PO.ET01.04 · 实验与分子治疗
STX-478与lorlatinib联合治疗PIK3CA突变型结直肠癌
The combination of STX-478 and lorlatinib for the treatment of PIK3CA mutant colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：超过15%的结直肠癌（CRC）患者为PIK3CA突变，使其成为药物开发的关键通路。最近，我们使用STX-478（一种突变选择性PIK3CA抑制剂）进行了高通量药物筛选，并鉴定出lorlatinib（一种ALK抑制剂）作为增强治疗疗效的潜在手段。在此，我们证实了这一疗效并评估潜在的作用机制，包括lorlatinib对MYC蛋白丰度和靶基因表达的潜在影响。
方法：CRC细胞系SW48和SW48PK（Horizon Discovery；携带PIK3CA H1047R突变的SW48）用于细胞活力实验。对SW48、SW48PK和患者来源癌症类器官（PDCO）进行了STX-478、lorlatinib和联合处理的免疫印迹。使用ImageJ定量这些印迹以评估PI3K蛋白表达。对PIK3CA H1047R突变型PDCO进行处理、成像，并测量任何类器官的最长直径。使用Glass's delta（GD）确定各治疗组相比对照的效应量。通过定量PCR（qPCR）测量五个MYC靶基因的基因表达。
结果：在SW48和SW48PK细胞中，lorlatinib单药治疗后细胞活力无显著差异。然而，STX-478与lorlatinib联合处理在两个细胞系中均显著降低了细胞活力（lorlatinib 500 nm，STX-478 500 nm；SW48 p=0.001，SW48PK p<0.001）。在SW48PK中，免疫印迹分析显示联合处理下核糖体蛋白S6（RPS6）和4EBP1的磷酸化水平明显降低，表明下游PI3K信号受到抑制（pRPS6：p=0.02，4EBP1：p=0.03）。在任何治疗组中，c-MYC和pAKT的蛋白表达均无显著变化。RT-qPCR分析总体上未显示MYC靶基因表达的变化。LDHA（一种在缺氧条件下上调的关键糖酵解基因）的表达在处理后的SW48PK细胞中显著降低，表明联合治疗可能降低糖酵解活性（p=0.005）。用STX-478和lorlatinib联合处理的PIK3CA H1047R突变型PDCO相比对照有显著的生长减少（直径相对变化 对照 vs 联合 = 42.9% vs 12.3%；GD=1.37）。
结论：STX-478与lorlatinib联合已证实在PIK3CA突变型CRC模型中具有显著疗效。该活性似乎与对MYC的影响无关。进一步的研究旨在确定联合治疗的作用机制，并在体内证实疗效。
查看英文原文 English abstract
Background: Over 15% of colorectal cancer (CRC) patients are PIK3CA mutant, making it a critical pathway for drug development. Recently, we performed a high throughput drug screen with STX-478, a mutant-selective PIK3CA inhibitor, and identified lorlatinib, an ALK inhibitor, as a potential means to enhance therapeutic efficacy. Here we confirm this efficacy and evaluate potential mechanisms of action, including the potential impact of lorlatinib on MYC protein abundance and target gene expression.
Methods: CRC cell lines SW48 and SW48PK (Horizon Discovery; SW48 with a PIK3CA H1047R mutation) were used in a cell viability assay. Immunoblotting was performed on SW48, SW48PK and patient derived cancer organoids (PDCOs) with STX-478, lorlatinib, and combo treatment. ImageJ was used to quantify these blots to evaluate PI3K protein expression. PIK3CA H1047R mutant PDCOs were treated, imaged, and the longest diameter of any organoid was measured. Glass's delta (GD) was used to determine effect size for each treatment group compared to control. Gene expressions for five MYC target genes were measured by quantitative PCR (qPCR).
Results: In SW48 and SW48PK cells, there were no significant differences in cell viability following lorlatinib single-agent treatment. However, combo treatment with STX-478 and lorlatinib significantly reduced cell viability in both cell lines (lorlatinib 500 nm, STX-478 500 nm; SW48 p=0.001, SW48PK p<0.001) In SW48PK, immunoblot analysis demonstrated a marked decrease in phosphorylation levels of ribosomal protein S6 (RPS6) and 4EBP1 under combo treatment, indicating suppression of downstream PI3K signaling ( pRPS6: p=0.02, 4EBP1: p=0.03). There were no significant changes in protein expression of c-MYC and pAKT in any of the treatment groups. RT- qPCR analysis overall did not demonstrate a change in MYC target gene expression. The expression of LDHA, a key glycolytic gene upregulated under hypoxic conditions, was significantly decreased in SW48PK cells following treatment, indicating a potential reduction in glycolytic activity associated with the combined therapy (p=0.005). PIK3CA H1047R mutant PDCOs treated with the combo of STX-478 and lorlatinib had a significant reduction in growth compared to control (relative change in diameter control versus combo = 42.9% vs 12.3%; GD=1.37).
Conclusion: The combination of STX-478 and lorlatinib was confirmed to have significant efficacy in PIK3CA mutant CRC models. This activity does not seem to be related to effects on MYC. Further studies look to determine the mechanism of action for the combo treatment, and confirm efficacy in vivo.
利益披露 Disclosure
A. T. Zick, None..
A. E. Schmitz, None..
C. A. Pasch, None.
D. A. Deming,
Merck ).
Genentech ).
Bristol Myers Squibb ).
Aadi Biosciences ), Other, Consulting/Advisory Boards.
Pfizer ), Other, Consulting/Advisory Boards.
Curegenix ).
Promega ).
Natera ).
STRATA Oncology ).
Cornerstone Pharmaceuticals ).
Arcus ).
Guardant Health ).
Ipsen ).
Takeda ).
Eli Lilly ), Other, Consulting/Advisory Boards.
Revolution Medicine ).
Bayer Other, Consulting/Advisory Boards.
Seagen Other, Consulting/Advisory Boards.
Foundation Medicine Other, Consulting/Advisory Boards.
Regeneron Other, Consulting/Advisory Boards.