Targeting CDK 2 overcomes melanoma resistance against BRAF and Hsp90 inhibitors

Abstract

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy‐resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX‐derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF‐MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.

Thermal proteome profiling for interrogating protein interactions

“…More commonly, TPP experiments involve chemical [e.g., drug (Azimi et al, 2018;Becher et al, 2016;Hu et al, 2019;Huber et al, 2015;Kitagawa et al, 2017;Mateus et al, 2018Mateus et al, , 2016Reinhard et al, 2015;Savitski et al, 2014Savitski et al, , 2018 . Some of the perturbations can be applied in a dose-dependent manner (Becher et al, 2016) or time-dependent manner Dai et al, 2018) to improve data analysis or facilitate mechanistic understanding of the perturbation (Fig 2).…”

Impact of phosphorylation on thermal stability of proteins

“…Mass spectrometry data were processed with isobarQuant, 2,7 In parallel, the phosphopeptides-enriched raw data files were processed by the MaxQuant software 8 (version 1.6.2.3) to assess phosphorylation sites localization probabilities. Files were searched against a reviewed Homo sapiens database (UniProt, September 2018), with the following parameters: trypsin digestion (cleavage at the C-term of lysine and arginine, even when followed by a proline residue) with a maximum of 3 missed cleavages, TMT10plex labeling, fixed carbamidomethylation of cysteines, variable oxidation of methionines, as well as, variable phosphorylation of serine, threonine and tyrosine residues.…”

“…Search results for phosphopeptide-enriched samples from isobarQuant 2,7 and MaxQuant 8 were merged based on peptide MS/MS scan ID and kept only for further analysis when they had at least one phosphosite which could be localized with a probability higher than 0.75 (extracted from MaxQuant output)-to only include phosphosites with high localization confidence, generally termed class I phosphosites 9 -, signal to interference ratio (S2I) equal or higher than 0.5 and precursor to threshold ratio (P2T) equal or higher than 4 (both extracted from isobarQuant output)-to minimize ratio compression originating from co-isolated peptides 1 . Peptides with identical sequence and phosphorylation localization were merged by summing their signal intensities.…”

“…The first application of phospho-TPP was described by Azimi et al 7 , though a systematic comparison between protein thermal stabilities and phosphorylated protein forms was not performed. Recently, Huang et al 8 investigated the effect of 2,883 phosphorylation sites on protein thermal stability, and reported significant changes in the melting behavior for 719 (25%) of these sites (without multiple testing correction).…”

Inhibitors of HSP90 in melanoma

“…The most common adverse effects of grade 3 and 4 were rash and diarrhea, and cutaneous squamous cell carcinomas was developed in 14% of patients [143]. XL888 resistance mechanism involving CDK2 was identified in melanoma cells, and CDK2 expression was dependent on MITF (microphthalmia-associated transcription factor) [144]. XL888 is currently evaluated in phase I clinical trials ( Table 1).…”