PO.ET02.10 · 实验与分子治疗
肿瘤电场治疗与多选择性RAS(ON)抑制剂RMC-6236在胰腺导管腺癌中的协同抗肿瘤活性
Synergistic antitumor activity of tumor-treating fields and multiselect-RAS (ON) inhibitor RMC-6236 in pancreatic ductal adenocarcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
RMC-6236(daraxonrasib)是一种新型多选择性RAS(ON)抑制剂，正在针对胰腺导管腺癌(PDAC)和其他RAS突变癌症进行研究。该化合物与RMC-7977具有结构相似性，并在活性RAS(RAS(ON))蛋白与亲环蛋白A(CypA)形成的三元复合物内表现出保守的结合位点和相互作用模式。肿瘤电场治疗(TTFields)是一种低强度交变电场，可破坏有丝分裂，并已在实体瘤中显示出临床获益。PANOVA-3试验表明，TTFields与吉西他滨(gemcitabine)加白蛋白结合型紫杉醇(nab-paclitaxel)联用可改善局部晚期PDAC患者的生存、疼痛控制和生活质量。我们此前的工作表明，TTFields可消除致癌性KRAS通路。因此我们假设该联合有可能增强疗效。本研究中，转移性PDAC细胞系AsPC-1接受了TTFields(150 kHz，1.8 V/cm)、RMC-6236(15 nM；亚IC₅₀)或两者联合的治疗。基于MTS实验结果，选择了亚IC₅₀浓度(15 nM)的RMC-6236，后续实验均在此剂量下进行。MTS实验显示，TTFields和RMC-6236均可独立降低AsPC-1细胞活力，且72小时相比48小时降低更为明显。与对照(48 h为100.0±8.4%；72 h为100.0±8.2%)相比，RMC-6236单药在48小时(85.82±13.9%)或72小时(90.48±15.4%)时效应甚微。TTFields治疗在48小时显著将活力降至74.42±21.4%，在72小时降至50.17±26.5%。联合治疗(TTFields+RMC-6236)进一步将活力在48小时降至63.8±25.2%、在72小时降至40.93±18.1%，相对于单药治疗表现出协同细胞毒性。进行了克隆形成实验以评估RMC-6236、TTFields及其联合治疗后的细胞存活分数。采用划痕实验评估细胞迁移，结果显示未处理细胞的迁移率为91.49±0.85%，RMC-6236处理的细胞为89.48±1.3%，TTFields单药将迁移降至61.33±16.3%，而联合治疗显著将迁移抑制至24.6±22.13%。所有情况下均将72 h细胞迁移率与0 h迁移率进行比较。这些发现表明，TTFields单药可显著削弱细胞运动能力，而其与RMC-6236联用则产生深远的抗迁移效应，大幅抑制伤口闭合。此外，进行了蛋白质印迹分析以评估关键PDAC生物标志物KRAS蛋白表达的变化。总体而言，我们的结果表明，TTFields与RMC-6236联用在亚IC₅₀浓度下即可显著抑制PDAC细胞的生长、迁移和存活，提示这是一种有前景的PDAC治疗替代策略。
查看英文原文 English abstract
RMC-6236 (daraxonrasib) is a novel multiselect RAS(ON) inhibitor under investigation for pancreatic ductal adenocarcinoma (PDAC) and other RAS-mutant cancers. This compound shares structural similarities with RMC-7977 and exhibits a conserved binding site and interaction pattern within the tri-complex formed between active RAS (RAS(ON)) proteins and cyclophilin A (CypA). Tumor Treating Fields (TTFields) are low-intensity, alternating electric fields that disrupt mitosis and have shown clinical benefit in solid tumors. The PANOVA-3 trial demonstrated that TTFields combined with gemcitabine plus nab-paclitaxel improved survival, pain control, and quality of life in locally advanced PDAC. Our previous work demonstrated that TTFields abrogate oncogenic KRAS pathway. We thus hypothesize a potential for increase in effectievness with the combination. In this study, the metastatic PDAC cell line AsPC-1 was treated with TTFields (150 kHz, 1.8 V/cm), RMC-6236 (15 nM; sub-IC₅₀), or combination. A sub-IC₅₀ concentration (15 nM) of RMC-6236 was selected based on MTS assay results, and subsequent experiments were performed at this dose. The MTS assay revealed that both TTFields and RMC-6236 independently reduced AsPC-1 cell viability, with a more pronounced reduction observed at 72 hours compared to 48 hours. RMC-6236 alone produced minimal effects at either 48 hours (85.82 ± 13.9%) or 72 hours (90.48 ± 15.4%) compared with control (100.0 ± 8.4%, 48 h; 100.0 ± 8.2%, 72 h). TTFields treatment significantly reduced viability to 74.42 ± 21.4% at 48 hours and 50.17 ± 26.5% at 72 hours. The combination treatment (TTFields + RMC-6236) further decreased viability to 63.8 ± 25.2% at 48 hours and 40.93 ± 18.1% at 72 hours, demonstrating synergistic cytotoxicity relative to monotherapy. Clonogenic assays were performed to evaluate cell survival fractions following treatment with RMC-6236, TTFields, and their combination. Cell migration was assessed using scratch assays, revealing that untreated cells exhibited a migration rate of 91.49 ± 0.85%, RMC-6236-treated cells showed 89.48 ± 1.3%, TTFields alone reduced migration to 61.33 ± 16.3%, and combination treatment markedly suppressed migration to 24.6 ± 22.13% . For every case we were compared with 72h cell migration rate with 0 h migration rate. These findings indicate that TTFields alone significantly impair cellular motility, while their combination with RMC-6236 results in a profound anti-migratory effect, substantially inhibiting wound closure. Additionally, western blot analysis was performed to assess changes in KRAS protein expression, a key PDAC biomarker. Overall, our results demonstrate that the combination of TTFields and RMC-6236 significantly inhibits PDAC cell growth, migration, and survival at sub-IC₅₀ concentrations, suggesting a promising alternative therapeutic approach for PDAC management.
利益披露 Disclosure
I. Saha, None..
D. Mukhopadhyay, None.