A Novel Phthalimide-Derivative, TC11, Induced G2/M Arrest and Apoptosis of High-Risk Myeloma Cells By Interacting with Nucleophosmin1 and α-Tubulin, but Not By Binding to Celeblon

Abstract Background and Purpose: Emergence of novel drugs including immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide and pomalidomide have markedly improved the survival of multiple myeloma (MM) patients. However, IMiDs have substantial limitation in the treatment of MM; little effect on MM patients with high-risk cytogenetic changes and their teratogenicity. Recently it was reported that IMiDs showed anti-tumor activity as well as teratogenicity by directly binding to cereblon (CRBN). We have previously reported that a novel phthalimide-derivative, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), induced apoptosis of high-risk MM cells in vivo and also showed anti-osteoclastogenic activity. At the same time, we observed abnormal α-tubulin fragmentation in TC11-treated MM cell (Matsushita et. al. PLoS One 2015). We have also demonstrated that TC11 bound to nucleophosmin 1(NPM1) and α-tubulin using in vitro virus (IVV) method (Shiheido et. al. PLoS One 2012). Interestingly, lenalidomide also bound to NPM1. The purpose of this study is to clarify molecular mechanisms of anti-myeloma effects of TC11. Results and Discussion: (1) Effect of TC11 is CRBN-independent: Recently it was reported that lenalidomide bound to CRBN, resulting in degradation of its substrates, IKZF1/3 and CK1α. In this study, we found that lenalidomide caused degradation of IKZF1/3 and CK1α in the high-risk MM cell line, KMS34. However, TC11 didn't down-regulate CRBN or its substrates, IKZF1/3 and CK1α. Taken together with the result of Biacore assay, TC11 induced MM cell death via CRBN-independent pathway. (2) Cell cycle arrest by TC11 vs. lenalidomide: Then we tried to elucidate molecular mechanisms of TC11 by the microarray analysis of KMS21, which is highly sensitive to TC11 and lenalidomide. Microarray Gene Set Enrichment Analysis (GSEA) indicated that TC11 upregulated the gene set of G2/M checkpoint, however lenalidomide did not. These results were also confirmed by analyzing the DNA contents of MM cells using flow cytometry and by western blot analysis of cell cycle regulators, phosphorylated cdc2 and p21. These data suggest that TC11 induced apoptosis of MM cells by distinct molecular mechanism from lenalidomide probably by regulating G2/M cell cycle. TP53 gene regulates G1/S cell cycle and occasionally deleted in high-risk MM cells. However, TC11 is able to induce apoptosis of TP53 -deleted high-risk MM cells by causing G2/M arrest. (3) Inhibition of tubulin assembly by TC11: Since we have previously showed that TC11 bound to α-tubulin by IVV method, we speculated that TC11 affected tubulin polymerization or depolymerization. Treatment of MM cells with TC11 significantly reduced polymerized fraction of α-tubulin, indicating TC11 inhibited tubulin polymerization. In contrast, lenalidomide did not inhibit either tubulin polymerization or depolymerization. In this regard, the mechanism of TC11 is different from that of lenalidomide. (4) Inhibition of oligomerization of NPM1 by TC11: We have also showed that TC11 bound to NPM1 by IVV method (Shiheido et. al. PLoS One 2012). It has been reported that oligomerized NPM1 had pleiotropic biological functions. Thus we examined whether TC11 affected NPM1 oligomerization using blue native polyacrylamide gel electrophoresis. As a result, TC11 treatment significantly reduced the amount of oligomerized NPM1 fraction. At the same time, we found that TC11 significantly increased in phosphorylation of NPM1 at Ser4, Thr95 and Thr199. It has been reported that phosphorylation of NPM1 inhibited its oligomerization (Mitrea et. al. Proc Natl Acad Sci U S A 2014). Thus, we speculated that TC11-induced phosphorylation resulted in inhibition of oligomerization of NPM1. Conclusion: It was shown that TC11 bound to α-tubulin and NPM1, but not to CRBN. TC11 inhibited tubulin assembly as well as NPM1 oligomerization, leading to G2/M arrest and mitotic catastrophe. These novel molecular events by TC11 induced apoptosis of TP53 -deleted high-risk MM cells. Download : Download high-res image (41KB) Download : Download full-size image Figure . Disclosures Yonemura: IDAC Theranostics Inc.: Employment. Tabata: IDAC Theranostics Inc.: Employment. Yanagawa: IDAC Theranostics Inc.: Employment. Hattori: Cosmic Corporation Co., Ltd.: Research Funding; Merck Sharp & Dohme: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding.