PO.ET09.05 · 实验与分子治疗
MTORC1/2抑制增强STX-478（一种PIK3CA H1047R突变体抑制剂）在PIK3CA H1047R突变型结直肠癌中的作用
MTORC1/2 inhibition enhances STX-478, a PIK3CA H1047R mutant inhibitor, in PIK3CA H1047R mutant colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：STX-478是一种PIK3CA突变体选择性抑制剂，我们此前已证明其在治疗PIK3CA H1047R突变型结直肠癌（CRC）模型中具有前景。我们进行了高通量药物筛选，发现AZD2014（一种MTORC1/2抑制剂）与STX-478联合具有增强的应答。在此，我们研究PI3K抑制与MTORC1/2抑制在PIK3CA H1047R突变型结直肠癌（CRC）中的联合作用。
方法：用不同浓度的STX-478（250-750 nM）和AZD2014（20-250 nM）处理CRC细胞系SW48和SW48PK（Horizon Discovery公司；SW48携带PIK3CA H1047R突变），并通过WST检测细胞活力。对PIK3CA H1047R突变型PDCO进行处理、成像，并通过测量类器官最长直径随时间的变化来确定应答。使用Glass's Delta作为统计检验来测量不同治疗组的效应量。小鼠来源癌症类器官（MDCO）来源于Fc 1 Apc fl/+ Pik3ca H1047R小鼠结肠肿瘤。MDCO/PDCO和CRC细胞系分别用750 nM或500 nM STX-478和100 nM AZD2014处理，并收集用于凋亡和PI3K蛋白的免疫印迹分析。
结果：SW48和SW48PK在100 nM单药AZD2014处理后细胞活力均显著降低（SW48 p<0.001，SW48PK p=0.005）。当用STX-478和AZD2014联合处理时，两种细胞系的细胞活力均显著降低（500 nM STX-478联合100 nM AZD2014；SW48 p=0.002，SW48PK p<0.001）。对于SW48PK，联合处理后观察到核糖体蛋白S6（RPS6；p<0.001）、AKT（p<0.001）和4EBP1（p=0.002）的磷酸化显著降低，而SW48未见显著差异。与对照和单药STX-478相比，MDCO的RPS6显著降低（p<0.001）。在PIK3CA H1047R突变型PDCO中，与对照相比，联合治疗观察到直径相对变化的显著减少（对照与联合的直径相对变化：30.2%对-0.96%；Glass's Delta = 1.67）。PIK3CA H1047R突变型PDCO的4EBP1（p<0.001）和RPS6（p<0.001）磷酸化也显著降低。
结论：STX-478和AZD2014的联合在2D和3D PIK3CA H1047R突变型CRC模型中均增强了应答，显示出将MTORC1/2抑制与PIK3CA抑制联合用于PIK3CA突变型CRC的前景。未来工作应在体内研究STX-478与MTORC1/2抑制在PIK3CA突变型CRC中的作用。
查看英文原文 English abstract
Background: STX-478, a PIK3CA mutant selective inhibitor, that we have previously shown to have promise in treating PIK3CA H1047R mutant colorectal cancer (CRC) models. A high throughput drug screen was performed and identified AZD2014, an MTORC1/2 inhibitor, to have enhanced response with STX-478. Here, we examine the combination of PI3K inhibition with MTORC1/2 inhibition in PIK3CA H1047R mutant colorectal cancer (CRC).
Methods: CRC cell lines SW48 and SW48PK (Horizon Discovery; SW48 with a PIK3CA H1047R mutation) were treated with varying concentrations of STX-478 (250-750 nM) and AZD2014 (20-250 nM) and viability measured by WST was performed. PIK3CA H1047R mutant PDCOs were treated, imaged, and response was determined by measuring the change in longest diameter of the organoids over time. Glass's Delta was used as a statistical test to measure the effect size of the different treatment groups. Mouse derived cancer organoids (MDCOs) were derived from Fc 1 Apc fl/+ Pik3ca H1047R murine colon tumors. MDCOs/PDCOs and CRC cell lines were treated with 750 nM or 500 nM STX-478 and 100 nM AZD2014, respectively, and collected for immunoblotting for apoptosis and PI3K proteins.
Results: Both SW48 and SW48PK had a significant reduction in cell viability with treatment of single agent AZD2014 at 100 nM (SW48 p<0.001, SW48PK p=0.005). When treated with combination of STX-478 and AZD2014, both cell lines had a significant reduction in cell viability (500 nM STX-478 with 100 nM AZD2014; SW48 p=0.002, SW48PK p<0.001). For SW48PK, a significant reduction of phosphorylation of ribosomal protein S6 (RPS6; p<0.001), AKT (p<0.001), and 4EBP1 (p=0.002) were observed after treatment with the combination, while no significant difference was seen in SW48. MDCOs had a significant decrease in RPS6 compared to control and single agent STX-478 (p<0.001). In PIK3CA H1047R mutant PDCOs, a significant reduction in relative change in diameter was observed in the combination treatment compared to control (relative change in diameter control versus combination: 30.2% vs -0.96%; Glass's Delta = 1.67). PIK3CA H1047R mutant PDCOs also had a significant decrease in phosphorylation of 4EBP1 (p<0.001) and RPS6 (p<0.001).
Conclusions: The combination of STX-478 and AZD2014 enhanced response in both 2D and 3D PIK3CA H1047R mutant CRC models and shows promise for combining MTORC1/2 with PIK3CA inhibition for PIK3CA mutant CRC. Future work should investigate STX-478 and MTORC1/2 inhibition in PIK3CA mutant CRC in vivo .
利益披露 Disclosure
A. E. Schmitz, None..
A. Zick, None..
C. A. Pasch, None.
D. A. Deming,
Merck ).
Genentech ).
Bristol Myers Squibb ), Other, Consulting/ Advisory Board.
Immunoscientific ).
Pfizer ), Other, Consulting/ Advisory Board.
Aadi Biosciences Other, Consulting/ Advisory Board.
Promega ).
Taiho Other, Consulting/ Advisory Board.
Inocras Other, Consulting/ Advisory Board.
DoMoreDx Other, Consulting/ Advisory Board.
Arcus ).
Fortvita Other, Consulting/ Advisory Board.
Ipsen ).
Takeda Other, Consulting/ Advisory Board.
Eli Lilly ), Other, Consulting/ Advisory Board.
Transthera ).
Exelixis Other, Consulting/ Advisory Board.
Illumina Other, Consulting/ Advisory Board.
Foundation Medicine Other, Consulting/ Advisory Board.
Regeneron Other, Consulting/ Advisory Board.