Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary

Abstract

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

Keywords: CUP; FGFR2; FGFR2 inhibitor; MEK inhibitor; cancer of unknown primary; infigratinib; liquid biopsy; occult primary tumors; patient-derived xenografts; trametinib.

Graphical abstract

Figure 1

Immunophenotypic characterization of CUP samples (A) Immunophenotypic characterization of tumor tissue, cell line, and PDX of CUP#55. Hematoxylin-eosin (HE), keratin7 (KRT7), and keratin20 (KRT20) IHC staining of CUP#55 tumor tissue, cell line, and PDX (40×). The staining showed that the cell line and PDX tumor recapitulate the histology of the patient's tumor tissue. (B) Immunophenotypic characterization of tumor tissue, cell line, and PDX of CUP#96. HE, KRT7, and KRT20 IHC staining of CUP#96 tumor tissue, cell line, and PDX (40x). CUP#96 cell line grows forming tumoroid structures. The staining showed that the cell line and PDX tumor recapitulate the histology of the patient's tumor tissue. (C) Immunophenotypic analysis of CUP #55 and #96 cell lines demonstrating the expression of cancer stem cell markers CD44 and EPCAM. Staining: nucleus (DAPI, blue), CD44 and EPCAM (green). Scale bars, 20 μm. (D) Gene expression analysis of stemness genes in CUP#55 and CUP#96. Bars represent the relative expression of genes using HPRT as reference gene and the 2^−ΔCt method. The expression in CUP cell lines was compared with other cancer cell lines from lung and melanoma origin.

Figure 2

Single-cell transcriptome analysis of CUP#55 (A) Scores computed by the scType method for putative tissues of origin for CUP#55 cells. (B) Cell clustering based on the similarity among expression profiles, obtained through the scLCA method; cells are separated in two distinct clusters. (C) The panels show the expression and distribution in CUP#55 single cells of the FGFR2 gene and other selected markers expressed in liver cancer stem cells and involved in the EMT process.

Figure 3

FGFR2 amplification in CUP models (A) FISH analysis shows the different nature of FGFR2 amplification in the two CUP cell models: double minutes for CUP#96 and HSR for CUP#55; FGFR2 gene probe is in red, Chr10 centromere probe in green, nucleus in blue. (B) Copy number variation (CNV) analysis of FGFR2 gene detected by ddPCR using a probe-based assay. (C) FGFR2 gene expression in the two amplified models and a control not amplified cell line. Bars represent the relative expression of genes using HPRT as reference genes and the 2^−ΔCt method. Data are represented as mean ± SD. Ordinary one-way ANOVA was used, ∗∗∗p <0.001. (D) Evaluation by western blot analysis of total and phosphorylated forms of FGFR2 in CUP#55 and #96, compared with the H1581 NSCLC cell line.

Figure 4

In vitro FGFR2 targeting and synergic activity of FGFR2 and MEK inhibitors in CUP#55 (A) CUP#55 cells were treated with BGJ398 at the indicated concentrations; after 24 h, protein extracts were analyzed by western blotting for the indicated proteins. The results are representative of two independent experiments. (B) CUP#55 cells were incubated with BGJ398 1 μM, trametinib 100 nM, or the combination; after 24 h, protein extracts were analyzed by western blotting for the indicated proteins. The results are representative of two independent experiments. (C) CUP#55 cells were treated with increasing concentrations of BGJ398 in combination with trametinib 100 nM. After 96 h, cell proliferation was assessed by MTS assay. The data are expressed as percent inhibition vs. control. The asterisks indicate the statistical significance vs. the corresponding points of the Bliss Theoretical curve. The results are representative of three independent experiments. (D) CUP#55 cells were incubated with BGJ398 1 μM and/or trametinib 100 nM; after 96 h, the percentage of cell death was evaluated by fluorescence microscopy after Hoechst 33342/PI staining. The data are mean values ± SD of three independent experiments. (E) CUP#55 cells were incubated with BGJ398 1 μM and/or trametinib 100 nM; after 72 h, the cleavage of caspase-3 was assessed by western blot analysis. The result is representative of two independent experiments. ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5

In vitro FGFR2 targeting and synergic activity of FGFR2 and MEK inhibitors in CUP#96 (A) CUP#96 cells were treated with BGJ398 at the indicated concentrations; after 24 h, protein extracts were analyzed by western blotting for the indicated proteins. The results are representative of two independent experiments. (B) CUP#96 cells were incubated with BGJ398 100 nM, trametinib 10 nM, or the combination; after 24 h, protein extracts were analyzed by western blotting for the indicated proteins. The results are representative of two independent experiments. (C) CUP#96 cells were treated with increasing concentrations of BGJ398 in combination with trametinib 10 nM. After 96 h, cell proliferation was assessed by MTS assay. The data are expressed as percent inhibition vs. control. The asterisks indicate the statistical significance vs. the corresponding points of the Bliss Theoretical curve. The results are representative of three independent experiments. (D) CUP#96 cells were incubated with BGJ398 100 nM and/or trametinib 10 nM; after 96 h, the percentage of cell death was evaluated by fluorescence microscopy after Hoechst 33342/PI staining. The data are mean values ± SD of three independent experiments. (E) CUP#96 cells were incubated with BGJ398 100 nM and/or trametinib 10 nM; after 72 h, the cleavage of caspase-3 was assessed by western blot analysis. The result is representative of two independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 6

In vivo activity of FGFR2 and MEK inhibitors In vivo treatment of (A) PDX#96 PDXs and (B) PDX#55 PDXs. The timeline shows the day of tumor volume measurement, and the color bars represent the scheme of each treatment. The line graph shows that the combination of trametinib+BGJ398 treatments (purple) is more effective than the single ones (trametinib in red and BGJ398 in blue) in both PDX models. n = 5 mice per group (mixed-effects analysis and unpaired t test were used). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.