ALK激酶域耐药突变的研究进展及未来应对策略

2022

10.26

15:00:00

作者

好医术

ALK激酶域耐药突变的研究进展及未来应对策略

间变性淋巴瘤激酶（anaplastic lymphoma kinase，ALK）是非小细胞肺癌（non-small cell lung cancer，NSCLC）中常见的致癌驱动基因，发生率约为5%，常见于无吸烟史、女性和年轻（中位确诊年龄为51岁）患者中^［1-2］。目前，第三代ALK酪氨酸激酶抑制剂（tyrosine kinase inhibitors，TKI）（包括克唑替尼、塞瑞替尼、阿来替尼、布加替尼、恩沙替尼和劳拉替尼）在ALK融合基因阳性的NSCLC患者中均取得了优异的治疗效果。近年来的研究数据表明，TKI序贯治疗能极大程度地延长患者的生存期，ALK基因突变也因此有“钻石突变”美誉^［3-4］。尽管靶向治疗疗效显著，患者最终会对靶向治疗产生获得性耐药^［5］。ALK融合基因阳性NSCLC患者的耐药机制包括依赖ALK信号转导通路（ALK基因扩增和激酶域突变）、不依赖ALK信号转导通路（旁路激活和组织类型转化）以及伴随基因（EGFR、KRAS和TP53等）改变。本文聚焦于ALK耐药突变谱探索后续治疗方案和应对策略。

ALK基因背景与耐药

ALK基因位于染色体2p23位点，正常状态下可编码受体酪氨酸激酶（receptor tyrosine kinase，RTK）。RTK包含三部分结构，即细胞外区（接受外界信号）、跨膜区及位于细胞内的功能结构域。ALK基因主要在胚胎时期表达（合成蛋白质），促进神经系统的发育，之后进入休眠状态^［6］。ALK基因变异（包括融合突变、点突变和基因扩增）导致激酶域异常激活，出现病理性ALK信号，诱发体细胞增殖和凋亡抑制，常出现在间变性大细胞淋巴瘤（anaplastic large cell lymphoma，ALCL）、炎症性肌成纤维细胞瘤（inflammatory myofibroblastic tumor，IMT）及NSCLC等恶性肿瘤^［7-9］。在NSCLC中，ALK融合突变是最常见的变异类型，以棘皮动物微管相关蛋白样因子4（echinoderm microtubule associated protein like 4，EML4）-ALK融合为主，约占85%，其他伴侣分子包括KIF5B、HIP1、GCC2及PLB1等^［10-13］。

ALK点突变和基因扩增是导致NSCLC对ALK-TKI耐药的重要机制。所有接受TKI治疗的患者在耐药后都能发现ALK基因扩^{增［14-15］}。点突变在初治患者中并不常见^［16］，绝大多数都在癌细胞对TKI耐药的情况下出现^［14］。ALK基因的常见突变位点于RTK细胞内激酶结构域，突变后导致相应部位氨基酸改变，蛋白质构象变化，形成空间位阻，干扰TKI与靶点的结合，导致获得性耐药^{［14，17］}。

由于肿瘤发展的异质性，ALK基因突变位点和类型丰富而复杂^{［14，18-19］}。首先，第一/二代TKI治疗失败后ALK基因的耐药变异以单点突变为主。约20%的患者在接受第一代TKI治疗进展后出现耐药突变，以L1196M、G1269A、C1156Y和F1174L为主；第二代TKI耐药后点突变的发生率更高（＞50%），类型更丰富，例如G1202R/del、F1174C/V和I1171T/N/S等^{［14，18， 20］}。其次，第二代TKI耐药后ALK双重突变和“脱靶”比例显著增加^{［14，21］}。第三代TKI耐药后几乎均为复合突变，并且耐药程度更高，如G1202R+L1196M^［22］。多代ALK-TKI治疗进展后，原有耐药位点发生变化，野生型的比列升高，耐药机制可能更为复杂^［18］。

1.1 变异体与耐药突变

EML4-ALK变异体是影响获得性耐药的重要因素。已有研究^［23-24］发现，V3变异体发生继发性耐药突变的概率高于V1、V2，突变类型及复合程度相对耐药性更高、更独特，如在V3中常见的ALK突变是C1156Y、F1174C/V、G1269A、I1171T、L1152R/V和G1202R，而L1196M突变在V1变异体中更常见。尤其， G1202R在V3变异体的发生率较V1变异体高（ 32% vs 0%，P = 0.001）^［23］，对G1/G2 ALK-TKI呈现普遍的高度耐药性，只有劳拉替尼对其效果明显^{［18，22］}。

1.2 共存突变与耐药突变

在ALK融合基因阳性的NSCLC患者中，最常见的伴随突变是肿瘤蛋白P53（tumor protein P53，TP53）基因突变（＞20%）^［25-27］。基线时具有TP53突变（TP53-mutant，TP53-MUT）患者的中位总生存期（median overall survival，mOS）显著劣于TP53野生型肿瘤（TP53-wild type，TP53-WT）的患者（44个月 vs 62个月，P = 0.018）^［28］。TP53共突变患者的突变负荷（tumor mutation burden，TMB）明显高于野生型患者（7.07±1.25 vs 3.20±0.33，P = 0.003），且耐药突变的发生率也高于TP53野生型患者，表明TP53突变可能导致其肿瘤抑制功能受损，影响基因组稳定性，促进基因进化，最终诱发耐药突变^［29］。

ALK-TKI获得性ALK激酶域耐药机制和治疗策略

ALK基因激酶结构域由较小的N瓣和较大的C瓣构成，通过“铰链区”链接^［30］。ATP结合位点位于N瓣和C瓣之间，核苷酸定位环（P环）的下方。ALK-TKI通过与 ALK激酶结构域的ATP结合位点竞争性结合来达到抗肿瘤疗效^［30］。ALK激酶域突变可分为5个热点区域：① ATP结合域（ATP-binding pocket）；② 核糖体结合口袋区域（ribose binding pocket）；③ α-C螺旋的N段或C段（N/C-terminal to the αC-helix）；④ 激酶铰链区（solvent front）；⑤ DFG基序（Asp-Phe- Gly）^［17］。常见的具有较强耐药性突变位点是“门控”的L1196、溶剂前沿的G1202和L1198以及DFG 基序附近的G1269等^［31］。

2.1 克唑替尼耐药谱

研究^［14-15］发现，克唑替尼（crizotinib）治疗失败后，30%~45%的患者经克唑替尼治疗后会发生ALK激酶区基因突变和基因扩增。2010年，Chio等^［31］首次发现L1196M、C1156Y两种突变，并且确认是克唑替尼的耐药机制。ALK-1196基因位于ATP结合域的底部，属“门控”关键位置，突变后形成空间位阻，影响药物与靶点结合，是克唑替尼治疗出现进展后常见的耐药突变之一^［31］。体外实验通过构建克唑替尼耐药细胞系时发现，耐药细胞不仅有获得性L1196M突变，还有EML4-ALK基因的拷贝数增加，提示获得性耐药可能是个动态过程，涉及基因扩增和点突变^［15］。随着NGS研究不断深入，ALK-TKI的耐药突变谱逐渐清晰，L1152R/P、F1174L/V、G1269S、D1203N、I1171T、S1206Y及1151Tins等耐药性突变被陆续发现（表1）。

ALK激酶域耐药突变的研究进展及未来应对策略

2.2 第二代ALK-TKI

在克唑替尼治疗失败后，超过70%的患者选择接受后代ALK-TKI进行治疗，继续延长生存期^［5］。相对于克唑替尼，第二代ALK-TKI（塞瑞替尼、阿来替尼、布加替尼和恩沙替尼）对ALK激酶具有更低的半数最大抑制浓度（median inhibition concentration，IC₅₀），可覆盖大部分的ALK耐药突变（表2），并显示出更好的中枢神经系统（central nervous system，CNS）渗透性^［25-40］（表2）。

塞瑞替尼（ceritinib）与ALK的结合位置是由铰链区、P环、α-C螺旋和激活环组成的ATP结合口袋，主要限制野生型和突变型复合物中P环的构象改变^［30］。在ASCEND-4研究^［51］中，一线塞瑞替尼的中位无进展生存期（ median progression-free survival，mPFS）显著优于化疗（16.6个月 vs 10.6个月，P＜0.001）；在没有基线CNS转移的患者中，塞瑞替尼的mPFS长达26.3个月。对比化疗，克唑替尼进展和1/2 线化疗失败的患者仍然能从塞瑞替尼治疗中获益（客观缓解率，overall response rate，ORR 42.6% vs 6.0%），mPFS（5.4个月 vs 1.7个月）更长^［52］。在临床前模型中，塞瑞替尼能有效地抑制克唑替尼常见的耐药性突变，如L1196M、G1269A、I1171T及S1206Y等^{［30，40］}。而且L1196M和I1171T/N/S突变点是塞瑞替尼独特的治疗位点^［41］，ALK F1245C突变也对塞瑞替尼非常敏感^［49］。而C1156Y、1151Tins、L1152R/P、R1275Q、G1202R/del、F1174V/C/L、T1151K及G1123S等突变会影响塞瑞替尼的结合位点产生耐药^{［14，32，34，40，50］}。其中，G1202R和F1174C/L是塞瑞替尼治疗失败后的主要耐药突变位点^{［14，53］}。G1123S 、T1151K和F1174C突变可导致P环出现波动，影响塞瑞替尼和P环之间较强的相互作用，导致耐药性的产生^{［32，34］}。而D1203、C1156、R1275、L1198及L1152等突变位点主要是由于影响塞瑞替尼与ALK的结合环境，干扰靶点的结合从而导致耐药性。ALK T1151Sins突变导致患者对塞瑞替尼、阿来替尼产生耐药性，对劳拉替尼敏感^［36］。

阿来替尼（alectinib）是经优化溶剂相互作用以及调节ATP结合位点后的高效选择性TKI，与野生型ALK结合位点主要在铰链区、P环及附近结构^［54］。在ALEX研究中，阿来替尼组的mPFS为34.8个月，显著优于克唑替尼组的10.9 个月（HR = 0.43，P＜0.000 1），并且显著改善ORR ^［55］。阿来替尼目前已成为ALK⁺晚期NSCLC患者的一线治疗优先选择。对于克唑替尼难治性患者，是否携带ALK耐药突变基因影响阿来替尼的治疗效果，不携带耐药突变的患者的mPFS（9.1个月 vs 5.6个月）和ORR（45% vs 35%）更好^［46］。在体外实验中，阿来替尼对大部分常见耐药突变有较高活性，如C1156Y、F1174L、G1269A及D1203N等^{［22，50］}。而研究^{［14，46］}发现，阿来替尼治疗失败后最常见的突变是G1202R和I1171N/S/T。ALK G1202R/del/ K突变会导致阿来替尼耐药性^{［22，56-57］}。由于L1198、V1180、I1171N和L1196位点突变会影响ATP结合入口和铰链区的构象变化，诱导阿来替尼与ALK之间的“锁扣”作用发生变化^［48］。诸如两种“门控”突变V1180L、ALK L1196M/Q及I1171T/N/S突变，对阿来替尼和克唑替尼均会产生耐药，但对塞瑞替尼、布加替尼及劳拉替尼等后代抑制剂敏感^{［22，42，45-47，58］}。

布加替尼（brigatinib）是ALK/EGFR双重抑制剂，对ALK L1196M突变和EGFR T790M突变高度敏感^［59］。ALTA-1L研究^［60］显示，布加替尼的mPFS优于克唑替尼（24.0个月 vs 11.0个月， P ＜0.001）。在克唑替尼耐药后患者中，布加替尼的mPFS为16.7个月^［61］。在塞瑞替尼、阿来替尼甚至经历多线治疗（至少2种ALK-TKI）中，布加替尼仍然能获得7个月的mPFS^［62-64］，从而确立了布加替尼在一代或其他二代ALK-TKI进展后的治疗地位。在临床前模型中，布加替尼对于ALK⁺和ALK^-细胞均有更强的抑制活性，对于其耐药突变谱均有更高的抑制效力，对G1202R突变有中度抑制能力^［38］。尽管对G1202R有抑制活性，高达70%的患者在接受布加替尼治疗失败后检测到新发的G1202R突变^［14］。临床研究显示，布加替尼确实对许多继发性ALK突变显示出抗肿瘤活性，包括L1196M、F1174L/V、G1269A、I1171N、L1198F和 V1180L等，与临床前试验显示的广谱活性相似^［64］。布加替尼主要通过氢键与ALK-K1150、L1196、L1198和E1210残基产生相互作用，能耐受ATP结合口袋中的任何单一突变，但G1202R除外^［65］。由于G1202R突变的存在，导致患者对布加替尼和神经营养性酪氨酸激酶（neurotrophic tyrosine kinase，NTRK）抑制剂的反应较差。因此，G1202R突变可能会导致布加替尼原发性耐药^［66］。此外，有研究^［14］显示，S1206C也会导致布加替尼耐药。

恩沙替尼（ensartinib）已获批用于治疗克唑替尼治疗后进展的或不耐受的ALK阳性的局部晚期或转移性NSCLC患者。在TKI初治患者中，恩沙替尼的mPFS（25.8个月 vs 12.7 个月；P＜0.001）和12 个月CNS进展率（23.9% vs 4.2%；P = 0.001）均优于克唑替尼^［67］。在克唑替尼难治性患者中，恩沙替尼的mPFS为9.6个月，颅内ORR可达70%^［68］。无论有无ALK激酶域耐药突变，恩沙替尼都表现出相似的ORR，对F1174L/V、C1156Y、T1151、G1123S和L1198F突变敏感性较高，再进展患者中最常见的继发性耐药突变是G1269A、G1202R和E1210K，而G1269A对其他第二代抑制剂敏感，G1202R对劳拉替尼的治疗效果最明确^{［27，29，68］}。

2.3 第三代ALK-TKI

劳拉替尼（lorlatinib）是第三代ALK-TKI，拥有更高的CNS渗透性，且可更广泛有效地克服已知的耐药性突变（表2）。CROWN研究^［69］显示，劳拉替尼12个月无进展生存率（78% vs 39%）和ORR（76% vs 58%）显著优于克唑替尼。在克唑替尼耐药的患者中，劳拉替尼的ORR为73%（95% CI：60%~84%），mPFS为11.1个月（95% CI：8.2 个月~未达到），而且无论是否存在可检测的ALK突变，患者的ORR是相似的^［18］。在二代ALK-TKI耐药背景下，劳拉替尼的抗肿瘤反应在不同ALK基因激酶域位点突变状态下产生差异，基于组织检测的ALK基因突变阳性患者的ORR（69% vs 27%）和PFS（11.0个月 vs 5.4 个月）明显优于突变阴性患者^［18］。因此，在曾接受过至少一种ALK-TKI的患者中，对肿瘤组织进行再次检测是很有必要的，ALK基因突变情况可能提示患者接受劳拉替尼的疗效及持久获益程度。

劳拉替尼对几乎所有已发现的耐药性突变有良好治疗效果，包括已知的高耐药性突变1151Tins、G1202R、I1171N和F1174L等（表2）。特别是对ALK-TKI普遍耐药的G1202R/del，劳拉替尼显示明显的抑制效果，ORR可达到57%，mPFS为8.2个月^{［18，22］}。而ALK L1256F会导致劳拉替尼耐药，但该位点对阿来替尼敏感性很高。此外，劳拉替尼耐药后发生的L1198F突变会重新对克唑替尼敏感^［22］。既往研究^［19］还发现，劳拉替尼进展后更容易出现双重或多重复合突变，某些复合突变对第一代/第二代TKI会重新敏感，如V1185L+L1196M复合突变，然而很多高度耐药性的复合突变依然得不到有效抑制，如G1202R+L1196M，这种复合突变是目前劳拉替尼程度最高的耐药突变（IC₅₀= 1 000 nmol/L），对所有的ALK-TKI都具有抗性^［19］。

Solvent-front区域突变及治疗对策

Solvent-front区域是ALK激酶域的溶剂前沿，由突变诱导的构象变化和ALK-TKI与结合口袋之间的快速解离过程导致耐药性^［54］。第二代TKI治疗失败后的耐药突变更容易出现于Solvent-front区域，比如G1202R/del、D1203N及L1198F^{［18，27］}。ALK L1198F突变导致蛋白ATP结合位点的构象变化，导致ALK激酶域结合位点与克唑替尼和劳拉替尼之间的亲和力的变化，使得癌细胞对劳拉替尼耐药，而对克唑替尼再次敏感^［70］。顽固性的溶剂前沿G1202R突变对一代/二代TKI保持普遍高水平耐药，是二代TKI治疗失败后最常见的耐药突变。这与克唑替尼的结合位点毗邻Arg-1202，塞瑞替尼、阿来替尼的结合位点位于铰链区相关^{［14，53］}。除了劳拉替尼能有效抑制^{［18，22］}，新型抑制剂ZX-29也能克服G1202R突变导致的耐药性^［71］。针对溶剂前沿区域（G1202R）合成的嘧啶-2,4-二胺衍生物对部分耐药突变有抑制活性，“半游离尿素” 基团（仅有1个-NH部分）对最常见的L1196M 突变（IC₅₀ = 0.91 nmol/L）以及G1202R（IC₅₀ =4.3 nmol/L）有显著的抗肿瘤效果^［72］。优化“尾巴”吡啶酮基序列，得到的2-氨基吡啶衍生物对L1196M（IC₅₀= 45 nmol/L）、G1202R（IC₅₀ = 22 nmol/L）和ROS1（2.3 nmol/L）也表现出令人满意的抗肿瘤活性^［73］。这些研究表明某些ALK特定区域的化学衍生物将是克服临床ALK激酶域耐药突变的有效方法。

复合耐药突变和治疗对策

ALK-TKI的多次序贯治疗后出现的双重复合突变已逐渐被认知。第二代ALK-TKI耐药后已出现一定比例的双重突变。V1185L+L1196M 双重突变对阿来替尼反应较差，但是V1185L、L1196M和复合突变均对塞瑞替尼敏感^［22］。I1171T+E1210K复合突变在塞瑞替尼耐药后出现^［21］。E1210K+D1203N和F1174C+D1203N复合突变赋予克唑替尼和第二代抑制剂耐药性，劳拉替尼可克服^{［14，22］}。而劳拉替尼治疗后更容易出现多重复合突变^［^19，22］。

研究^［19］显示，接受连续靶向治疗后，有35%的患者在劳拉替尼治疗失败后出现ALK复合突变。由于ALK G1202R突变是第二代ALK抑制剂耐药后最常见的激酶结构域突变，含G1202R的复合突变可能成为接受序贯第二代和第三代TKI后再次进展的患者中最常见的靶向耐药机制^［19］。Okada等^［22］在劳拉替尼治疗失败后的患者中发现，存在I1171N- 双重突变（I1171N = L1198F、+L1196M、+T1151K、+C1156Y、+F1174I、+L1198H、+L1256F和+G1269A）及G1202R-双重突变（G1202R = L1198F、+G1269A、+L1196M、+F1174C、+F1174L和S1206Y）。此外，还有I1171S+G1269A、C1156Y+G1269A和高水平耐药的L1196M+D1203N等复合耐药突变^{［20，74-75］}。

G1202R+G1269A对劳拉替尼、塞瑞替尼耐药，虽然对布加替尼中度敏感，但是由于G1202R顽固耐药，临床应用布加替尼的效果可能受限^{［14，22］}。布加替尼对I1171N复合突变（ +L1198F、+L1196M、+L1256F和+G1269A ）有抑制效果；塞瑞替尼可抑制I1171N+L1196M和+G1269A；阿来替尼可抑制I1171N+L1256F^［22］。值得注意的是，由于克唑替尼对L1198F的敏感性较高^［59］，对劳拉替尼耐药的L1198F突变可再次从克唑替尼治疗中获益，也能克服L1198F-复合突变，如I1171N/G1202R/ C1156Y+L1198F突变^{［19，22］}。其他复合突变类型，如I1171S+G1269A、G1202R+S1206Y、L1196M+D1203N和C1156Y+G1269A，再次应用第二代TKI可能对肿瘤有抑制效果，如塞瑞替尼、布加替尼^{［20，74-75］}。此外，第三代FMS样酪氨酸激酶-3（FMS-like tyrosine kinase-3，FLT3）抑制剂吉尔替尼（gilteritinib）对于I1171T/N/S单突变及I1171N-双重突变（+L1198F、+L1256F、+L1196M及+F1174I）有显著的效果^［76］。

第四代TKI（TPX-0131和NVL-655）表现出显著的“双突变活性” ，并且具有更强的CNS渗透性和对野生型ALK的抑制能力^［77-78］。除了对已知单发耐药突变的广谱活性TPX-0131对L1198F+G1202R、L1198F+L1196M、L1198F+C1156Y、E 1210K+S1206C 、T1151I+L1152ins 和G1202R+L1196M 有抑制能力。然而，G120 2R+G1269A和G1202R+G1269A+L1204V复合突变仍会导致TPX-0131耐药^［78］。目前第四代TKI已进入临床试验阶段，有望突破复合耐药突变的瓶颈。

展望

目前，第三代ALK-TKI在ALK融合基因阳性的NSCLC患者中均取得了显著的治疗效果，然而，耐药所致靶向治疗失败不可避免。ALK激酶域突变是ALK-TKI的重要耐药机制之一，认识和探索ALK激酶域耐药突变的发生机制和优化未来应对策略是ALK+NSCLC治疗中亟待解决的问题。迄今为止，ALK激酶域耐药突变大多为是临床前试验、小样本检测和个案报道，缺乏大规模临床研究数据明确TKI治疗后耐药突变的发生机制和对后代TKI的应答反应。目前，在克唑替尼进展后有第二代或第三代TKI可供选择，而第二代TKI治疗出现疾病进展后，仅有劳拉替尼作为“最后手段”。新一代TPX-0131和NVL-655在临床前研究中以表现出优异的抑瘤效果，尤其是能够克服ALK复合耐药突变，但仍需要临床试验的验证。同时，考虑到肿瘤的时空异质性以及个体耐药机制差异，仍建议在ALK-TKI治疗出现疾病进展后再次检测（包括活检和基因检测），有助于识别难治性和重敏性ALK基因激酶域突变，明确TKI的耐药机制，开展更具针对性的基础和临床转化研究，确立更加精准有效的治疗策略。

利益冲突声明：所有作者均声明不存在利益冲突。

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