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 of TMB, stage, MSI-status and significantly altered genes are shown on the y-axis, with frequency of each alteration annotated on the left of the waterfall plot. (B) Pie chart displaying the proportion of FGFR2 alterations in TCGA STAD cohort (n=295). (C) FGFR2 mRNA expression levels were analyzed between FGFR2-amplified group (n=13) and FGFR2-unamplified group (n=250) from TCGA-STAD cohort. (D) Correlation between PTPN11 and FGFR2 mRNA expressions among samples from TCGA-STAD hohort. Data are shown as Mean±SEM. ****p < 0.0001. P values are detemined by unpaired t test or linear regression t test.

SHP099 enhances the antitumor effects and overcomes the resistance of FGFR2 inhibitors in FGFR2-amplified GC.

Sensitivity of (A) KATOIII and (B) SNU-16 to SHP099, AZD4547 or combination therapy with different concentration gradients. Effects of different treatments on cell apoptosis of (C) KATOIII and (D) SNU-16 after 48-hour drugs incubation. (E) KATOIII and (F) SNU-16 were incubated with vehicle, SHP099 10 μM, AZD4547 3nM 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 detemined by ordinary one-way ANOVA or two-way ANOVA.

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) Representive CT images displaying the occurrence and progression of ascites. Ascites are indicatied 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. Data are shown as Mean±SEM. *p < 0.05, ****p < 0.0001. P values are detemined 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.

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) Body weights, (C) tumor volumes and (D) tumor weights of different groups. (E) Representative images of tumors. (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 detemined by ordinary one-way ANOVA or two-way ANOVA.

SHP099 activates CD8+ T cells and promotes its 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. (C) Proportions of MSI-H or MSS in FGFR2-amplified group (n=12) and FGFR2-unamplified group (n=237) from TCGA-STAD cohort. 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) PD-1+/CD8+ cells and (E) IFN-γ/CD8+ cells were detected by flow cytometry assay after 24-hour of drugs incubation. (F) Expression levels of IFN-γ in cellular supernatant were measured by Cytometric Bead Array (CBA) assay after 24-hour of drugs incubation. (G, H) After 48-hour 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. (I) Cell viability of human PBMCs after 48-hour of drugs incubation. Data are shown as Mean±SEM. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. P values are detemined by Welch’s t test, Fisher’s exact test, ordinary one-way ANOVA or two-way ANOVA.

Schematic of the mechasims 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.