103 (Phospho)proteomics biomarkers for sunitinib response in RCC On protein expression level, gene ontology mining of primary resistant tumors revealed that processes related to vesicle mediated transport and excretion were enriched (Figure 2c). One could hypothesize that this possibly reflects enhanced ability of these tumors for drug efflux, contributing to drug resistance66,67. Alternatively, this vesicle mediated transport may reflect activation of immune processes, for example degranulation of mast cells. This would corroborate our phosphoproteomics data, with post-translational modification signatures indicative of enhanced immune processes in resistant patients (Figure 1e), which is in line with previously published work linking upregulation of cellular immune pathways and inflammatory markers to an unfavorable response to anti-VEGFR TKI’s in ccRCC44,68,69. Shifting our view towards the group of sensitive patients, we found a different biological profile. At the kinase level, INKA analysis showed significantly increased inferred kinase activity of MAPK3 and EGFR (Figure 1d). EGFR is known for its activating effect on the MAPK signaling cascade70. Also the downstream substrates MAP2K1 and MAP2K2 were enriched in sensitive patients (Supplementary Figure 5), pointing towards MAPK as a contributing signaling pathway in this group. In line with these findings, two MAPK1 sites (T185 and Y187) that are known to induce the activity of the MAPK pathway71 were differentially phosphorylated in sensitive patients, as well as a uniquely identified EGFR site (Y1138) that is a known regulator of this pathway72. Several phosphorylated sites on different peptides identified in sensitive patients are being directly regulated by EGFR (PEAK1, EPHA2, TNK2, RPS27 and CAVIN1)72, supporting EGFR activation in sensitive patients. Based on these results, we propose that EGFR-driven MAPK signaling plays an important role in sensitivity to sunitinib in RCC, and may present an alternative target for (combination) treatment73. This corroborates the findings of Li et al who found their P3 phosphoproteomic subtype to be associated with the EGFR pathway and other kinases including MAPK3, that plays a role in VEGF/angiogenesis signaling58. PTM signatures associated with sunitinib sensitivity showed enrichment of VEGF, KIT, Thrombin signaling, vanadate and FGF2 treatment signatures (Figure 1e), pointing towards the anti-angiogenic effects of sunitinib74,75. Acknowledging the limited sample size of the sensitive (n = 16) and resistant (n = 7) tumors, our analyses may have been influenced by a number of other factors: (i) differences in pre-analytical handling of the frozen, archival specimen may have resulted in different cold ischemia times, potentially altering the phosphorylation profile76,77, (ii) the use of mostly primary tumor tissue, whereas treatment benefit is evaluated based on response of metastases and (iii) the range of intervals (median 6 months) between resection and start of systemic therapy may suggest indolent biology as a cause of longer PFS. However, we found no significant correlation between the time to start sunitinib and the PFS (Spearman’s rho -0.018). Also, the influence of longer storage time at -80 °C of samples on the phosphorylation profile is unknown. Our data are internally consistent based on reproducibly identified phosphosites and –peptides (see Figure 1b and Supplementary Figure 2b) as well as identified kinase-substrate relations (e.g. for INSR/IGF1R and INSULIN treatment; Figure 1c and 1d). Lacking an external data4
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