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PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
When the tumour size of xenografts reached ~ 2000 mm, biopsies were obtained and the mice were castrated.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The tumour growth inhibition (TGI) was calculated according to actual tumour weight using the formula: (W vehicle − W Test)/W Vehicle × 100% in which W is defined as actual tumour weight.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Standard procedures of immunohistochemistry staining have been described previously .
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Briefly, tumour tissue samples were processed for paraffin embedding.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The sections were stained with haematoxylin and eosin (H&E), human Ki‐67 (ZSGB‐BIO, Beijing, China) and in situ Cell Death Detection Kit (POD; Roche, Mannheim, Germany), respectively.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
We first tested the antiproliferative effects of nintedanib, sunitinib, imatinib, avapritinib and ripretinib against a panel of BaF3 cells transformed by KIT kinase; their proliferation depends on the KIT kinase (Fig. 1A, Table S1).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The results demonstrated that nintedanib displayed better potency than sunitinib or imatinib in KIT wt BaF3 cells (GI50 values were 0.058, 0.59 and 0.11 μm, respectively).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
For primary mutations in the juxtamembrane domain, including L576P and V559D/G, nintedanib exhibited better activity than sunitinib, imatinib, avapritinib and ripretinib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib, sunitinib and ripretinib were highly effective in BaF3 cells with the KIT V654A mutation and the mixed V654A/V559D and T670I/V559D mutations, which are secondary imatinib‐resistant mutations in the ATP‐binding pocket.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Interestingly, for BaF3 cells with the imatinib‐resistant gatekeeper mutation T670I, nintedanib was more potent than sunitinib, avapritinib and ripretinib (GI50 < 0.0003 μm vs 0.005 μm; 0.039μm; 0.017μm).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In addition, nintedanib showed similar potencies to avapritinib and ripretinib, and the efficacy is better than sunitinib or imatinib in activating KIT loop mutations, such as D816H/V and D820E/G/Y. Overall, these results suggested that most of the secondary imatinib‐resistant mutations in the activation loop and ATP‐binding pocket of KIT are sensitive to nintedanib, especially the gatekeeper mutation T670I, compared with sunitinib and imatinib, avapritinib and ripretinib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib inhibits KIT wt and KIT mutant in vitro. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The heatmap shows GI50 values (growth inhibitory activity) of nintedanib, imatinib, sunitinib, avapritinib and ripretinib in BaF3 isogenic cell lines whose proliferation were depend on KIT wt and KIT mutant kinase.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The cell lines were treated with drugs (0–10 μm) for 3 days (n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The heatmap shows that EC50 (effective concentrations) values was calculated by quantifying the levels of KIT Y703/Y719/Y823 relative to KIT after a various dose of nintedanib treatment for 2 h. (C) Relativity between GI50s and EC50s on KIT wt and mutants of BaF3 isogenic cell lines panel. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib inhibits KIT wt and KIT mutant proteins using ADP‐Glo biochemical assay.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Data are shown as mean ± SD (n = 2, independent experiments).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
High GI50 (EC50) and low GI50 (EC50) values were corresponded by red colour and green colour, respectively.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
To ensure that the antiproliferative effects of nintedanib in engineered BaF3 cells were due to mutations in on the KIT signalling pathway, we then examined its inhibitory effects on KIT Y703, Y719 and Y823 phosphorylation (Fig. 1B, Fig. S1 and Table S2).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Western blotting showed that nintedanib potently inhibited the autophosphorylation of KIT wt and KIT with V559A/D/G, L576P, T670I, D820E, Y823D or A829P mutations but was much less potent on the autophosphorylation of KIT with D816V and N822K mutations, which showed similar trends to the growth inhibition of the KIT BaF3 cells (Fig. 1C).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In addition, to test the inhibitory activity of nintedanib in cells with KIT wt and mutant proteins, we used an ADP‐Glo biochemical assay (Fig. 1D, Table S3).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The data revealed that nintedanib was potent against KIT wt (IC50 = 3.85 nm), KIT V654A (IC50 = 7.94 nm), KIT T670E (IC50 = 59.4 nm), KIT T670I (IC50 = 1.65 nm), KIT D816H and D816V (IC50 = 1.14 nm and 2.38 nm, respectively), KIT D820E (IC50 = 3.14 nm), KIT N822K (IC50 = 2.6 nm), KIT Y823D (IC50 = 3.5 nm), and KIT A829P (IC50 = 2.64 nm).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
It was more potent against the mixed mutation T670I/V559D (IC50 < 0.5 nm) but relatively less potent against the KIT T670E mutation.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
These data suggested that nintedanib can suppress the proliferation of a panel of BaF3 cells due to its inhibitory effect on primary and secondary KIT kinase mutations.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
We next assessed the antiproliferative effects of nintedanib against a series of GIST cell lines and human primary GIST cells derived from three GIST patients expressing KIT wt, KIT‐V559D or KIT‐K642E mutations.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The study showed that compared with imatinib and sunitinib, nintedanib was more potent against GIST‐T1 cells (GI50 = 1.7 nm; Fig. 2A), which harbour the Δ560–578 mutation in the juxtamembrane region of KIT.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib also showed potency similar to sunitinib against GIST882 cells, which harbours the primary K642E mutation in the KIT c‐helix, but it was more potent than imatinib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In addition, nintedanib was more effective than sunitinib in the GIST‐T1‐T670I and GIST‐5R cell lines, which are both imatinib resistant and harbour gatekeeper T670I and Δ560–578 KIT mutations.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
For GIST‐48B cells, which are KIT‐independent cells, none of the three compounds showed activity.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Similarly, in human primary GIST cells harbouring KIT wt and the KIT‐V559D and KIT‐K642E mutations, nintedanib displayed high potency in a dose‐dependent manner (Fig. 2B, Table S4).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib on human GIST cell lines and human primary GIST cells. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Antiproliferative effects of nintedanib against GIST‐T1, GIST‐882, GIST‐5R, GIST‐48B and GIST‐T1‐T670I cell lines.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The cell lines were treated with drugs (0–10 μm) for 3 days using CellTiter‐Glo assay.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Data are shown as mean ± SD (n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Anti‐proliferation of human primary GIST cells after treatment with nintedanib for 6 days using CellTiter‐Glo assay.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Data are shown as mean ± SD (n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Inhibition of signalling pathways of KIT in GIST‐T1, GIST‐882, GIST‐5R and GIST‐48B cell lines after treatment with nintedanib for 4 h (immunoblotting; n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib on cell cycle progression after treatment for 24–72 h (flow cytometry).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
This experiment was carried out once. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib induced GIST‐T1, GIST‐882, GIST‐5R and GIST‐48B cell line apoptosis.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
This experiment was conducted once.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
To further ensure on‐target efficacy in GIST cell lines, we tested the response of the KIT‐mediated signalling pathway upon nintedanib treatment (Fig. 2C).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib potently inhibited the phosphorylation of KIT, such as Y703, Y719 and Y823, and downstream mediators, such as AKT and STAT3, which was also observed in cell lines with high expression RTK drug targets , in GIST‐T1 and GIST‐882 (imatinib sensitive) cells.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Interestingly, in GIST‐5R (imatinib resistant) cells, nintedanib was more potent than sunitinib against KIT Y719 phosphorylation, which regulates SCF‐mediated cell migration , and both nintedanib and sunitinib displayed profound inhibitory effects on KIT Y703 and Y823 phosphorylation.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
As expected, in the GIST‐48B cell line, which is KIT independent, neither compound showed an effect on the downstream mediators of the KIT signalling pathway.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
We then evaluated the effects of nintedanib on cell cycle progression.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The results showed that nintedanib induced cell cycle arrest in the G0‐G1 phase starting at 30 nm in GIST‐T1 and GIST‐882 (imatinib sensitive) and GIST‐5R (imatinib resistant) cells but not in GIST‐48B (KIT independent) cells (Fig. 2D).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Nintedanib induced the apoptosis of KIT‐mutant GIST cell lines but not the KIT‐independent GIST‐48B cell line (Fig. 2E).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Together, these data demonstrated that nintedanib suppressed proliferation and KIT pathway signalling in imatinib‐resistant cells harbouring a KIT‐dependent resistance mutation and preclinical GIST models in vitro.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
GIST‐T1, KIT‐T670I/BaF3, GIST‐T1‐T670I and GIST‐5R mouse xenograft models were used to assess the in vivo antitumour efficacy of nintedanib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
At doses as high as 100 mg·kg·day, nintedanib caused no significant body weight changes in any treatment group.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In the GIST‐T1 mouse xenograft models, tumour growth was suppressed by nintedanib in a dose‐dependent manner, and the tumour growth inhibition (TGI) rate was 72.3% at the 50 mg·kg dose and 78.2% at the 100 mg·kg dose (Fig. 3A).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In the KIT‐T670I/BaF3‐ and GIST‐T1‐T670I‐inoculated mouse models, 100 mg·kg nintedanib nearly completely suppressed tumour growth, with TGI values of 93.3% and 80.3%, respectively, while imatinib showed a limited effect on tumour growth at the same doses (Fig. 3B,C).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
As expected, nintedanib inhibited the KIT signalling pathway in KIT‐T670I/BaF3 tumour tissues in a dose‐dependent manner (Fig. S2).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In the GIST‐5R mouse xenograft models, tumour growth was inhibited by nintedanib in a dose‐dependent manner, and the TGI rate was 71.6% at 100 mg·kg, while imatinib had no effect on tumour growth (Fig. 3D).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Immunohistochemical (IHC) staining showed that nintedanib inhibited cell proliferation and induced cell apoptosis in the GIST‐T1 and GIST‐5R mouse xenograft models in a dose‐dependent manner (Figs S3 and S4).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
These results confirmed that nintedanib elicited robust antitumour efficacy in KIT Δ560–578 and T670I mutation‐dependent tumour models at well‐tolerated doses.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In vivo antitumour efficacy of Nintedanib against GIST‐T1, KIT‐T670I/BaF3, GIST‐T1‐T670I and GIST‐5R mouse xenograft model. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib on GIST‐T1 mouse xenograft model (n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib on BaF3‐KIT‐T670I mouse allograft model (n = 5, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib on GIST‐T1‐T670I mouse xenograft model (n = 5, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib on GIST‐5R mouse xenograft model (n = 3, independent experiments).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Animals were treated orally once a day with a various of dose of the drugs.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Data are shown as mean ± SEM, n.s.,
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
not significant; *P‐value < 0.05; **P‐value < 0.01; ****P < 0.0001 (one‐way ANOVA).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
As previously reported , imatinib resistance in GISTs in which FGF2 is universally highly expressed can be induced by MAP kinase activation through FGF signalling.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Therefore, we next investigated whether FGFR‐mediated reactivation of the MAPK pathway can attenuate the antiproliferative effect of nintedanib in GISTs (Fig. 4A).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The results showed that nintedanib was more potent than imatinib and sunitinib against the GIST‐T1 and GIST‐882 cells with FGF signalling pathway activated by FGF2 ligand.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
To address the molecular mechanism underlying the growth inhibition of the GIST‐T1 and GIST882 cell line by nintedanib in the presence of 20 ng·mL FGF2, we tested the activated states of downstream mediators of the KIT and FGF signalling pathways (Fig. 4B).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
As expected, nintedanib strongly inhibited the KIT downstream signalling pathway and phospho‐FRS2α, the major downstream substrate of FGFRs.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
By comparison, imatinib inhibited only the autophosphorylated KIT and downstream signalling pathways but not ERK, which was due to reactivated FGFR signalling.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
To test whether nintedanib can overcome the resistance induced by imatinib, which enables FGFR signalling activation, we examined the effects of nintedanib on KIT and FGFR‐mediated signalling pathways after imatinib treatment (Fig. 4C).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The results demonstrated that nintedanib could strongly inhibit FGFR signalling.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Interestingly, the phosphorylation of histone 2A family member X (γ‐H2AX) , which is an indication for DNA injury, was remarkably increased in this experiment (Fig. S5).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
We then tested the effects of nintedanib on human primary GIST cells that expressed the KIT‐V559D, KIT‐K642E and KIT‐wt in KIT (Fig. 4D).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The results showed that nintedanib could strongly inhibit the autophosphorylated KIT, phospho‐FRS2α and KIT downstream signalling pathways with 20 ng·mL FGF2.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In addition, nintedanib increased the phosphorylation of H2AX in the primary tumour cells derived from KIT‐V559D and KIT‐K642E GIST patients, which suggested that nintedanib could induce apoptosis in part through DNA double‐strand breaks (Fig. S6).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Anti‐proliferation of human GIST cancer cell lines and patient‐derived primary cells after treating with nintedanib and imatinib within FGF‐2. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib, imatinib and sunitinib on proliferation of GIST‐T1 and GIST‐882 cell lines within 20 ng·mL FGF2 for 3 days using CellTiter‐Glo assay.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Data are shown as mean ± SD (n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib and imatinib on the FGFR and KIT signalling pathways in GIST‐T1 and GIST‐882 cell lines within 20 ng·mL FGF2 for 4 h (immunoblotting; n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
GIST‐T1 and GIST‐882 cell lines were treated with imatinib for 4 h to activate FGF/FGFR signalling pathway, after which imatinib was removed and the cells were treated with nintedanib for 4 h for immunoblotting analysis.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
This experiment was conducted once. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib and imatinib on the FGFR and KIT signalling pathways in three GIST patients within 20 ng·mL FGF2 for 4 h (immunoblotting).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Considering a previous report showing that the effect of imatinib was reduced in a GIST882 xenograft model by inducing activation of FGFR signalling , we tested the antitumour efficacy of nintedanib on GIST882 tumour‐bearing mice.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
A GIST882 xenograft model was treated with imatinib or nintedanib twice daily for 1 day and 4 days.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
The phosphorylation of FRS2α was increased after treatment with imatinib for 4 days, a finding not found with nintedanib treatment and was consistent with the imatinib‐induced activation of the FGF signalling pathway (Fig. 5A).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
We next determined whether nintedanib was more effective than imatinib in vivo.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
In the GIST882 cell‐inoculated mouse xenograft model, nintedanib at doses as high as 100 mg·kg·day showed no apparent toxicity (Fig. 5B).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Tumour growth was inhibited by nintedanib in a dose‐dependent manner, and the TGI rate was 60.8% at 100 mg·kg·day, whereas imatinib showed a limited effect on tumour growth at the same dose.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
After drug withdrawal, nintedanib displayed a long‐lasting response (Fig. 5C,D).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
As expected, nintedanib, but not imatinib, reduced the phosphorylation of FRS2α, which is a downstream substrate of FGFR (Fig. 5E).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
These results support the notion that the application of nintedanib may be a new strategy for enhancing the treatment for GIST patients with de novo or acquired resistance to imatinib.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effect of Nintedanib and Imatinib in GIST‐882 mouse xenograft models in vivo. (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib and Imatinib on the FGFR signalling pathways in GIST‐882 mouse xenograft models (control, n = 2, independent experiments; treated, n = 3, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
B‐D) Effect of nintedanib and imatinib on GIST‐882 mouse xenograft models (n = 5, independent experiments). (
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Effects of nintedanib and imatinib on the FGFR signalling pathways in GIST‐882 mouse xenograft models after treated by 40 days (n = 3, independent experiments).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Animals were treated orally once a day with a various of dose of the drugs.
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Data are shown as mean ± SEM, *P‐value < 0.05; **P‐value < 0.01; ***P‐value < 0.001 (one‐way ANOVA).
PMC9019892
Nintedanib overcomes drug resistance from upregulation of FGFR signalling and imatinib‐induced KIT mutations in gastrointestinal stromal tumours
Because of advancements in next‐generation sequencing technology, it has been found that secondary mutations of kinases are not the only mechanisms of drug resistance and that the activation of alternative pathways can also contribute to drug resistance.