Blocking SHP2 benefits FGFR2 inhibitor and overcomes its resistance in FGFR2-amplified gastric cancer
Figures
Figure 1 with 4 supplements
Recurrent FGFR2 gene amplification in Chinese GC patients.
(A) Overview of genomic alterations in GC patient samples collected in Nanjing Drum Tower Hospital (n=161). The patient samples are shown on the x-axis. Information on TMB, stage, MSI-status, and significantly altered genes is shown on the y-axis, with frequency of each alteration annotated on the left of the waterfall plot. Pie chart displaying the proportion of FGFR2 alterations in (B) TCGA STAD cohort (n=295) and (C) Nanjing Drum Tower Hospital cohort (n=161).
Figure 1—figure supplement 1
Overview of genomic alterations in GC patient samples collected in Nanjing Drum Tower Hospital (n=161).
The patient samples are shown on the x-axis. Information on TMB, stage, MSI-status, CPS score, and significantly altered genes is shown on the y-axis, with frequency of each alteration annotated on the right of the waterfall plot.
Figure 1—figure supplement 2
Proportions of different AJCC stages and TNM stages among FGFR2-amplified (n=9) group and FGFR2-unamplified group (n=124) from Nanjing Drum Tower hospital cohort.
Ns, not significant. p-Values of AJCC stages and T stages are determined by Pearson’s chi-square test; p-value of N stages is determined by Fisher’s exact test.
Figure 1—figure supplement 3
FGFR2 mRNA expression levels were analyzed between FGFR2-amplified group (n=13) and FGFR2-unamplified group (n=250) from TCGA-STAD cohort (unpaired t-test).
Data are shown as mean ± SEM. ns, not significant, ****p<0.0001. p-Value is determined by unpaired t-test.
Figure 1—figure supplement 4
Correlation between PTPN11 and FGFR2 mRNA expressions among samples from TCGA-STAD cohort (n=263; linear regression t-test).
p-Value is determined by linear regression t-test.
Figure 2 with 1 supplement
SHP099 enhances the anti-tumor effects and overcomes the resistance of FGFR2 inhibitors in FGFR2-amplified GC.
Sensitivity of KATOIII and SNU-16 to (A) SHP099, (B) AZD4547, or combination therapy with different concentration gradients (n=4; two-way ANOVA). Effects of different treatments on cell apoptosis of (C) KATOIII and (D) SNU-16 after 48-hour drugs incubation (n=3; one-way ANOVA). (E) KATOIII and (F) SNU-16 were incubated with vehicle, SHP099 10 μM, AZD4547 3 nM or combination therapies for 1 hour or 48 hours, then cell lysates were immunoblotted for phospho-FGFR and total-FGFR2, phospho-SHP2 and total-SHP2, phospho-Erk and total-Erk, phospho-p38 and total-p38, phospho-AKT and total-AKT, and phospho-mTOR and total-mTOR. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p-Values are determined by ordinary one-way ANOVA or two-way ANOVA.
-
Figure 2—source data 1
PDF file containing original western blots for Figure 2E and F, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104060/elife-104060-fig2-data1-v1.pdf
-
Figure 2—source data 2
Original file for western blots displayed in Figure 2E and F.
- https://cdn.elifesciences.org/articles/104060/elife-104060-fig2-data2-v1.zip
Figure 2—figure supplement 1
Expression levels of total-FGFR2 in different human GC cell lines were detected by western blotting.
Figure 3
The synergistic efficacy of combining AZD4547 with SHP099 in primary tumor cells derived from a FGFR2 inhibitor-resistant GC patient.
(A) Schematic of therapeutic process of the FGFR2-amplified GC patient who was previously sensitive to FGFR2 inhibitors but quickly became resistant. (B) Representative CT and MRI images of recurrent liver lesions at different time points. Liver lesions are indicated by yellow arrows. (C) Representative CT images displaying the occurrence and progression of ascites. Ascites are indicated by yellow arrows. (D) Pathology of malignant asites from this FGFR2 inhibitor-resistant patient. Tumor cells are indicated by red arrows. (E) AFP concentration variations of the patient during FGFR2 inhibitors medication. (F) Sensitivity of cancer cells from FGFR2 inhibitor-resistant patient’s asites to FGFR2 inhibitor AZD4547, SHP2 inhibitor SHP099 or combined administration with different concentration gradients (n=4; two-way ANOVA). Data are shown as mean ± SEM. *p<0.05, ****p<0.0001. p-Values are determined by two-way ANOVA. (G) Tumor cells from asites were incubated with vehicle, SHP099 5 μM, AZD4547 3 μM or combination therapy for 1 hour, then cell lysates were immunoblotted for phospho-FGFR and total-FGFR2, phospho-SHP2 and total-SHP2, phospho-Erk and total-Erk, phospho-AKT and total-AKT, and phospho-mTOR and total-mTOR.
-
Figure 3—source data 1
PDF file containing original western blots for Figure 3G, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104060/elife-104060-fig3-data1-v1.pdf
-
Figure 3—source data 2
Original file for western blots displayed in Figure 3G.
- https://cdn.elifesciences.org/articles/104060/elife-104060-fig3-data2-v1.zip
Figure 4 with 2 supplements
The combination of SHP099 and AZD4547 has significant anti-tumor effects in SNU-16 xenograft nude mice.
BALB/c nude mice were injected with 1×107 SNU-16 cancer cells and received different formulations by oral gavage every day upon tumor volumes reached 100–150 mm3. (A) Schematic of SHP099 and AZD4547 therapeutic schedule in FGFR2-amplified SNU-16 subcutaneous xenograft model. (B) Representative images of tumors (n=5). (C) Tumor volumes (n=5; two-way ANOVA), (D) tumor weights (n=5; one-way ANOVA) and (E) body weights (n=5; two-way ANOVA) of different groups. (F) Tumors were harvested 6 hours after the last dose of drugs, and cell lysates from tumor tissues were immunoblotted for phospho-FGFR and total-FGFR2, phospho-SHP2 and total-SHP2, phospho-Erk and total-Erk, phospho-AKT and total-AKT, and phospho-mTOR and total-mTOR. Data are shown as mean ± SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p-Values are determined by ordinary one-way ANOVA or two-way ANOVA.
-
Figure 4—source data 1
PDF file containing original western blots for Figure 4F, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/104060/elife-104060-fig4-data1-v1.pdf
-
Figure 4—source data 2
Original file for western blots displayed in Figure 4F.
- https://cdn.elifesciences.org/articles/104060/elife-104060-fig4-data2-v1.zip
Figure 4—figure supplement 1
Tumor volume of individual mice in control, SHP099, AZD4547, SHP099+AZD4547 group.
Figure 4—figure supplement 2
SHP2 inhibition combined with FGFR2 inhibition is safe in SNU-16 xenograft nude mice model.
Safety evaluations of different administrations of mice organs, including heart, liver, spleen, lung, and kidney, are shown by hematoxylin and eosin (H&E) staining (Scale bars, 100 µm).
Figure 5 with 5 supplements
SHP099 activates CD8+ T cells and promotes their tumor-killing capacity in vitro.
(A) Overview of FGFR2 alterations individually in GC patients from Nanjing Drum Tower hospital cohort. (B) PD-L1 mRNA expression levels were analyzed between FGFR2-amplified group (n=13) and FGFR2-unamplified group (n=250) from TCGA-STAD cohort (Welch’s t-test). (C) Proportions of MSI-H or MSS in FGFR2-amplified group (n=12) and FGFR2-unamplified group (n=237) from TCGA-STAD cohort (Fisher’s exact test). Human peripheral blood mononuclear cells (PBMCs) were incubated with different administrations in the presence of 0.25 μg/ml human anti-CD3 and 1 μg/ml human anti-CD28. Proportions of (D) IFN-γ/CD8+ cells were detected by flow cytometry assay after 24 hours of drugs incubation (n=3; one-way ANOVA). (E) Expression levels of IFN-γ in cellular supernatant were measured by Cytometric Bead Array (CBA) assay after 24 hours of drugs incubation (n=3; one-way ANOVA). (F) Cell viability of human PBMCs after 48 hours of drugs incubation (n=4; one-way ANOVA). (G, H) After 48 hours of advance drugs stimulation in human PBMCs, cytotoxicities of human PBMCs against SNU-16 at different E:T ratios of 10:1, 20:1, 40:1 were analyzed by flow cytometry assay after CFSE/PI staining (n=3; two-way ANOVA). Data are shown as mean ± SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. p-Values are determined by Welch’s t-test, Fisher’s exact test, ordinary one-way ANOVA or two-way ANOVA.
Figure 5—figure supplement 1
PD-L1 CPS outcomes among FGFR2-amplified group (n=6) and FGFR2-unamplified group (n=78) from Nanjing Drum Tower hospital cohort (Pearson’s Chi-square test).
Ns, not significant, p-value is determined by Pearson’s Chi-square test. CPS ≥ 1 was defined as CPS-positive and CPS＜1 was defined as CPS-negative.
Figure 5—figure supplement 2
MSI status among FGFR2-amplified group (n=10) and FGFR2-unamplified group (n=150) from Nanjing Drum Tower hospital cohort (Fisher’s exact test).
Ns, not significant, p-value is determined by Fisher’s exact test.
Figure 5—figure supplement 3
TMB levels were analyzed between FGFR2-amplified group (n=15) and FGFR2-unamplified group (n=274) from TCGA-STAD cohort (Wilcoxon test).
Data are shown as mean ± SEM. p-Value is determined by Wilcoxon test.
Figure 5—figure supplement 4
Proportions of IFN-γ/CD8+ cells were detected by flow cytometry assay after 24 hours of drugs incubation.
Human peripheral blood mononuclear cells (PBMCs) were incubated with different administrations in the presence of 0.25 μg/ml human anti-CD3 and 1 μg/ml human anti-CD28.
Figure 5—figure supplement 5
Proportions of IFN-γ/CD4+ cells were detected by flow cytometry assay.
Human peripheral blood mononuclear cells (PBMCs) were incubated with different administrations in the presence of 0.25 μg/ml human anti-CD3 and 1 μg/ml human anti-CD28 for 24 hours (n=3; one-way ANOVA). Data are shown as Mean ± SEM. ****p<0.0001. p-Values are determined by ordinary one-way ANOVA.
Figure 6
Schematic of the mechanisms of blocking SHP2 and FGFR2 in inhibiting tumor progression by targeted therapy and immune intervention.
On one hand, SHP2 inhibitor can boost the tumor-cytotoxicity effects of FGFR2 inhibitor by inhibiting PI3K/AKT and RAS/ERK pathways in FGFR2-amplified GC, and overcome FGFR2 inhibitor resistance caused by feedback activation. On the other hand, SHP2 inhibitor can activate CD8+ T cells to kill tumor cells, suggesting its synergizing effects with FGFR2 inhibitor by enhancing T cell-mediated anti-tumor immune responses. Our results demonstrate the utility and feasibility of combining SHP2 inhibitor to FGFR2 inhibitor in GC patients with FGFR2 amplification.
Author response image 1
Author response image 2
Author response image 3
Author response image 4
Author response image 5
Author response image 6
Author response image 7
Author response image 8
Author response image 9
Author response image 10
Author response image 11
Author response image 12
Author response image 13
Author response image 14
Author response image 15
Additional files
-
Supplementary file 1
Clinical characteristics of GC patients in Nanjing Drum Tower Hospital cohort with and without FGFR2 amplification.
- https://cdn.elifesciences.org/articles/104060/elife-104060-supp1-v1.pptx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/104060/elife-104060-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Blocking SHP2 benefits FGFR2 inhibitor and overcomes its resistance in FGFR2-amplified gastric cancer
eLife 14:RP104060.
https://doi.org/10.7554/eLife.104060.3
