Approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations and no effective therapeutic strategy exists for these patients. The use of Heat shock protein 90 (Hsp90) inhibitors in KRAS mutant NSCLC appeared to be a promising approach since these inhibitors target many KRAS downstream effectors, however, limited clinical efficacy has been observed due to resistance. Here, we examined the mechanism(s) of acquired resistance to the Hsp90 inhibitor, ganetespib, and identified novel and rationally devised Hsp90 inhibitor combinations which may prevent and overcome resistance to Hsp90 inhibitors. We derived KRAS mutant NSCLC ganetespib resistant (GR) cell lines to identify the resistance mechanism(s) and identified hyperactivation of RAF/MEK/ERK/RSK and PI3K/AKT/mTOR pathways as key resistance mechanisms. Furthermore, we found that GR cells are “addicted” to these pathways as ganetespib resistance lead to synthetic lethality to a dual PI3K/mTOR, a PI3K, or an ERK inhibitor. Interestingly, the levels and activity of a key activator of the mTOR pathway and an ERK downstream target, p90 ribosomal S6 kinase (RSK) were also increased in the GR cells. Genetic or pharmacologic inhibition of p90RSK in GR cells restored sensitivity to ganetespib, whereas p90RSK overexpression induced ganetespib resistance in naïve cells, validating p90RSK as a mediator of resistance and a novel therapeutic target. Our studies offer a way forward for Hsp90 inhibitors through the rational design of Hsp90 inhibitor combinations that may prevent and/or overcome resistance to Hsp90 inhibitors providing an effective therapeutic strategy for KRAS mutant NSCLC.

A subset of non-small cell lung cancers (NSCLC) are dependent upon oncogenic driver mutations including the most frequently observed driver mutant KRAS which is associated with a poor prognosis. As direct RAS targeting in the clinic has been unsuccessful to date, use of Heat shock protein 90 (Hsp90) inhibitors appeared to be a promising therapy for KRAS mutant NSCLC, however limited clinical efficacy was observed due to rapid resistance. Furthermore, the combination of the Hsp90 inhibitor (Hsp90i), ganetespib and docetaxel was tested in a phase III clinical trial and failed to demonstrate benefit. Here, we investigated the mechanism(s) of resistance to ganetespib and explored why the combination with docetaxel failed in the clinic. We have not only identified a critical role for the bypass of the G2/M cell cycle checkpoint as a mechanism of ganetespib resistance (GR) but have also found that GR leads to cross-resistance to docetaxel. Reactivation of p90RSK and its downstream target, CDC25C was critical for GR and mediated the bypass of a G2/M arrest. Overexpression of either p90RSK or CDC25C lead to bypass of G2/M arrest and induced ganetespib resistance in vitro and in vivo. Moreover, resistance was dependent on p90RSK/CDC25C signaling, as synthetic lethality to ERK1/2, p90RSK or CDC25C inhibitors was observed. Importantly, the combination of ganetespib and p90RSK or CDC25C inhibitors was highly efficacious in parental cells. These studies provide a way forward for Hsp90 inhibitors through the development of novel rationally designed Hsp90 inhibitor combinations that may prevent or overcome resistance to Hsp90i.

Lung cancer is the leading cause of cancer death in the United States and worldwide. A large fraction of non-small cell lung cancers (NSCLC) are dependent upon oncogenic driver mutations of which the most commonly observed driver is mutant KRAS. Unfortunately, NSCLC patients with oncogenic KRAS mutations have no effective therapies and prognosis is poor. As direct RAS targeting has been unsuccessful in the clinic to date, use of Heat shock protein 90 (Hsp90) inhibitors in KRAS mutant NSCLC appeared to be promising approach for targeting KRAS mutant NSCLC through its downstream effectors. However, limited clinical efficacy as monotherapy was observed due to rapid resistance. Furthermore, the combination of ganetespib and docetaxel was recently tested in a large phase III clinical trial (Galaxy-2) in advanced lung cancer and failed to demonstrate benefit. Here, we investigated the mechanism(s) of resistance to the Hsp90 inhibitor (Hsp90i), ganetespib by generating NSCLC cells with acquired resistance to Hsp90i’s including ganetespib (GR cells). We have not only identified the mechanism of acquired resistance to ganetespib but have also found that this mechanism induces cross resistance to docetaxel. Finally, we have identified novel Hsp90i combinations that can overcome and prevent this resistance. We report that hyperactivation of ERK and p90RSK and its downstream target, CDC25C leads to acquired resistance to ganetespib and docetaxel. Moreover, this resistance is mediated via bypass of a G2/M arrest. Overexpression of either p90RSK or CDC25C in naïve cells was sufficient to induce the bypass of this G2/M arrest as well induced resistance to both ganetespib and docetaxel. Remarkably, p90RSK or CDC25C overexpression also led to ganetespib resistance in vivo. The observed resistance was dependent on continued p90RSK/CDC25C signaling, as synthetic lethality to specific ERK, p90RSK or CDC25C inhibitors was observed. Importantly, we have found that the combination of ganetespib with inhibitors of ERK1/2, p90RSK, or CDC25C was highly efficacious. In summary, we propose that the hyperactivation of p90RSK induces CDC25C overexpression and activity which then induces G2/M progression via CyclinB1/cdc2 regulation resulting in ganetespib resistance. Despite two decades of testing in the clinic, either as monotherapy or in combination with chemotherapy, Hsp90 inhibitors have been ineffective due to acquired resistance. Our preclinical analyses provide a way forward for Hsp90 inhibitors through the development of novel rationally designed Hsp90 inhibitor combinations that may prevent or overcome resistance to Hsp90 inhibitors. Citation Format: Suman Chatterjee, Eric H. Huang, Ian Christie, Timothy F. Burns. The p90RSK-CDC25C signaling pathway leads to bypass of the ganetespib induced G2/M arrest and mediates acquired resistance to ganetespib in KRAS mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2017-4152