Evaluating Melanoma Drug Response and Therapeutic Escape with Quantitative Proteomics

Abstract

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 g of total protein). We further showed MEK inhibition to be associated with signaling through the NFB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor ␤ signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, creno- 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 ϳ5.3 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). Alth...

Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma

“…Our data adds to this and suggest that amuvatinib may be of utility in some NRAS -mutant melanomas, particularly those that are dependent upon the targets of this drug. The emerging evidence suggests that NRAS -mutant melanomas are more diverse than BRAF -mutant melanoma, potentially requiring the development of highly personalized therapeutic strategies on a patient-by-patient basis (20). Enhanced genetic profiling integrated with quantitative proteomic profiling is likely to play a key role in future therapy selection that allows durable responses to be achieved.…”

Proteomic interrogation of HSP90 and insights for medical research

“…From a different angle, aiming at developing a method for routine evaluation of personalized treatments against melanoma, Rebecca et al [76] studied the effect of HSP90 inhibition by targeted proteomics measuring specifically about 80 signaling proteins and a few heat shock proteins. HSP90 inhibition resulted in increase of HSP71 (an inducible cytosolic HSP70 isoform) and both HSP90 isoforms levels in a NRAS mutant cell line.…”

PDGFR-alpha inhibits melanoma growth via CXCL10/IP-10: a multi-omicsapproach

“…As recently highlighted, the specific role such receptors play in cancer development and progression has still to be largely clarified [35]. The stimulatory role of PDGF-BB and PDGFR-beta has been demonstrated in several cells and tissues, including melanoma [36–40]. Non-selective inhibitors of both PDGFR-beta and PDGFR-alpha, such as imatinib and dasatinib, are known to partially inhibit melanoma growth, but the role of the alpha or beta receptors has not been fully clarified yet [41–43].…”