産婦人科教室抄読会平成 26 年 12 月 8 日 NAME 高橋一広 TITLE TOPK 阻害薬：夢の抗腫瘍薬か？ BACKGROUND がん細胞の持つ特異的な性質を分子レベルで捉えて、それを標的に効率良く狙い撃ちする薬を「分子標的薬」という。その分子標的薬の新しい有力候補となる化合物を発見したという報告を、平成 26 年 10 月 22 日にシカゴ大学の中村祐輔教授の研究チームが Science Translational Research に発表した 1)。その分子標的が TOPK というタンパク質である。この TOPK は 2000 年に、ほぼ同時に米国と日本で発表された MAPKK 様の蛋白キナーゼであり 2)3)、癌細胞の増殖や移動に深く関与している。中村研究室のゲノム包括的遺伝子解析により、乳がん、肺がんなどを含めた多くのがんで TOPK 発現は高く、逆に正常な組織ではほとんど発現が見られないことがわかった 4)。そのため、彼らはこの TOPK に作用する薬剤が、多様ながんに適応すると考え、30 万種類の化合物の中から TOPK の働きを阻害するという新物質を探し出した(http://credo.asia/2014/10/27/cancer/)。 SUMMARY 1. 肺がん細胞株 (A549)移植により形成された腫瘍(200 mm3)に対し、TOPK 阻害薬 OTS514 を 2 週間連日静注すると投与量依存的に腫瘍増殖が抑制された。しかし、同時に WBC, RBC の減少および血小板の増加が認められた。より増殖能の高い肺がん細胞株 (LU-99)による腫瘍に対し、リポソーム化 OTS514 はパクリタキセルと同様に腫瘍増殖抑制効果を示した。OTS514 とパクリタキセルでみられた WBC 減少が、リポソーム化 OTS514 では認めなかった。(Fig. 2) 2. LU-99 腫瘍 (150 mm3)に対して OTS514 の誘導体である OTS964 も抗腫瘍効果を示した。リポソーム化 OTS964 を投与すると、6 匹中 5 匹のヌードマウスで腫瘍が完全消失した。また、リポソーム化 OTS964 では WBC 減少は認められなかった。(Fig. 4) 3.リポソーム化 OTS964 は、TOPK 、histone H3 のリン酸化を抑制し、Ki-67 陽性細胞を減少させ、cleaved caspase-3 陽性細胞を増加させた。(Fig. 5) 4.OTS964 を 2 週間経口投与すると、投与終了後も腫瘍退縮効果がみられ、投与後 2 週間で 6 匹すべてのヌードマウスで腫瘍が完全消失した。投与後にみられた WBC の減少は、 2 週後には自然回復した。(Fig. 6) REFERENCES 1. Sci Transl Med 2014;6:250 2. Proc Natl Acad Sci USA 2000;10:5167 3. J Biol Chem 2000;275:21525 4. Cancer Res 2006;66:9186 P1 図1 日本人の生涯がん罹患率 http://gansupport.jp/article/drug/drug06/3400.html 図2 抗がん剤と分子標的薬の作用概念 http://gansupport.jp/article/drug/drug06/3402.html 図3 分子標的薬の標的部位図4 日本で承認されている分子標的治療薬 http://www.cancertherapy.jp/targeted_therapy/2013_08/01.html P2 TOPK: T-lymphokine-activated killer cell-originated protein kinase (PDZ-binding kinase, PBK) ・TOPKはp38MAPKをリン酸化するMAPKKであり、in vitroにおいて細胞有糸分裂時に活性化される。 TOPKはhistone H3,PRC1 (protein regulator of cytokinesis), GPSM2 (G protein signaling modulator 2), p97など細胞分裂に重要な蛋白群をリン酸化する。・TOPKの活性化には、M期CDK1/サイクリンBキナーゼ複合体と他の未知のキナーゼによるリン酸化が必要である。・TOPKは増殖や分化といった細胞周期関連プロセスに関与していると考えられる。 MAPKK様の蛋白キナーゼであるTOPKは癌細胞の増殖に深く関与している乳がん細胞正常組織 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, colon; 16, peripheral blood leukocyte; 17, stomach; 18, thyroid; 19, spinal cord; 20, lymph node; 21, trachea; 22, adrenal gland; and 23, bone marrow. ACTB served as a loading control. Park JH, et al. Cancer Res 2006；66：9186 図5 TOPKは精巣、胸腺以外の正常組織では発現していない TOPK inhibitor induces complete tumor regression in xenograft models of human cancer through inhibition of cytokinesis P3 Yo Matsuo,1* Jae-Hyun Park,2* Takashi Miyamoto,1 Shinji Yamamoto,1 Shoji Hisada,1 Houda Alachkar,2 Yusuke Nakamura2† TOPK (T–lymphokine-activated killer cell–originated protein kinase) is highly and frequently transactivated in various cancer tissues, including lung and triple-negative breast cancers, and plays an indispensable role in the mitosis of cancer cells. We report the development of a potent TOPK inhibitor, OTS964 {(R)-9-(4-(1(dimethylamino)propan-2-yl)phenyl)-8-hydroxy-6-methylthieno[2,3-c]quinolin-4(5H)-one}, which inhibits TOPK kinase activity with high affinity and selectivity. Similar to the knockdown effect of TOPK small interfering RNAs (siRNAs), this inhibitor causes a cytokinesis defect and the subsequent apoptosis of cancer cells in vitro as well as in xenograft models of human lung cancer. Although administration of the free compound induced hematopoietic adverse reactions (leukocytopenia associated with thrombocytosis), the drug delivered in a liposomal formulation effectively caused complete regression of transplanted tumors without showing any adverse reactions in mice. Our results suggest that the inhibition of TOPK activity may be a viable therapeutic option for the treatment of various human cancers. 1OncoTherapy Science Inc., Kawasaki, Kanagawa 213-0012, Japan. 2Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. *These authors contributed equally to this work. †Corresponding author. E-mail: [email protected] 誘導体(メチル化) H3 C H3 C 大腸がん IC50 7.6-73.0 nM 膵臓がん前立腺がん肝臓がん胃がん胃がん大腸がんリンパ腫膀胱がん乳がん肺がん乳がん肺がん乳がん IC50 1.5-14.0 nM Fig. 1. In vitro antiproliferative activity of OTS514. (A) Chemical structure of OTS514. (B) Western blot shows expression of TOPK in cancer cell lines. (C) Graph indicates IC50 values of OTS514 in 13 TOPK-positive cancer cell lines and a TOPK-negative cancer cell line, HT29. *P = 5.92 × 10−11. Fig. 3. In vitro antiproliferative activity of OTS964. (A) Chemical structure of OTS964. (B) Graph indicates IC50 values of OTS964 in 13 TOPK-positive cancer cell lines and a TOPK-negative cancer cell line, HT29. *P = 1.45 × 10−4. 肺がん細胞株 A549 連日静注14日間 Xenograftモデル http://www.miracure.co.jp/sub5.html から引用肺がん細胞株 LU-99 OTS514 2日毎静注 7回パクリタキセル Day1,4,8,11 静注 Fig. 2. In vivo efficacy of OTS514 in lung cancer xenograft models. (A to C) Nude mice bearing A549 (n = 8) were intravenously treated with vehicle control or free OTS514 once every day for 2 weeks. Mean tumor volumes ± SD (A) and mean relative body weights ± SD (B) in comparison with the mean body weight just before the administration (day 1) are shown. Counting of WBCs, platelets, and RBCs was performed with Sysmex XT-1800iV Analyzer (C). (D) After treatment with OTS514, differentiation of HSCs to megakaryocytes was assessed by staining with CD41a. (E to G) Nude mice bearing LU-99 (n = 6) were intravenously treated with vehicle control, free OTS514, or liposomal OTS514 every 2 days for 2 weeks or paclitaxel at days 1, 4, 8, and 11. Mean tumor volumes ± SD (E) and mean relative body weights ± SD (F) in comparison with the mean body weight just before the administration (day 1) are shown. WBCs were counted with a cell counting chamber after administration of vehicle control, free OTS514 (5 mg/kg), or liposomal OTS514 (5 mg/kg) every 2 days for 2 weeks, or paclitaxel (24 mg/kg) at days 1, 4, 8, and 11 (G). *P < 0.05, **P <0.01, or ***P < 0.001 by t test. Original data and exact P values are provided in table S2. P4 P5 6匹中5匹のマウスで腫瘍完全消失肺がん細胞株 LU-99 Free OTS964 Day 1, 4, 8, 11, 15, 18 Liposomal OTS964 Day 1, 4, 8, 11, 15 パクリタキセル Day 1, 4, 8, 11, 15, 18 Fig. 4. In vivo efficacy of OTS964 in lung cancer xenograft models. (A to D) Nude mice bearing LU-99were intravenously treated with vehicle control, free OTS964, liposomal OTS964, or paclitaxel. The administration doses were 40 mg/kg for free OTS964 at days 1, 4, 8, 11, 15, and 18 (A and B), 40 mg/kg for liposomal OTS964 at days 1, 4, 8, 11, and 15 (C and D), and 24 mg/kg for paclitaxel at days 1, 4, 8, 11, 15, 18, and 22 (C and D). (A and C) Mean tumor volumes ± SD (n = 6 per treatment group) are shown. (B and D) Mean relative bodyweights ± SD(n = 6 per treatment group) in comparison with the mean body weight just before the administration (day 1) are shown. (E) Counting of WBCs was performed with a cell counting chamber after final administration of free OTS964 (40 mg/kg), liposomal OTS964 (40 mg/kg), or paclitaxel (24mg/kg). *P<0.05or ***P<0.001byt test. Original data and exact P values are provided in table S2. P6 Fig. 5. Suppression of cancer cell proliferation and increase in cancer cell death after treatment with liposomal OTS964. Immunohistochemical staining and quantification of cancer cells were performed on tumor sections from LU-99 xenografts (n = 3 per group) intravenously treated with liposomal OTS964 or vehicle control. (A) Tumor sections were stained with methyl green to clarify cell morphology. Scale bar, 100 mm. (B) LU-99 cells were photographed 48 hours after treatment with 10 nM OTS964 or dimethyl sulfoxide (DMSO). Scale bar, 100 mm. (C to F) Each dot indicates the percentage of 3,3′diaminobenzidine (DAB)– positive cells per field of interest (FOI) stained with antibodies against phospho-TOPK (p-TOPK) at Thr9 [30 FOIs per group, (C)], phosphohistone H3 at Ser10 [11 FOIs per group, (D)], Ki-67 [30 FOIs per group, (E)], and cleaved caspase-3 [15 FOIs per group, (F)]. Horizontal lines represent the means ± SD. Exact percentages of DAB-positive cells and P values are listed in table S2. 肺がん細胞株 LU-99 Free OTS964 経口投与 14日間パクリタキセル Day1,4,8,11 静注全て(6匹)のマウスで腫瘍完全消失 2週後自然回復 Fig. 6. Oral administration of OTS964 in lung cancer xenograft models. Nude mice bearing LU-99 were orally treated with vehicle control or free OTS964 or intravenously treated with paclitaxel. The free OTS964 was dosed at 50 and 100 mg/kg orally once every day for 2 weeks, whereas paclitaxel was given at 24mg/kg on days 1, 4, 8, and11. (A) Mean tumor volumes ± SD (n = 6 per treatment group) are shown. (B) Mean relative body weights ± SD (n = 6 per treatment group) in comparison with the mean body weight just before the administration (day 1) are shown. (C and D) Counting of WBCs was performed with a cell counting chamber on day 15 (C) and on day 29 (D), 2 weeks after the final administration of free OTS964 (100 mg/kg). **P <0.01 or ***P<0.001by t test. Original data and exact P values are provided in table S2.