ALK Mutants in the Kinase Domain Exhibit Altered Kinase Activity and Differential Sensitivity to Small Molecule ALK Inhibitors
ALK Mutants in the Kinase Domain Exhibit Altered Kinase Activity and Differential Sensitivity to Small Molecule ALK Inhibitors
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DOI：
10.1021/bi8020923
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发表时间：
2009-04-28
期刊：
影响因子：
2.9
通讯作者：
Cheng, Mangeng
中科院分区：
文献类型：
--
作者：
Lu, Lihui;Ghose, Arup K.;Cheng, Mangeng
在间变性大细胞淋巴瘤（ALCL）的发病机制中，组成型活性间变性淋巴瘤激酶（ALK）嵌合蛋白的异常表达已得到充分证实。最近的小分子激酶抑制剂研究提供了坚实的概念验证，即抑制ALK足以减弱ALK（+）ALCL细胞的生长和增殖。在本研究中，在磷酸锚和门控区产生了几种ALK错义突变体，并测定了其激酶活性。NPM-ALK L182 M、L182 V和L256 M突变体在细胞中显示的激酶活性与NPM-ALK野生型（WT）相当或更高，并使BaF 3细胞进入IL-3非依赖性生长，而NPM-ALK L182 R、L256 R、L256 V、L256 P和L256 Q在细胞中显示的激酶活性弱得多或几乎没有。通过体外激酶试验，使用相应的GST-ALK突变体获得了相似的激酶活性。关于抑制剂应答，NPM-ALK L182 M和L182 V对稠合吡咯并咔唑（FP）衍生的ALK抑制剂的敏感性与NPM-ALK WT相当，但对二氨基嘧啶（DAP）衍生的ALK抑制剂的敏感性显著降低。另一方面，NPM-ALK L256 M对FP衍生和DAP衍生ALK抑制剂的敏感性均降低> 30倍。ALK抑制剂诱导的BaF 3/NPM-ALK突变细胞的生长抑制和细胞毒性与细胞NPM-ALK自磷酸化的抑制一致。在小鼠存活模型中，经口生物利用度DAP-ALK抑制剂治疗显著延长了接种BaF 3/NPM-ALK WT细胞的小鼠的存活期，但未延长接种BaF 3/NPM-ALK L256 M细胞的小鼠的存活期。使用ALK同源性模型分析ALK抑制剂与ALK WT和突变体的结合。总之，鉴定了几种潜在的活性ALK突变体，我们的数据表明其中一些突变体对选定的小分子ALK抑制剂具有耐药性。对这些突变体的进一步表征可能有助于鉴定和开发对ALK的WT和耐药突变体均具有活性的强效ALK抑制剂。
Abnormal expression of constitutively active anaplastic lymphoma kinase (ALK) chimeric proteins in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well established. Recent studies with small molecule kinase inhibitors have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. In this study, several missense mutants of ALK in the phosphate anchor and gatekeeper regions were generated and their kinase activity was measured. NPM-ALK L182M, L182V, and L256M mutants displayed kinase activity in cells comparable to or higher than that of NPM-ALK wild type (WT) and rendered BaF3 cells into IL-3-independent growth, while NPM-ALK L182R, L256R, L256V, L256P, and L256Q displayed much weaker or little kinase activity in cells. Similar kinase activities were obtained with corresponding GST-ALK mutants with in vitro kinase assays. With regard to inhibitor response, NPM-ALK L182M and L182V exhibited sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor comparable to that of NPM-ALK WT but were dramatically less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK L256M exhibited > 30-fold lower sensitivity to both FP-derived and DAP-derived ALK inhibitors. The growth inhibition and cytotoxicity of BaF3/NPM-ALK mutant cells induced by ALK inhibitors were consistent with inhibition of cellular NPM-ALK autophosphorylation. In a mouse survival model, treatment with the orally bioavailable DAP-ALK inhibitor substantially extended the survival of the mice inoculated with BaF3/NPM-ALK WT cells but not those inoculated with BaF3/NPM-ALK L256M cells. Binding of ALK inhibitors to ALK WT and mutants was analyzed using ALK homology models. In summary, several potential active ALK mutants were identified, and our data indicate that some of these mutants are resistant to select small molecule ALK inhibitors. Further characterization of these mutants may help to identify and develop potent ALK inhibitors active against both WT and resistant mutants of ALK.