Drug Design for Overcoming High-Risk Myeloma and Identification of Novel Binding Proteins to Immune-Modulatory Drugs
Authors: Hozumi Masashi , Ichikawa Daiju , Matsushita Maiko , Kamiyama Eriko , Yanagawa Hiroshi , Tabata Noriko , Kitabatake Shotaro , Ueda Arimi , Yamaguchi Takashi , Sato Mariko , Hattori Yutaka 
Published: October 2019

Abstract
PURPOSE: Recent progress of the treatment of multiple myeloma (MM) has significantly improved prognosis. However, the MM patients with high-risk cytogenetic abnormalities still showed significantly shorter survival. The purpose of this study is to find compounds effective for high-risk myeloma with lower toxicity by developing chemical structure of the phthalimide. We also tried to isolate their binding molecules to understand molecular pharmacology of the compounds.
RESULTS: (1) Screening and optimization of phthalimide derivatives: We screened library of synthetic phthalimide derivatives by their ability to induce apoptosis of MM cell line, KMS34, which has high-risk cytogenetic abnormalities such as t(4;14) and TP53 gene deletion. We finally found TC11, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione, which showed the most potent tumor growth inhibition. Based on the results of screening of phthalimide library, we found the two important points of chemical structure: (i) A 4-amino group lacked growth inhibitory effect, and thus, 5-amino branch is necessary for strong anti-tumor activity. (ii) Modification of 2,6-diisopropylphenyl in phenyl ring of TC11 significantly decreased inhibitory effects of tumor cell growth.
(2) Anti-myeloma effects of phthalimide derivatives: TC11 showed significant growth inhibition of all myeloma cell lines (IC50=3-8μM) including those with high-risk cytogenetic changes. Lenalidomide also significantly inhibited growth of myeloma cell lines. However, 30μM of lenalidomide failed to inhibit growth of KMS34 and KMS28 cell lines that have high-risk cytogenetic alterations. TC11 also inhibited growth of bone marrow myeloma cells obtained from the MM patients.
(3) Anti-myeloma effects of TC11 in vivo: Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous plasmacytoma in human myeloma cell (KMS34 and KMS11)-bearing SCID xenografts. In the pharmacokinetic analyses, the Cmax was 2.1μM at 1 h after the injection of TC11, with 1.2 h as the half-life. Cmax was 18.1μM at 1.5hr (Tmax), and T1/2 was 4.5hr, when 100mg/kg of TC11 was injected. Cmax was 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr, if 20mg/kg was injected. In oral administration of 200mg/kg of TC11 in lcr mice, Cmax was 3.81μM at 8.0hr (Tmax), and T1/2 was 2.77hr.
(4) Toxicity of TC11 and lenalidomide: When 20mg/kg of TC11 was injected to SCID-mice for two weeks, weight loss compared with control mice was not observed. Clonological assay using mice bone marrow cells showed that 10μM TC11 did not suppress the colony formation while 5μM lenalidomide significantly decreased the colony out put. Therefore, TC11 in the concentrations that induced apoptosis of myeloma cells unlikely caused systemic and hematological toxicity.
(5) Binding molecules: To understand the molecular mechanism, we employed our unique in vitro screening system using mRNA display, in vitro virus (IVV) method. It was found that TC11 directly bound to nucleophosmin 1 (NPM1) and α-tubulin. Knock-down of NPM1 gene in myeloma cells significantly delayed tumor cell growth. However, cereblon (CRBN), which was considered to be associated with teratogenicity of thalidomide, was not included in TC11-binding molecules in IVV assay. TC11 lacked glutarimide moiety to which CRBN was reported to bind. Thus, TC11 revealed anti-myeloma activity in a CRBN-independent pathway and is conceptionally expected as a non-teratogenic thalidomide-related compound. Interestingly, lenalidomide also directly bound to NPM1. Further analyses were needed to elucidate biological significance of interaction of IMiDs with NPM1 and α-tubulin.
CONCLUSION: Our results suggested the possibility that phthalimide derivatives induce tumor cell death independent of CRBN pathway. It was also suggested that drug design and modification of chemical structure of phthalimide enable us to develop further new thalidomide derivatives which have more potent antitumor activity and less toxicity.

Disclosures
Ichikawa: Takeda Pharmaceutical Company: Research Funding. Matsushita:Takeda Pharmaceutical Company: Research Funding. Hattori:MSD company: Research Funding; Ono Pharmaceutical company: Research Funding; Takeda Pharmaceutical Company: Consultancy; Novo Nordisk company: Research Funding; Mitsubisi Tanabe Pharm: Research Funding; Cosmic Corporation: Research Funding.