Elucidation of the IMiDs-Resistant Mechanism and Development of the Overcoming Drugs Inducing CRBN Independent G2/M Cell Cycle Arrest in Myeloma Cells
Authors: Uozaki Ryo , Aida Shuji , Yamagughi Takasi , Yamamoto Tomofumi , Kashiwazaki Sho , Yanagawa Hiroshi , Ichikawa Daiju , Matsushita Maiko , Hattori Yutaka 
Published: October 2019

Abstract
[Introduction]
Recent development of novel drugs significantly improved prognosis of Multiple Myeloma (MM). Immunomodulatory drugs (IMiDs) and proteasome inhibitors play central roles in MM therapy. However expanded usage of lenalidomide (Len) has increased the number of Len-resistant patients. And limited information is obtainable with regand to Len-resistant mechanism, such as overexpression and genetic mutation of IMiDs-binding protein, celeblon (CRBN); thus, elucidating the Len-resistant mechanism and development of drugs overcoming the Len-resistance are very important for improving the outcome of MM. The purposes of this study are [1] to clarify the molecular mechanism of Len-resistance using Len-resistant MM cell lines [2] to confirm the mechanism of CRBN independent myeloma cell death by novel phthalimide-derivatives, TC11 and PEG(E)-TC11.
[Method]
[1] In our laboratory, Len-resistant cell lines, KMS21R, KMS27R and MUM24R have been established by long-term co-culture with low-dose Len. Using these cell lines, we examined expression of CRBN and the downstream molecules, IKZF1/3, IRF4 and c-MYC by western blotting. We also examined the mutation of CRBN in KMS27R. [2] We have originally developed a novel phthalimide-derivative, TC11 and PEG(E)-TC11 synthesized for improving water solubility. We examined whether TC11 and PEG(E)-TC11 induced cell death to Len-resistance MM or not.
[Result]
[1] First, we validated expression of CRBN and the down-stream molecules, which mediate pharmacological action of Len. Decreased expression of CRBN and subsequent up-regulation of down-stream IKZF1 were confirmed in KMS21R cell. In KMS27R cell, IKZF 1/3 expressions are increased without alteration of CRBN expression level. Thus, genetic mutation in CRBN or IKZF1 is suspected in KMS27R cells. In MUM24R cell, no significant change in the expression levels of the CRBN pathway molecules was confirmed, suggesting other molecular alternation than CRBN pathway.
[2] TC11 significantly induced apoptosis of Len-resistant cells. We have previously reported that TC11 didn't bind to CRBN and TC11 directly bound to nucleophosmin1 (NPM1) and α-tubulin. It was found that TC11 induced G2/M arrest and subsequent apoptosis by inhibition of tubulin polymerization and NPM1 oligomerization. Fluorescence microscopy observation showed that TC11 treatment induced hyper duplication of centrosomes in MM cells. Water solubility and blood absorption of PEG(E)-TC11 were significantly improved compared with those of TC11. As a consequence, PEG-(E)TC11 significantly delayed tumor growth in xenograft model mice.
[Discussion & Conclusion]
[1] Our present data suggested diversity of Len-resistant mechanism in MM patients. For example, in KMS21R, decreased expression of CRBN was likely the cause of Len-resistance. In KMS27R, genetic mutation in CRBN-IKZF1 pathway caused inhibition of IKZF1 degradation. In MUM24R, the Len-resistant mechanism didn't relate to the CRBN pathway but to unknown molecular mechanism. Len-resistant cell lines are useful tools for studying Len-resistant mechanisms and developing drugs overcoming Len-resistance.
[2]TC11 abrogated tubulin polymerization and NPM1 oligomerization, induced centrosome disruption and G2/M arrest. Since G2/M check point doesn't closely rely on p53. TC11 was able to induce apoptosis of MM cells with high-risk cytogenetic mutations such as deletion of TP53 gene. TC11 and PEG-(E)TC11 are expected as a candidate compound overcoming Len-resistance and high-risk MM.

Disclosures
Matsushita: Amgen: Research Funding. Hattori:Takeda: Research Funding; IDAC inc.: Research Funding.