Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.Despite excitement about the development of targeted therapy strategies for cancer, few cures have been achieved. In patients with BRAF mutant melanoma, treatment with small molecule BRAF inhibitors typically follows a course of response and tumor shrinkage followed by eventual relapse and resistance (mean progression-free survival is ∼5.3 months) (1). Resistance to BRAF inhibitors is typically accompanied by reactivation of the MAPK signaling pathway, an effect mediated through activating mutations in NRAS and MEK1/2, genomic amplification of BRAF, increased expression of CRAF and Cot, and the acquisition of BRAF splice-form mutants (2–5). There is also evidence that increased PI3K/AKT signaling, resulting from the genetic inactivation of PTEN and NF1 and increased receptor tyrosine kinase (RTK)¹ signaling, may be involved in acquired BRAF inhibitor resistance (5–7). Many of the signaling proteins implicated in the escape from BRAF inhibitor therapy are clients of heat shock protein (HSP)-90 (8). Preclinical evidence now indicates that HSP90 inhibitors can overcome acquired and intrinsic BRAF inhibitor resistance, and clinical trials have been initiated to evaluate the BRAF/HSP90 combination in newly diagnosed patients (8, 9).Although targeted therapy strategies have been promising in BRAF mutant melanoma, few options currently exist for the 15–20% of melanoma patients whose tumors harbor activating NRAS mutations (10). Although there is some evidence that MEK inhibitors have activity in NRAS mutant melanoma patients, responses tend to be short-lived (mean progression-free survival ∼3 months) and resistance is nearly inevitable (11). Our emerging experience suggests that oncogene-driven signaling networks are highly robust with the capacity to rapidly adapt (12, 13). The future success of targeted therapy for melanoma and other cancers will depend upon the development of strategies that identify and overcome these adaptive escape mechanisms.The evaluation of targeted therapy responses in patients has proved to be challenging. The clinical development of HSP90 inhibitors has been hampered in part by the lack of a good pharmacodynamic assay for measuring HSP90 inhibition within tumor specimens (14). Additionally, very little is known about the adaptive changes that occur following the inhibition of MEK/ERK signaling in NRAS mutant melanoma. To address these issues, the optimal technique is liquid chromatography-multiple reaction monitoring mass spectrometry, which been shown to be highly reproducible and portable across laboratories (15–18).In addition to these technical developments, LC-MRM has also been shown to have excellent application to the study of biological pathways, including phosphotyrosine signaling, β-catenin signaling in colon cancer, and the evasion of apoptosis following BRAF inhibition in PTEN null melanoma (19–21). This technique can also be readily translated from cell line models to patient specimens. Here, we have developed a novel multiplexed LC-MRM assay to quantify the expression of >80 key signaling proteins in cell line models and fine needle aspirates from accessible melanoma lesions (22). In this study, we present the proof-of-principle for monitoring multiple signaling proteins in melanomas treated with either HSP90 or MEK inhibitors. Through this method, we identify the degradation of key HSP90 client proteins in vivo and elucidate a novel mechanism of adaptation to MEK inhibition through increased RTK signaling.