Rearranged during transfection (RET) rearrangements occur in 1% to 2% of lung adenocarcinomas as well as other malignancies and are now established targets for tyrosine kinase inhibitors. We developed three novel RET fusion–positive (RET+) patient–derived cancer cell lines, CUTO22 [kinesin 5B (KIF5B)–RET fusion], CUTO32 (KIF5B-RET fusion), and CUTO42 (echinoderm microtubule-associated protein-like 4–RET fusion), to study RET signaling and response to therapy. We confirmed each of our cell lines expresses the RET fusion protein and assessed their sensitivity to RET inhibitors. We found that the CUTO22 and CUTO42 cell lines were sensitive to multiple RET inhibitors, whereas the CUTO32 cell line was >10-fold more resistant to three RET inhibitors. We discovered that our RET+ cell lines had differential regulation of the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B (AKT) pathways. After inhibition of RET, the CUTO42 cells had robust inhibition of phosphorylated AKT (pAKT), whereas CUTO22 and CUTO32 cells had sustained AKT activation. Next, we performed a drug screen, which revealed that the CUTO32 cells were sensitive (<1 nM IC₅₀) to inhibition of two cell cycle–regulating proteins, polo-like kinase 1 and Aurora kinase A. Finally, we show that two of these cell lines, CUTO32 and CUTO42, successfully establish xenografted tumors in nude mice. We demonstrated that the RET inhibitor BLU-667 was effective at inhibiting tumor growth in CUTO42 tumors but had a much less profound effect in CUTO32 tumors, consistent with our in vitro experiments. These data highlight the utility of new RET+ models to elucidate differences in response to tyrosine kinase inhibitors and downstream signaling regulation. Our RET+ cell lines effectively recapitulate the interpatient heterogeneity observed in response to RET inhibitors and reveal opportunities for alternative or combination therapies.

中文翻译：

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新型人源 RETFusion NSCLC 细胞系对 RET 抑制剂和下游信号传导的差异调节具有异质反应


转染期间重排 ( RET ) 重排发生在 1% 至 2% 的肺腺癌以及其他恶性肿瘤中，现已成为酪氨酸激酶抑制剂的既定靶标。我们开发了三种新型RET融合阳性 ( RET +) 患者来源的癌细胞系：CUTO22 [驱动蛋白 5B ( KIF5B )– RET融合]、CUTO32 ( KIF5B - RET融合) 和 CUTO42（棘皮动物微管相关蛋白样 4） – RET融合），研究 RET 信号传导和治疗反应。我们确认了每个细胞系都表达 RET 融合蛋白，并评估了它们对 RET 抑制剂的敏感性。我们发现 CUTO22 和 CUTO42 细胞系对多种 RET 抑制剂敏感，而 CUTO32 细胞系对三种 RET 抑制剂的耐药性高出 3E10 倍。我们发现我们的RET + 细胞系对丝裂原激活蛋白激酶和磷酸肌醇 3-激酶/蛋白激酶 B (AKT) 途径具有差异调节。抑制 RET 后，CUTO42 细胞对磷酸化 AKT (pAKT) 具有强烈抑制作用，而 CUTO22 和 CUTO32 细胞则持续激活 AKT。接下来，我们进行了药物筛选，结果显示 CUTO32 细胞对两种细胞周期调节蛋白（polo 样激酶 1 和 Aurora 激酶 A）的抑制敏感 (<1 nM IC ₅₀ )。最后，我们表明其中的两种蛋白这些细胞系 CUTO32 和 CUTO42 在裸鼠中成功建立异种移植肿瘤。我们证明 RET 抑制剂 BLU-667 可有效抑制 CUTO42 肿瘤中的肿瘤生长，但对 CUTO32 肿瘤的影响要小得多，这与我们的体外实验一致。 这些数据强调了新的RET+模型在阐明酪氨酸激酶抑制剂反应和下游信号调节差异方面的实用性。我们的RET+细胞系有效地概括了 RET 抑制剂反应中观察到的患者间异质性，并揭示了替代或联合疗法的机会。