Tumor Drug Concentration and Phosphoproteomic Profiles After Two Weeks of Treatment With Sunitinib in Patients with Newly Diagnosed Glioblastoma

Myra E van Linde, Mariette Labots, Cyrillo G Brahm, Koos E Hovinga, Philip C De Witt Hamer, Richard J Honeywell, Richard de Goeij-de Haas, Alex A Henneman, Jaco C Knol, Godefridus J Peters, Henk Dekker, Sander R Piersma, Thang V Pham, William P Vandertop, Connie R Jiménez, Henk M W Verheul, Myra E van Linde, Mariette Labots, Cyrillo G Brahm, Koos E Hovinga, Philip C De Witt Hamer, Richard J Honeywell, Richard de Goeij-de Haas, Alex A Henneman, Jaco C Knol, Godefridus J Peters, Henk Dekker, Sander R Piersma, Thang V Pham, William P Vandertop, Connie R Jiménez, Henk M W Verheul

Abstract

Purpose: Tyrosine kinase inhibitors (TKI) have poor efficacy in patients with glioblastoma (GBM). Here, we studied whether this is predominantly due to restricted blood-brain barrier penetration or more to biological characteristics of GBM.

Patients and methods: Tumor drug concentrations of the TKI sunitinib after 2 weeks of preoperative treatment was determined in 5 patients with GBM and compared with its in vitro inhibitory concentration (IC50) in GBM cell lines. In addition, phosphotyrosine (pTyr)-directed mass spectrometry (MS)-based proteomics was performed to evaluate sunitinib-treated versus control GBM tumors.

Results: The median tumor sunitinib concentration of 1.9 μmol/L (range 1.0-3.4) was 10-fold higher than in concurrent plasma, but three times lower than sunitinib IC50s in GBM cell lines (median 5.4 μmol/L, 3.0-8.5; P = 0.01). pTyr-phosphoproteomic profiles of tumor samples from 4 sunitinib-treated versus 7 control patients revealed 108 significantly up- and 23 downregulated (P < 0.05) phosphopeptides for sunitinib treatment, resulting in an EGFR-centered signaling network. Outlier analysis of kinase activities as a potential strategy to identify drug targets in individual tumors identified nine kinases, including MAPK10 and INSR/IGF1R.

Conclusions: Achieved tumor sunitinib concentrations in patients with GBM are higher than in plasma, but lower than reported for other tumor types and insufficient to significantly inhibit tumor cell growth in vitro. Therefore, alternative TKI dosing to increase intratumoral sunitinib concentrations might improve clinical benefit for patients with GBM. In parallel, a complex profile of kinase activity in GBM was found, supporting the potential of (phospho)proteomic analysis for the identification of targets for (combination) treatment.

Trial registration: ClinicalTrials.gov NCT02239952.

©2022 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.
Figure 1.
Sunitinib effect in vitro. Proliferation (MTT) assay of GBM and colorectal cancer cell lines incubated with increasing sunitinib concentrations, showing the percentage of proliferation compared with untreated controls (left) and calculated IC50 in micromoles per liter (right). Sunitinib inhibited proliferation of GBM tumor cells in vitro at concentrations 2.8 times higher than achieved intratumorally in patients (Table 1). GBM, glioblastoma; CRC, colorectal cancer.
Figure 2.
Figure 2.
Supervised clustering analysis of the tyrosine phosphoproteome. Supervised hierarchical clustering of differential phosphopeptides (P < 0.05) identified by tyrosine-phosphoproteomics shows separation of sunitinib-treated and control tumor tissues from 11 patients with glioblastoma. The heatmap shows the relative phosphopeptide intensities (z-score) in these samples based on log10-transformed values (orange, high abundance; blue, low abundance). SUN, sunitinib-treated; CON, control.
Figure 3.
Figure 3.
Protein interaction network. Protein interaction network of differential phosphopeptides (P < 0.05) between sunitinib-treated and control samples. Regulated phosphopeptides are mapped to proteins and visualized as protein interaction network. Green, Down- and Red, Upregulated in sunitinib-treated patients. Colored subdivisions indicate identification of multiple upregulated phosphopeptides mapping to the same protein. Note EGFR was identified with three upregulated phosphopeptides in the sunitinib-treated group.
Figure 4.
Figure 4.
Ranking of top 20 active kinases in tumors from 11 patients with glioblastoma. Ranked kinase activities in 7 control and 4 sunitinib-treated tumors. For each tumor, bar graphs depict kinase ranking based on combined INKA scores of kinase- and substrate-centric analysis of tyrosine-phoshoproteomics (12). For patient SUN-04, three biological replicates have been analyzed. Top bars of potential hyperactive kinases are highlighted by dark coloring (MAPK10 blue, EGFR red). Note the very high INKA score for MAPK10 in patient CON-08. Light blue and red colored bars indicate lower ranked MAPK10 and EGFR kinase activities, respectively. Green colors highlight kinases with “outlier activity”, i.e., higher activity in one versus the remaining 10 tumors (see Supplementary Fig. S3). SUN, sunitinib-treated; CON, control.

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