Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors
Ashvinikumar V. GavaiClaude QuesnelleDerek NorrisWen-Ching HanPatrice GillWeifang ShanAaron BalogKe ChenAndrew J. TebbenRichard RampullaDauh‐Rurng WuYingru ZhangArvind MathurRonald J. WhiteAnne RoseHaiqing WangZheng YangAsoka RanasingheCelia D’ArienzoVictor R. GuarinoLan XiaoChing SuGerry EverlofVinod AroraDing Ren ShenMary Ellen CvijicKrista MenardMei-Li WenJere E. MeredithGeorge L. TrainorLouis J. LombardoRichard E. OlsonPhil S. BaranJohn T. HuntGregory D. ViteBruce S. FischerRichard A. WesthouseFrancis Y. Lee
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Abstract:
Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.Keywords:
Solid tumor
本研究においては,マウス精巣細胞へのクロラムフェニコールアセチルトランスフェラーゼ遺伝子のトランスフェクション効率をin vivoおよびin vitroで行ったリポフェクション法,エクレクトロポーレーション法ならびにパーティクルガン法にっいて比較した.併せて,in vivoエレクトロポーレーションによってマウスの生体精巣における大腸菌βガラクトシダーゼ遺伝子の三次元空間的な発現の獲得を試みた.4週齢のICR系統雄マウスを用いて全部で3回実験を行った.その結果,用いた3種類のトランスフェクション法のうちではin vivoエレクトロポーレーション法とin vivoパーティクルガン法が最も効率が良いことが判明した.in vivoエレクトロポーレーションを用いて大腸菌βガラクトシダーゼ遺伝子を導入したところ,マウス精巣において三次元空間的な明白な遺伝子発現がX-gal染色によって確認され,X-gal染色された細胞の中には精母細胞もしくは精子細胞に類似したものも含まれていた.最大導入可能DNA量,組織の損傷およびトランスフェクション効率を考慮に入れると, in vivoエレクトトポーレーションはマウスの生体精巣細胞への外来遺伝子導入に対して優れている方法であると結論された.
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The strength of Notch signaling contributes to pleiotropic actions of Notch; however, we do not yet have a full understanding of the molecular regulation of Notch-signaling strength. We have investigated the mode of Notch activation in binary fate specification in the
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皮膚血管は冷却刺激に応答して収縮し，体表面からの熱放散を制限する．この反応は，交感神経の興奮を介する全身性メカニズムと，局所的に皮膚血管の収縮反応性が増大する局所性メカニズムとが相乗的に機能して引き起こされる．局所性メカニズムの存在は，摘出血管を用いたin vitro実験によって証明されてきた．我々は，ラットやマウスを使ってin vivoで皮膚血流調節を解析する実験方法を確立した．皮膚循環は，様々な因子に起因する神経性の影響を強く受けるため，in vivoで皮膚血流を定量的に測定することは難しい．そこで，電位依存性Na+チャネル阻害薬であるテトロドトキシン（TTX）処置により神経伝導を遮断した条件下で皮膚血流を測定することを試みた．この条件下においても，ラットおよびマウスの後肢を局所冷却することにより，足底部の皮膚血流量が減少することを証明し，局所性メカニズムによる反応をin vivoで定量的に評価できることを示した．興味深いことに，マウスとラットでは，この反応の主要なメカニズムが異なっていた．すなわち，ラットでは，冷却刺激により血管あるいは周囲の細胞からATPが遊離され，これが交感神経終末のP2受容体に作用することでノルアドレナリン遊離を誘発し，平滑筋細胞の主にα1受容体の活性化を介して皮膚血管が収縮するのに対し，マウスでは，冷却刺激はRhoキナーゼの活性化を介して血管平滑筋のα2C受容体を介した収縮反応性を増大させることで皮膚血管を収縮させることが示唆された．In vivoでの解析は，レイノー病はもちろん，糖尿病やホルモン異常など末梢循環障害を来たす病態と皮膚循環調節との関係を解析する際に不可欠であり，この解析方法はこうした病態の治療薬や予防薬の開発にも役立つであろう．
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Notchシグナル伝達経路は胎生発生段階に機能をしている．遺伝子分析の結果，形態発生におけるNotchシグナルは重要な役割を果たしている事が判明した．最近の研究において，様々な悪性腫瘍においてこのNotch pathwayが活性化されているとの報告があるが，ヒトの骨腫瘍において活性化しているかどうかは不明である．我々は，Notchシグナル経路について，RT-PCR法，免疫染色法検討した．γ-secretase inhibitorをHOSに添加しMTT法にてcell viavilityを検討した．すべての検体にてNotch1, 2, Jagged1のmRNAの発現上昇を認めた．免疫染色法にてHOS及び臨床検体においてNotch-ICの核内局在を認めた．γ-secretase阻害薬はNotchシグナル経路を遮断し細胞増殖が有意に抑制されている事を確認した．本研究は，Notch経路が骨肉腫の新たな治療的ターゲットとなり得る事を示唆している．
Notch proteins
Cyclin-dependent kinase 8
Notch 1
Hes3 signaling axis
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The Delta-Notch pathway is a signal exchanger between adjacent cells to regulate numerous differentiation steps during embryonic development. Blood vessel formation by sprouting angiogenesis requires high expression of the Notch ligand DLL4 in the leading tip cell, while Notch receptors in the trailing stalk cells are activated by DLL4 to achieve strong Notch signaling activity. Upon ligand binding, Notch receptors are cleaved by ADAM proteases and gamma-secretase. This releases the intracellular Notch domain that acts as a transcription factor. There is evidence that also Notch ligands (DLL1, DLL4, JAG1, JAG2) are processed upon receptor binding to influence transcription in the ligand-expressing cell. Thus, the existence of bi-directional Delta-Notch signaling has been proposed. We report here that the Notch ligands DLL1 and JAG1 are processed in endothelial cells in a gamma-secretase-dependent manner and that the intracellular ligand domains accumulate in the cell nucleus. Overexpression of JAG1 intracellular domain (ICD) as well as DLL1-ICD, DLL4-ICD and NOTCH1-ICD inhibited endothelial proliferation. Whereas NOTCH1-ICD strongly repressed endothelial migration and sprouting angiogenesis, JAG1-ICD, DLL1-ICD and DLL4-ICD had no significant effects. Consistently, global gene expression patterns were only marginally affected by the processed Notch ligands. In addition to its effects as a transcription factor, NOTCH1-ICD promotes cell adhesion to the extracellular matrix in a transcription-independent manner. However, JAG1-ICD, DLL1-ICD and DLL4-ICD did not influence endothelial cell adhesion. In summary, reverse signaling of Notch ligands appears to be dispensable for angiogenesis in cellular systems.
JAG1
Notch proteins
Hes3 signaling axis
Cyclin-dependent kinase 8
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NOTCH signaling induced by Delta1 (DLL1) and Jagged1 (JAG1) NOTCH ligands is modulated by the β3N-acetylglucosaminyl transferase Fringe. LFNG (Lunatic Fringe) and MFNG (Manic Fringe) transfer N-acetylglucosamine (GlcNAc) to O-fucose attached to EGF-like repeats of NOTCH receptors. In co-culture NOTCH signaling assays, LFNG generally enhances DLL1-induced, but inhibits JAG1-induced, NOTCH signaling. In mutant Chinese hamster ovary (CHO) cells that do not add galactose (Gal) to the GlcNAc transferred by Fringe, JAG1-induced NOTCH signaling is not inhibited by LFNG or MFNG. In mouse embryos lacking B4galt1, NOTCH signaling is subtly reduced during somitogenesis. Here we show that DLL1-induced NOTCH signaling in CHO cells was enhanced by LFNG, but this did not occur in either Lec8 or Lec20 CHO mutants lacking Gal on O-fucose glycans. Lec20 mutants corrected with a B4galt1 cDNA became responsive to LFNG. By contrast, MFNG promoted DLL1-induced NOTCH signaling better in the absence of Gal than in its presence. This effect was reversed in Lec8 cells corrected by expression of a UDP-Gal transporter cDNA. The MFNG effect was abolished by a DDD to DDA mutation that inactivates MFNG GlcNAc transferase activity. The binding of soluble NOTCH ligands and NOTCH1/EGF1–36 generally reflected changes in NOTCH signaling caused by LFNG and MFNG. Therefore, the presence of Gal on O-fucose glycans differentially affects DLL1-induced NOTCH signaling modulated by LFNG versus MFNG. Gal enhances the effect of LFNG but inhibits the effect of MFNG on DLL1-induced NOTCH signaling, with functional consequences for regulating the strength of NOTCH signaling. NOTCH signaling induced by Delta1 (DLL1) and Jagged1 (JAG1) NOTCH ligands is modulated by the β3N-acetylglucosaminyl transferase Fringe. LFNG (Lunatic Fringe) and MFNG (Manic Fringe) transfer N-acetylglucosamine (GlcNAc) to O-fucose attached to EGF-like repeats of NOTCH receptors. In co-culture NOTCH signaling assays, LFNG generally enhances DLL1-induced, but inhibits JAG1-induced, NOTCH signaling. In mutant Chinese hamster ovary (CHO) cells that do not add galactose (Gal) to the GlcNAc transferred by Fringe, JAG1-induced NOTCH signaling is not inhibited by LFNG or MFNG. In mouse embryos lacking B4galt1, NOTCH signaling is subtly reduced during somitogenesis. Here we show that DLL1-induced NOTCH signaling in CHO cells was enhanced by LFNG, but this did not occur in either Lec8 or Lec20 CHO mutants lacking Gal on O-fucose glycans. Lec20 mutants corrected with a B4galt1 cDNA became responsive to LFNG. By contrast, MFNG promoted DLL1-induced NOTCH signaling better in the absence of Gal than in its presence. This effect was reversed in Lec8 cells corrected by expression of a UDP-Gal transporter cDNA. The MFNG effect was abolished by a DDD to DDA mutation that inactivates MFNG GlcNAc transferase activity. The binding of soluble NOTCH ligands and NOTCH1/EGF1–36 generally reflected changes in NOTCH signaling caused by LFNG and MFNG. Therefore, the presence of Gal on O-fucose glycans differentially affects DLL1-induced NOTCH signaling modulated by LFNG versus MFNG. Gal enhances the effect of LFNG but inhibits the effect of MFNG on DLL1-induced NOTCH signaling, with functional consequences for regulating the strength of NOTCH signaling.
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Somitogenesis
Notch proteins
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Notch proteins
Hes3 signaling axis
Cell fate determination
Cell Signaling
Cyclin-dependent kinase 8
Lateral inhibition
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3 cases of leukemia after solid tumor and 2 cases of solid tumors after leukemia were reported. The features of leukemia after solid tumor and the relationship between the solid tumor and leukemia, as well as the possible pathogenetic factors of the secondary malignancies were discussed.
Solid tumor
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