PO.ET01.06 · 实验与分子治疗

多靶点激酶抑制剂LCI139通过利用内源性和外源性凋亡途径克服患者来源非小细胞肺癌类器官的化疗耐药

Multitargeted kinase inhibitor LCI139 overcomes chemotherapy resistance in patient-derived non small cell lung cancer organoids by harnessing intrinsic and extrinsic apoptosis

编号 3171 展板 6 时间 4/20 02:00–05:00 区域 Section 19 主讲 Anna Ivanina Foureau, PhD

分会场 Targeting Cell Surface Vulnerabilities to Overcome Therapeutic Resistance

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作者与单位 Authors & Affiliations

Anna Ivanina Foureau¹, Nadeem Wajih², Cody C. McHale³, Hailey L. Dryden⁴, Sara Muhiczukic², Dhananjaya Pal¹, Bharath Yada¹, David Foureau¹, Fei Guo¹, Raj Dhupar⁵, Donald Durden¹, Konstantinos Votanopoulos⁴, Shay Soker⁶, Eleftherios Markis², Kathryn Mileham³

¹Atrium Health, Charlotte, NC,²Wake Forest Organoid Research Center, Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC,³Levine Cancer Institute, Charlotte, NC,⁴Wake Forest University, Winston-Salem, NC,⁵Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC,⁶Wake Forest Organoid Research Center, Wake Forest Institute of Regenerative Medicine,, Wake Forest School of Medicine, Winston Salem, NC

摘要 Abstract

中文摘要

引言：计算机辅助设计的多靶点抑制剂LCI139（PI3K-CDK4/6-CDK9-AURKA/B）经工程改造，通过同时靶向肿瘤代谢并诱导强效凋亡，克服单靶点药物固有的耐药性。该化合物在体外对NSCLC表现出纳摩尔级效力。我们评估了其在NSCLC细胞系和患者来源肿瘤类器官（PTO）中的疗效——PTO为生物制备的球形结构，重现了天然肿瘤组织的细胞基质。方法：在IRB批准的方案下，使用NSCLC细胞系（NCI-H1703、NCI-H1781）和由PEPGEL基质中未分选肺肿瘤悬液生成的PTO（n=7）。治疗包括LCI139（0.25-1μM）、化疗药物（卡铂、吉西他滨、培美曲塞、顺铂）以及单药抑制剂：PI3K（Buparlisib）、CDK4/6（Ribociclib）、CDK9（AZD4573）、AURKA（Barasertib）、AURKB（Alisertib）。细胞系与以下抑制剂共同处理：（carbenoxolone）、Caspase（Casp）-8（Z-IETD-FMK）、Casp-9（Z-LEHD-FMK），或AMPK激活剂（PF-06409577）。测定活力（碘化丙啶、CellTiter-Glo）、细胞周期（DNA染料）和凋亡（Annexin-V/7-AAD，Casp-3/7/8/9活性）。通过免疫印迹检测pAkt、pRb、pRNA Pol II、MCL-1、pAMPK、pFOXO3和p53，评估靶点抑制。结果：LCI139对H1703细胞（48小时）实现纳摩尔级IC50，并表现出剂量依赖性的PTO细胞毒性：0.25μM时降低49%，0.5μM时降低62%，1μM时降低78.4%（均p<0.0001）。相比之下，1μM单药抑制剂降低PTO活力如下：AURKA（34%）、AURKB（24%）、PI3K（29%）、CDK9（50.9%）均p<0.0001；CDK4/6（6.9%，p=0.43）。LCI139克服了显著的卡铂耐药（IC50：10.25-11.69μM），并优于标准化疗药物：吉西他滨（29.2%）、顺铂（28.2%）、培美曲塞（无活性）。LCI139在细胞和PTO中激活caspase 8、9、3/7。选择性抑制Casp-8或Casp-9减弱但未消除细胞毒性。Western印迹证实剂量依赖性的靶点抑制：pAKT、pRb、MCL-1、pRNAPol II降低及p53调节。LCI139通过pAMPK-pFoxO3激活诱导代谢应激。FoxO3抑制减弱了LCI139对敏感H1703细胞的效力。AMPK激活增强了对p53正常的H1703的效力，并使p53缺陷的H1781细胞敏感。结论：LCI139通过双重激活内源性（细胞周期阻滞诱导）和外源性（代谢应激诱导）凋亡途径，在耐药NSCLC中表现出优于标准化疗药物的抗肿瘤活性。在细胞系和PTO中的疗效验证了这一微生理平台在药物开发中的价值，支持多靶点激酶抑制在克服治疗耐药方面的转化潜力，与FDA指南一致。

查看英文原文 English abstract

Introduction: In-silico designed multitarget inhibitor LCI139 (PI3K-CDK4/6-CDK9-AURKA/B) was engineered to overcome resistance inherent to single-target agents by simultaneously targeting tumor metabolism and inducing robust apoptosis. This compound demonstrated nanomolar potency against NSCLC in vitro. We evaluated efficacy in NSCLC cell lines and patient-derived tumor organoids (PTOs)-biofabricated spherical constructs recapitulating cellular matrix of native tumor tissue. Methods: NSCLC cell lines (NCI-H1703, NCI-H1781) and PTOs generated from unsorted lung tumor suspensions in PEPGEL matrix (n=7) were utilized under IRB-approved protocol. Treatments included LCI139 (0.25-1μM), chemotherapeutics (carboplatin, gemcitabine, pemetrexed, cisplatin), and single-agent inhibitors: PI3K (Buparlisib), CDK4/6 (Ribociclib), CDK9 (AZD4573), AURKA (Barasertib), AURKB (Alisertib). Cell lines were co-treated with inhibitors of (carbenoxolone), Caspase (Casp)-8 (Z-IETD-FMK), Casp-9 (Z-LEHD-FMK), or AMPK activator (PF-06409577). Viability (propidium iodide, CellTiter-Glo), cell cycle (DNA dye) and apoptosis (Annexin-V/7-AAD, Casp-3/7/8/9 activity) were measured. Target inhibition was evaluated immunoblotting for pAkt, pRb, pRNA Pol II, MCL-1, pAMPK, pFOXO3, and p53. Results: LCI139 achieved nanomolar IC 50 against H1703 cells (48hr) and demonstrated dose-dependent PTO cytotoxicity: 49% reduction at 0.25μM, 62% at 0.5μM, 78.4% at 1μM (all p<0.0001). By comparison, 1 μM single-agent inhibitors reduced PTO viability by: AURKA (34%), AURKB (24%), PI3K (29%), CDK9 (50.9%) all p<0.0001; CDK4/6 (6.9%, p=0.43). LCI139 overcame significant carboplatin resistance (IC 50 : 10.25-11.69μM) and outperformed standard chemotherapeutics: gemcitabine (29.2%), cisplatin (28.2%), pemetrexed (no activity). LCI139 activated caspases 8, 9, 3/7 in cells and PTOs. Selective Casp-8 or Casp-9 inhibition diminished but didn't abrogate cytotoxicity. Western blotting confirmed dose-dependent target inhibition: decreased pAKT, pRb, MCL-1, pRNAPol II, and p53 modulation. LCI139 induced metabolic stress via pAMPK-pFoxO3 activation. FoxO3 inhibition diminished LCI139 potency against sensitive H1703 cells. AMPK activation enhanced potency in p53-proficient H1703 and sensitized p53-deficient H1781 cells. Conclusions: LCI139 demonstrates superior anti-tumor activity versus standard chemotherapeutics in resistant NSCLC through dual activation of intrinsic (cell cycle arrest-induced) and extrinsic (metabolic stress-induced) apoptotic pathways. Efficacy in both cell lines and PTOs validates this micro-physiological platform for drug development, supporting translational potential of multitargeted kinase inhibition for overcoming therapeutic resistance, as aligned with FDA guidance.

利益披露 Disclosure

A. Ivanina Foureau, None.. N. Wajih, None.. S. Muhiczukic, None.. R. Dhupar, None.. S. Soker, None.. E. Markis, None.

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