of cAMP-response element modulator (CREM) and blocked both trans-activation of CCND2 by various myeloma oncogenes and cis-activation of translocated CCND1, suggesting induction of an overriding repressor activity that blocks multiple oncogenic pathways targeting cyclin D genes. These data support targeted repression of cyclin D genes as a therapeutic strategy for human malignancies.

Authors

Rodger E. Tiedemann, Xinliang Mao, Chang-Xin Shi, Yuan Xiao Zhu, Stephen E. Palmer, Michael Sebag, Ron Marler, Marta Chesi, Rafael Fonseca, P. Leif Bergsagel, Aaron D. Schimmer, A. Keith Stewart

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Figure 1

Lentiviral shRNA suppression of cyclin D1 and D2 induces cell-cycle arrest and delayed apoptosis in H929 tumor cells.

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Lentiviral shRNA suppression of cyclin D1 and D2 induces cell-cycle arre...
(A) Immunoblot analysis of cyclin D1, D2, and D3 expression in H929 myeloma cells 65 hours after infection with lentiviruses expressing cyclin D1 or cyclin D2 shRNA, NT control shRNA, or GFP cDNA. α-Tubulin levels are shown as loading controls. (B) Cell-cycle profiles of H929 cultures 4 days after lentivirus infection, comparing the effects of suppression of cyclin D1 or D2 with control NT lentivirus infection and with lentivirus-induced suppression of both cyclin D1 and cyclin D2. To ensure equivalent exposure to lentivirus and shRNA, cells infected with both D1 lentivirus and D2 lentivirus (D1 + D2) are compared with cells infected with D1 lentivirus and NT lentivirus (D1 + NT), D2 lentivirus and NT lentivirus (D2 + NT), or 2 aliquots of NT lentivirus (NT + NT). (C) H929 apoptosis was examined serially by flow cytometry (in this example on day 11) after cyclin D suppression. Viable cells (left lower quadrants) are distinguished from early apoptotic (annexin V–positive) or late apoptotic (propidium iodide–positive) cells. (D) Time course of H929 viability after infection with D1 + NT, D2 + NT, D1 + D2, or control NT + NT lentiviruses. Viability was determined by annexin V and propidium iodide staining, as illustrated in C, and is normalized to uninfected H929; 5% error in viability is shown.