FGFR

 

Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured  using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

 

Activation of FGFR2 and MAPK by FGFR2-AHCYL1 and its suppression by FGFR inhibitors. Lysates from NIN3T3 cells expressing FGFR2-AHCYL1 or EZR-ROS1 (control) treated with vehicle (DMSO), 0.2 and 1 µM BGJ398, and 0.2 and 1 µM PD173074 were immunoblotted with the relevant antibodies. β-actin was used as a loading control.

BIBF 1120 inhibition of verapamil-stimulated ABCB1 ATPase activity. ABCB1 ATPase assays were performed according to the instruction of Pgp-Glo™ Assay Systems. Each point represents the mean±SDs for triplated independent determinations

Effect of HDIL-2/TKI on apoptosis of RCC cells. Three RCC cell lines treated with different concentrations of Pazopanib and HDIL-2 and incubated for 48 h. Microscopic images show apoptotic materials 48 h following treatment (arrows show the apoptotic materials in the pazopanib-treated cells).

FGFR inhibitors block signaling in FGFR2-fusion-expressing cells. Activation of FGFR2 and MAPK by FGFR2-AHCYL1 and its suppression by FGFR inhibitors. Lysates from NIH3T3 cells expressing FGFR2-AHCYL1 or EZR-ROS1 (control) treated with vehicle (DMSO), 0.2 and 1 uM BGJ398, and 0.2 and 1 uM PD173074 were immunoblotted with the relevant antibodies. β-Actin was used as a loading control.

 

Orthotopic xenografts of HNSCC cell lines (A) SCC-1 and (B) OSC-19 were treated with dovitinib (20 mg/kg/day). In addition, the OSC-19 xenografts were treated with +/ radiation therapy. Marker, mean for triplicate; bars, SE. Statistical significance by unpaired t-test, p < 0.05.

FCM evaluating lung CD11b+/Gr1+ myeloid cells isolated from 4T1 tumor-bearing mice after 20 days of treatment with AZD4547 or control. Statistical significance was assessed by unpaired t test.

Mice bearing subcutaneous allografts of conditional patched mutant tumor cells were treated twice weekly with vehicle (saline) or 30 mg/kg PHA-739358. (B)Images of tumors. (C) Tumor weights. Each point represents a single tumor, and grey lines represent mean tumor weights, which were significantly different between vehicle and PHA-739358 treated mice (p < 0.05, based on paired two-tailed t-test).

Dot Plot Distribution of Live, Preapoptotic and Apoptotic Cells after Administration of DuP-697 and E7080 Combination.

For MTT assays, cells (2,000 ~ 5,000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of Brivanib by adding 20 μl of 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution per 100 μl of growth medium. After incubating for 3-4 h at 37°C, the media were removed and 150 µl/well of MTT solvent (either absolute DMSO or isopropanol containing 4 μM HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells.

 

Breast cancer cells line MDA-MB-231 were treated with the indicated concentrations of ENMD-2076.

 

Three RCC cell lines treated with different concentrations of TKI and HDIL-2 and incubated for 48 h. Microscopic images show apoptotic materials 48 h following treatment (arrows show the apoptotic materials in the pazopanib-treated cells).

Both V-H cells and V-J cells are treated with MK-2461, XL-184 or ABT-869, respectively. (i-k) Western blot analysis of sFlt-1 expression after incubation with three VEGF-A receptor inhibitors or in the control.

After inoculation with KAT4 cells, administration of compounds including MK-2206 (100 mg/kg), tyrphostin AG 1296 (100 mg/kg), or a combination of MK-2206 (100 mg/kg) and tyrphostin AG 1296 (100 mg/kg) was performed. The combination of MK-2206 and tyrphostin AG 1296 induced significant apoptosis of KAT4 tumor cells in vivo measured by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay (green), and nuclei were stained with Hoechst (blue).

Western blot analysis of the expression and activation status (a) of pFGFR, pERK and Bim after treatment of API5-overexpressing cells (a) with either DMSO or SSR128129E.

A: Overall structure of LY2874455/FGFR4 complex. B: The diagram of LY2874455. C: Fo-Fc omit map of LY2874455 in the FGFR4/LY2874455 complex. The electron density is superimposed with the final model. D: The DFG motif conformation of FGFR4. Active ApoFGFR4 DFG-in conformation is shown in blue (PDB: 4QQT); FGFR4/Ponatinib DFG-out conformation is shown in yellow (PDB: 4UXQ); FGFR4/BLU9931 DFG-in conformation is shown in pink (PDB: 4XCU); FGFR4/LY2874455 DFG-in conformation is shown in grey (this work). LY2874455 is highlighted in brown.

Four FGFR inhibitors, namely PD-173074 (PD-74), PD-166866 (PD-66), SU5402 (SU54) and NVP-BGJ398 (BG-98), inhibit A673, SKNMC, POE, RDES and SKES Ewing cell growth in vitro in a dose-dependent manner, whereas normal cells (IMR90 fibroblasts) remained unaffected. PD-74 proved to be most effective in four out of five Ewing sarcoma cell lines tested. Cells were grown in 10% FBS conditions and cell proliferation was measured after 72 h using a Resazurin assay.

(c) Percent survival for the AGS cancer cell line is shown with FGFR2 inhibitors of varying specificity. (d) The KatoIII diffuse gastric cancer cell line was treated with FGFR2 inhibitors of varying specificity. The Y-axis depicts percent survival versus the X-axis with log concentrations. In all panels, error bars represent standard error of the mean. The difference in percent cell survival between KatoIII and AGS cells was statistically significant (P <0.05) at the three highest concentrations of all drugs, except Brivanib which was only significant at the highest concentration.

KYSE150 and KYSE450 cells were treated each day with 1 μM H3B‐6527 or left untreated for three days, and lysates were immunoblotted with indicated antibodies. Western blotting demonstrated that the expression of EMT‐related markers (E‐cadherin, N‐cadherin, Claudin‐1, Vimentin, and Snail) was altered with FGFR4 blockade.

E, Continuous ERK phosphorylation in FGFR inhibitor resistant cells under 24-hour treatment with 1 μmol/L BGJ398 assessed by immunoblotting. FGFR inhibitors: AZD4547, BGJ398, and JNJ-42756493

(E,G) Microscopic appearance of peritoneal dissemination of YTN16 (E) without BLU9931 and (G) with BLU9931 treatment for 3 weeks. (F,H) Microscopic features of s.c. tumor of YTN16 (F) without BLU9931 and (H) with BLU9931 treatment for 3 weeks. YTN16 tumor under treatment with BLU9931 does not form glandular structures.

 

Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured  using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

 

Activation of FGFR2 and MAPK by FGFR2-AHCYL1 and its suppression by FGFR inhibitors. Lysates from NIN3T3 cells expressing FGFR2-AHCYL1 or EZR-ROS1 (control) treated with vehicle (DMSO), 0.2 and 1 µM BGJ398, and 0.2 and 1 µM PD173074 were immunoblotted with the relevant antibodies. β-actin was used as a loading control.

BIBF 1120 inhibition of verapamil-stimulated ABCB1 ATPase activity. ABCB1 ATPase assays were performed according to the instruction of Pgp-Glo™ Assay Systems. Each point represents the mean±SDs for triplated independent determinations

Effect of HDIL-2/TKI on apoptosis of RCC cells. Three RCC cell lines treated with different concentrations of Pazopanib and HDIL-2 and incubated for 48 h. Microscopic images show apoptotic materials 48 h following treatment (arrows show the apoptotic materials in the pazopanib-treated cells).

FGFR inhibitors block signaling in FGFR2-fusion-expressing cells. Activation of FGFR2 and MAPK by FGFR2-AHCYL1 and its suppression by FGFR inhibitors. Lysates from NIH3T3 cells expressing FGFR2-AHCYL1 or EZR-ROS1 (control) treated with vehicle (DMSO), 0.2 and 1 uM BGJ398, and 0.2 and 1 uM PD173074 were immunoblotted with the relevant antibodies. β-Actin was used as a loading control.

 

Orthotopic xenografts of HNSCC cell lines (A) SCC-1 and (B) OSC-19 were treated with dovitinib (20 mg/kg/day). In addition, the OSC-19 xenografts were treated with +/ radiation therapy. Marker, mean for triplicate; bars, SE. Statistical significance by unpaired t-test, p < 0.05.

FCM evaluating lung CD11b+/Gr1+ myeloid cells isolated from 4T1 tumor-bearing mice after 20 days of treatment with AZD4547 or control. Statistical significance was assessed by unpaired t test.

Mice bearing subcutaneous allografts of conditional patched mutant tumor cells were treated twice weekly with vehicle (saline) or 30 mg/kg PHA-739358. (B)Images of tumors. (C) Tumor weights. Each point represents a single tumor, and grey lines represent mean tumor weights, which were significantly different between vehicle and PHA-739358 treated mice (p < 0.05, based on paired two-tailed t-test).

Dot Plot Distribution of Live, Preapoptotic and Apoptotic Cells after Administration of DuP-697 and E7080 Combination.

For MTT assays, cells (2,000 ~ 5,000 cells/well) were subcultured into 96-well plates according to their growth properties. Cell proliferation was assayed at 72 hr after treatment of Brivanib by adding 20 μl of 5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution per 100 μl of growth medium. After incubating for 3-4 h at 37°C, the media were removed and 150 µl/well of MTT solvent (either absolute DMSO or isopropanol containing 4 μM HCl and 0.1% Nonidet-40) was added to dissolve the formazan. The absorbance of each well was measured by ELx808 (BioTek, Winooski, VT) or Wallac Victor2 (Perkin-Elmer Life Sciences, Boston, MA) Microplate Reader. Viable cells are presented as percent of control, vehicle-treated cells.

 

Breast cancer cells line MDA-MB-231 were treated with the indicated concentrations of ENMD-2076.

 

Three RCC cell lines treated with different concentrations of TKI and HDIL-2 and incubated for 48 h. Microscopic images show apoptotic materials 48 h following treatment (arrows show the apoptotic materials in the pazopanib-treated cells).

Both V-H cells and V-J cells are treated with MK-2461, XL-184 or ABT-869, respectively. (i-k) Western blot analysis of sFlt-1 expression after incubation with three VEGF-A receptor inhibitors or in the control.

After inoculation with KAT4 cells, administration of compounds including MK-2206 (100 mg/kg), tyrphostin AG 1296 (100 mg/kg), or a combination of MK-2206 (100 mg/kg) and tyrphostin AG 1296 (100 mg/kg) was performed. The combination of MK-2206 and tyrphostin AG 1296 induced significant apoptosis of KAT4 tumor cells in vivo measured by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay (green), and nuclei were stained with Hoechst (blue).

Western blot analysis of the expression and activation status (a) of pFGFR, pERK and Bim after treatment of API5-overexpressing cells (a) with either DMSO or SSR128129E.

A: Overall structure of LY2874455/FGFR4 complex. B: The diagram of LY2874455. C: Fo-Fc omit map of LY2874455 in the FGFR4/LY2874455 complex. The electron density is superimposed with the final model. D: The DFG motif conformation of FGFR4. Active ApoFGFR4 DFG-in conformation is shown in blue (PDB: 4QQT); FGFR4/Ponatinib DFG-out conformation is shown in yellow (PDB: 4UXQ); FGFR4/BLU9931 DFG-in conformation is shown in pink (PDB: 4XCU); FGFR4/LY2874455 DFG-in conformation is shown in grey (this work). LY2874455 is highlighted in brown.

Four FGFR inhibitors, namely PD-173074 (PD-74), PD-166866 (PD-66), SU5402 (SU54) and NVP-BGJ398 (BG-98), inhibit A673, SKNMC, POE, RDES and SKES Ewing cell growth in vitro in a dose-dependent manner, whereas normal cells (IMR90 fibroblasts) remained unaffected. PD-74 proved to be most effective in four out of five Ewing sarcoma cell lines tested. Cells were grown in 10% FBS conditions and cell proliferation was measured after 72 h using a Resazurin assay.

(c) Percent survival for the AGS cancer cell line is shown with FGFR2 inhibitors of varying specificity. (d) The KatoIII diffuse gastric cancer cell line was treated with FGFR2 inhibitors of varying specificity. The Y-axis depicts percent survival versus the X-axis with log concentrations. In all panels, error bars represent standard error of the mean. The difference in percent cell survival between KatoIII and AGS cells was statistically significant (P <0.05) at the three highest concentrations of all drugs, except Brivanib which was only significant at the highest concentration.

KYSE150 and KYSE450 cells were treated each day with 1 μM H3B‐6527 or left untreated for three days, and lysates were immunoblotted with indicated antibodies. Western blotting demonstrated that the expression of EMT‐related markers (E‐cadherin, N‐cadherin, Claudin‐1, Vimentin, and Snail) was altered with FGFR4 blockade.

E, Continuous ERK phosphorylation in FGFR inhibitor resistant cells under 24-hour treatment with 1 μmol/L BGJ398 assessed by immunoblotting. FGFR inhibitors: AZD4547, BGJ398, and JNJ-42756493

(E,G) Microscopic appearance of peritoneal dissemination of YTN16 (E) without BLU9931 and (G) with BLU9931 treatment for 3 weeks. (F,H) Microscopic features of s.c. tumor of YTN16 (F) without BLU9931 and (H) with BLU9931 treatment for 3 weeks. YTN16 tumor under treatment with BLU9931 does not form glandular structures.