Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Lung Cancer. 2014 Jan;83(1):37-43. doi: 10.1016/j.lungcan.2013.09.019. Epub 2013 Oct 14.
The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK.
We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 μM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases.
All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as "bypass" tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib+erlotinib (reversible EGFR TKI) and crizotinib+afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib.
We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.
多靶点酪氨酸激酶抑制剂（TKI）克唑替尼对 ALK 易位的非小细胞肺癌（NSCLC）有效；然而，随着时间的推移，获得性耐药不可避免地会出现。ALK 突变以前仅与三分之一的耐药肿瘤有关。我们试图评估替代耐药机制，并在由 EML4-ALK 驱动的细胞系中寻找克服这些机制的临床前策略。
我们选择 NSCLC 细胞系 NCI-H3122（H3122：EML4-ALK E13；A20），并衍生出能够在 1 μM 克唑替尼存在下生长的耐药变体。分析这些变体的 ALK 突变、对克唑替尼与其他 TKI 联合治疗的敏感性以及替代酪氨酸激酶的激活情况。
所有 NCI-H3122 克唑替尼耐药（CR）克隆均缺乏 ALK 激酶结构域的扩增或突变。为了评估替代激酶是否可能作为这些耐药细胞中下游信号激活的“旁路”途径，我们进行了磷酸化受体酪氨酸激酶阵列分析，结果表明 CR 克隆在暴露于克唑替尼前后的磷酸-EGFR 信号高于 H3122 细胞。使用双重 ALK TKI（与克唑替尼联合）联合组合 TKI 抑制的功能方法作为可能靶点的二次筛选。克唑替尼+厄洛替尼（可逆 EGFR TKI）和克唑替尼+阿法替尼（不可逆 EGFR/ERBB2 TKI）能够抑制 H3122 CR 克隆的生长，证实 EGFR 激活是耐药的一种机制。从培养基中去除克唑替尼使 CR 细胞重新对克唑替尼敏感。
我们确定了 EGFR 的激活是 ALK 易位 NSCLC 临床前模型中对克唑替尼耐药的一种机制。如果 EGFR 激活被确认为患者来源肿瘤中 ALK TKI 诱导耐药的主要机制，那么可以探索使用 ALK 加 EGFR TKI 治疗这一重要的 NSCLC 患者群体。
