A Phthalimide Derivative That Inhibits Centrosomal Clustering Is Effective on Multiple Myeloma

Despite the introduction of newly developed drugs such as lenalidomide and bortezomib, patients with multiple myeloma are still difficult to treat and have a poor prognosis. In order to find novel drugs that are effective for multiple myeloma, we tested the antitumor activity of 29 phthalimide derivatives against several multiple myeloma cell lines. Among these derivatives, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3- dione (TC11) was found to be a potent inhibitor of tumor cell proliferation and an inducer of apoptosis via activation of caspase-3, 8 and 9. This compound also showed in vivo activity against multiple myeloma cell line KMS34 tumor xenografts in ICR/SCID mice. By means of mRNA display selection on a microfluidic chip, the target protein of TC11 was identified as nucleophosmin 1 (NPM). Binding of TC11 and NPM monomer was confirmed by surface plasmon resonance. Immunofluorescence and NPM knockdown studies in HeLa cells suggested that TC11 inhibits centrosomal clustering by inhibiting the centrosomal-regulatory function of NPM, thereby inducing multipolar mitotic cells, which undergo apoptosis. NPM may become a novel target for development of antitumor drugs active against multiple myeloma

A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing

TC11 inhibited both the maturation and function of bone marrow-derived osteoclasts.

<p>Mice bone marrow cells were cultured with M-CSF (10 ng/mL) for 3 days, then further cultured with M-CSF (10 ng/mL) and RANKL (10 ng/mL). After an additional 3–6 days of culture with the indicated concentration of TC11, thalidomide, bortezomib, or DMSO, the cells were fixed and stained for TRAP as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116135#sec002" target="_blank">Materials and Methods</a> section. TRAP-positive multinucleated cells containing more than three nuclei were considered TRAP⁺ multinuclear osteoclasts. (A) The number and size of TRAP⁺ multinuclear osteoclasts were decreased by treatment with 0.5–1µM of TC11. (B) The number of TRAP⁺ multinuclear osteoclasts in each well of a 96-well plate was counted. Bars: means±SD. *p<0.05, **p<0.01 (Student’s <i>t</i>-test). (C) RAW 264.7 cells were incubated for 5–8 days with RANKL (10 ng/mL) and seeded on multi-test slides. The indicated concentrations of TC11, thalidomide, bortezomib, or OPG were added every 2 days for 7 days, followed by Von Kossa staining. The resorption pits were observed by fluorescence microscopy and the number of the pits was quantified using Image J software. (B) Bone resorption is indicated by white spots (pit). Bone resorption was suppressed by treatment with 3–5 µM of TC11. (D) The pit area was measured by ImageJ software. The pit area of the DMSO-treated wells was used as the control ( = 1). Data shown are representative of three independent experiments. Bars: means±SD. *p<0.05, **p<0.01 (Student’s <i>t</i>-test)</p

NPM1 interacts with TC11 and lenalidomide.

<p>Recombinant NPM1 was expressed in <i>E. coli</i> and purified. The NPM1 obtained was immobilized onto the sensor chip NTA followed by an SPR analysis. Panels A and B show representative biosensorgrams of TC11 and lenalidomide, respectively.</p

<i>In vivo</i> growth inhibition of myeloma cells by intraperitoneal injections of TC11.

<p>A total of 3×10⁷ KMS34 or KMS11 cells were inoculated subcutaneously into ICR/SCID mice. When the tumors reached 50 mm³ (day 0), TC11 (20 mg/kg or 100 mg/kg) or vehicle was administered intraperitoneally twice every 3 days for 2 wks. (A) Growth of KMS34-derived subcutaneous plasmacytomas was examined (n = 7). (B) Sections of dissected KMS34-derived tumors were stained with hematoxylin and eosin. The arrows indicate nuclear fragmentation of TC11-treated cells. (C) The weights of KMS34-derived tumors dissected from the KMS34-innoculated mice were measured on day 14. Bars: means ±SD.**p<0.01 (Students <i>t</i>-test). (D) Growth of KMS11-derived subcutaneous plasmacytomas was examined. (E) Pathological examination of KMS11-derived tumors was shown. Immunohistochemical staining of the KMS11-derived tumors were also shown. (F) Pharmacokinetics of TC11. Plasma concentrations of TC11 in mice after a single injection of 20 mg/kg or 100 mg/kg of TC11 determined by HPLC.</p

TC11 altered the α-tubulin formation of myeloma cells.

<p>KMS34 was treated with 5µM TC11 for 4 hours and attached to a slide by cytospin. The sample was fixed and stained with antibody against α-tubulin followed by FITC-conjugated anti-mouse IgG (green). Nucleus was stained with DAPI (blue). A, Representative mitotic cells with or without TC11 treatment. B, The percentage of cells with fragmented α-tubulin in 2500 cells was calculated independently in 9 areas. **P<0.01 (Student <i>t</i>-test). A and B indicate representative data from 3 experiments.</p

TC11 induces apoptosis of multiple myeloma cells in a caspase-dependent manner.

<p>(A) HeLa or KMS34 cells were treated with 0–50 µM TC11 or thalidomide for 24 h. The whole cell lysates were analyzed by Western blotting with anti-PARP antibody. (B) KMS34 cells were treated with 0, 5 or 25 µM TC11 for 6 h. The whole cell lysates were analyzed by Western blotting with anti-caspase-3, 8 or 9 antibody, respectively. (C) KMS34 cells were treated with 0–50 µM TC11 or staurosporin A for 6 h. After DNA extraction, 1% agarose gel electrophoresis was performed. (D) KMS34 cells were treated with 50 µM thalidomide or TC11. After 96 h, cells were stained with FITC-coupled annexin V and propidium iodide, and induction of apoptosis was evaluated by flow cytometry.</p

TC11 inhibited the growth of MM cell lines.

<p>(A) The chemical structures of TC11, thalidomide, and lenalidomide. (B) The dose-dependent inhibition of MM cells by TC11. Cell lines KMS11, KMS26, KMS28, KMS34, MUM24 with del 17p13 and/or t(4;14) were cultured for 48 h with the indicated dose of TC11, followed by an assessment of cell viability using MTT assays. Mononuclear cells separated from bone marrow samples of three MM patients were also treated with TC11 and examined by MTT assay. (C) Synergistic effects of TC11 with dexamethasone were examined by MTT assay. Bars indicate means±SD. *p<0.05 (Student’s <i>t</i>-test). (D) The effect of TC11 on the number of hematopoietic progenitor cells was examined by colony assay. Mice bone marrow cells were plated in 1.2% methylcellulose medium containing 20 ng/mL mIL-3, 10 ng/mL mIL-6, 20 ng/mL mSCF and 1 U/mL hEPO in the presence or absence of TC11. The numbers of colony-forming cells were counted after 14 days. Bars indicate means±SD. *p<0.05, **p<0.01 (Student’s <i>t</i>-test).</p

NPM knockdown induces multipolar spindles.

<p>HeLa cells were transfected with siRNA for luciferase (control) or NPM. (A) After 48 h, the whole cell lysates were analyzed by western blotting with antibody against NPM (left) and the band intensities were quantified (right). (B) Immunofluorescence with anti-NPM (green) and anti-γ-tubulin (red) antibody was performed (left). White arrows indicate centrosomes. At least 50 mitotic cells were counted in three independent experiments. The ratio of cells with multipolar spindles under the indicated conditions was quantified (below). (C) Representative multinucleated cell under the indicated conditions. (D) After 72 h, caspase-9 activity was determined using luminescence-based assay. (E) At 48 h after siRNA transfection, the cells were treated with 0–2.5 µM TC11 and then incubated for a further 72 h. Cell viability was determined by means of WST-1 assay in three independent experiments. An asterisk denotes a statistically significant difference according to Student’s <i>t</i>-test (P<0.05). Bar; 10 µm.</p

Schematic representation of <i>in vitro</i> selection of TC11-binding protein using mRNA display.

<p>(A) The chemical structure of biotinylated TC11. (B) A cDNA library derived from KMS34 cells was transcribed, ligated with PEG-Puro spacer (1) and <i>in vitro</i> translated (2) to form a protein-mRNA conjugates library. The library is injected into micro fluidic chip on which TC11 is immobilized (3) and unbound molecules were washed away. The bound molecules were eluted and their mRNA portion is amplified by RT-PCR (4). The resulted DNA can be used for the next of round and analyzed by cloning and sequencing. (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038878#s4" target="_blank">Materials and Methods</a>.).</p