Targeting the Hsp90 Molecular Chaperone and Resistant Pathways in BRAF-mutant Melanoma
Sanchez, Jaquelyn
2020
Abstract
Melanoma remains the most aggressive and fatal type of skin cancer. In greater than 50% of cases, patients present with an activating BRAF mutation (BRAF+), leading to upregulated mitogen-activated protein kinase (MAPK) pathway signaling. Of these patients, 80-90% have a missense mutation at codon 600 (e.g., BRAFV600E), making the mutant form of the protein an attractive and druggable target. In 2011, the FDA approved combination therapy of BRAF+ and MEK inhibitors (BRAFi/MEKi), like vemurafenib and cobimetinib (Ve/Cb), for use in unresectable late-stage melanoma patients, drastically changing treatment options and initial outcomes. Still, the majority of patients become refractory to BRAFi/MEKi within the first year of treatment. The lack of treatment durability underscores the need for novel therapeutic strategies and drug candidate development, such as the utilization of molecular chaperone inhibitors. The 90-kDa heat shock protein (Hsp90) is a molecular chaperone and responsible for stabilizing the protein folding of “client” proteins that interact with the heterochaperone complex that Hsp90 forms with the 70-kDa heat shock protein (Hsp70) and other co-chaperones. These clients are involved in several cellular signaling pathways and processes, highlighting the significance of chaperone function in eukaryotic cells. Interestingly, Hsp90 expression increases several-fold in cancer cells to compensate for cellular stress and client protein dependence on chaperone function. To-date 18 small molecule Hsp90 inhibitors (Hsp90i) entered clinical trials, of which 94.4% target the N-terminus (NT-Hsp90i) but failed to get FDA approval. The NT-Hsp90i are effective and xvi potent, but pan-inhibitors of all four Hsp90 isoforms. In clinical trials, patients require dose-escalation of NT-Hsp90i to reach a therapeutic effect, ultimately leading to dose-limiting toxicities (DTL). Studies suggest a link between DTLs and the activation of the heat shock response (HSR), especially the cytoprotective role of Hsp70. Previously, our lab, with collaborators, developed novel C-terminal Hsp90i (CT-Hsp90i) and showed efficacy in several cancer models in vitro and in vivo while mitigating the HSR, suggesting a decreased toxicity profile compared to NT-Hsp90i. For this dissertation, I researched therapeutic resistance mechanisms in BRAF+ melanoma through various preclinical in vitro studies that targeted Hsp90. Specifically, I tested the hypothesis that several resistance-promoting processes require Hsp90 function and, therefore, could be targeted with an Hsp90i to simultaneously knockdown resistance pathways and oncogenic processes. First, I showed effective melanoma cell death using the CT-Hsp90i KU758 at potent micromolar concentrations (e.g., IC50 = 0.36 – 0.43 micromolar). Next, I demonstrated robust synergy (e.g., CI<0.5) of KU758 when combined with either a BRAFi or MEKi to target two resistance pathways effectively (e.g., MAPK/Erk and PI3K/Akt), significantly mitigate melanocyte migration, and downregulate key Hsps involved in HSR activation. Finally, I accessed publicly available genomic data via the National Cancer Institute and The Cancer Genome Atlas (TCGA) program to identify additional genes of interest in BRAF+ melanomas. Using a clustered heatmap of RNA expression data, I distinguished genes of interest based on common expression alterations amongst a subset of BRAF+ melanoma patients to provide a genetic perspective in the context of Hsp90i use in melanoma patients. Collectively, this work reviews the use of and development of several small xvii molecule inhibitors in melanomas (e.g., BRAFi/MEKi and Hsp90i), identifies a novel and effective KU758-combination approach in BRAF+ melanomas, and gives insight into future therapeutic directions based on various translational and genetic signatures in these difficult-to-treat tumors.Subjects
BRAF-mutant melanoma heat shock proteins Hsp90 combination therapy translational science MAPK pathway
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