Prospective identification of resistance mechanisms to HSP90 inhibition in KRAS mutant cancer cells [GenomeWideSNP_6]

Inhibition of the HSP90 chaperone results in depletion of many signaling proteins that drive tumorigenesis, such as downstream effectors of KRAS, the most commonly mutated human oncogene. As a consequence, several small-molecule HSP90 inhibitors are being evaluated in clinical trials as anticancer agents. To prospectively identify mechanisms through which HSP90-dependent cancer cells evade pharmacologic HSP90 blockade, we generated multiple mutant KRAS-driven cancer cell lines with acquired resistance to the purine-scaffold HSP90 inhibitor PU-H71. All cell lines retained dependence on HSP90 function, as evidenced by sensitivity to short hairpin RNA-mediated suppression of HSP90AA1 or HSP90AB1 (also called HSP90α and HSP90β, respectively), and exhibited two types of genomic alterations that interfere with the effects of PU-H71 on cell viability and proliferation: (i) a Y142N missense mutation in the ATP-binding domain of HSP90α that co-occurred with amplification of the HSP90AA1 locus, (ii) genomic amplification and overexpression of the ABCB1 gene encoding the MDR1 drug efflux pump. In support of a functional role for these alterations, exogenous expression of HSP90α Y142N conferred PU-H71 resistance to HSP90-dependent cells, and pharmacologic MDR1 inhibition with tariquidar or lowering ABCB1 expression restored sensitivity to PU-H71 in ABCB1-amplified cells. Finally, comparison with structurally distinct HSP90 inhibitors currently in clinical development revealed that PU-H71 resistance could be overcome, in part, by ganetespib (also known as STA9090) but not tanespimycin (also known as 17-AAG). Together, these data identify potential mechanisms of acquired resistance to small molecules targeting HSP90 that may warrant proactive screening for additional HSP90 inhibitors or rational combination therapies. SNP arrays were profiled in PU-H71-sensitive and resistant A549, MDA-MB-231 and SW480 cell lines.

热休克蛋白90（HSP90）分子伴侣的抑制可导致多种驱动肿瘤发生的信号蛋白耗竭，例如最常见的人类突变癌基因KRAS的下游效应因子。鉴于此，多款小分子HSP90抑制剂正作为抗癌药物开展临床试验。为前瞻性鉴定HSP90依赖型癌细胞逃避药物性HSP90阻断的潜在机制，我们构建了多株携带KRAS驱动突变、对嘌呤骨架型HSP90抑制剂PU-H71产生获得性耐药的癌细胞系。所有细胞系仍依赖HSP90功能，该特性可通过其对短发夹RNA（short hairpin RNA, shRNA）介导的HSP90AA1或HSP90AB1（分别又称HSP90α与HSP90β）基因沉默的敏感性得以证实；同时这些耐药细胞系存在两类可干扰PU-H71对细胞活力与增殖产生影响的基因组改变：（i）HSP90α的ATP结合结构域内发生Y142N错义突变，且该突变伴随HSP90AA1基因座扩增；（ii）编码多药耐药1（MDR1）药物外排泵的ABCB1基因发生基因组扩增与过表达。为验证上述改变的功能作用，我们通过外源表达HSP90α Y142N使HSP90依赖型细胞获得PU-H71耐药性；而使用他喹莫德（tariquidar）进行药物性MDR1抑制或下调ABCB1表达，可恢复ABCB1扩增细胞对PU-H71的敏感性。最后，与当前临床开发中的结构迥异的HSP90抑制剂对比发现，PU-H71耐药性可部分被加纳替布（ganetespib，又称STA9090）逆转，但无法被他尼霉素（tanespimycin，又称17-AAG）逆转。综上，本研究明确了靶向HSP90的小分子药物产生获得性耐药的潜在机制，这提示需对其他HSP90抑制剂开展前瞻性筛查，或制定合理的联合治疗策略。本研究对PU-H71敏感及耐药的A549、MDA-MB-231与SW480细胞系进行了单核苷酸多态性（Single Nucleotide Polymorphism, SNP）芯片检测。