Infigratinib and FGF401 synergistically inhibited tumor growth in HCC PDX models
We first compared the antitumor activity of combination infigratinib/FGF401 with infigratinib, FGF401, and sorafenib in HCC models that express either FGF19, FGFR2/3/4, or both (Supplementary Table 1). Both infigratinib and FGF401 had similar antitumor efficacy in the HCC09-0913 model (high FGF19 and FGFR2/3/4; Fig. 1A–C). Infigratinib showed better antitumor activity than FGF401 or sorafenib in the HCC01-0909 model (High FGFR2/3/4 but very low FGF19; Fig. 1D–F, p < 0.05). Sorafenib inhibited tumor growth by approximately 40% in HCC09-0913 and 50% in HCC01-0909 models (Fig. 1, p < 0.05). In contrast, combination infigratinib/FGF401 exerted nearly complete growth inhibition and showed superior antitumor activity than FGF401 or infigratinib alone in both models (p < 0.05). Supplementary Table 2 shows the T/C ratios for nine HCC models treated with infigratinib, FGF401, or combination infigratinib/FGF401. For the eight FGFR-dependent models, the response rates for infigratinib, FGF401 and infigratinib/FGF401were 50% (4/8 with T/C < 0.42), 50% (4/8 with T/C < 0.42) and 100% (8/8 with T/C ≤ 20%), respectively. Although the sorafenib-resistant HCC06-0606Sora87 model was modestly sensitive to infigratinib and completely resistant to FGF401, its growth was significantly suppressed by combination infigratinib/FGF401 (p < 0.001, Supplementary Table 2).
FGF401 significantly inhibited the growth of high FGF19/FGFR4-expressing HCC models (HCC09-0913, HCC13-0212, HCC26-1004, and HCC29-1104) but had no activity in high FGFR2/3-expressing but undetectable FGF19 models (HCC26-0808A, HCC06-0606, HCC06-0606Sor87) (Supplementary Table 2, Supplementary Data 1). Conversely, infigratinib significantly inhibited the growth of high FGFR2/3 but undetectable FGF19 expressing models. HCC01-0909 model was sensitive to infigratinib and modestly to FGF401 (Supplementary Table 2; Fig. 1D-F). FGF401 synergistically acted with infigratinib to enhance antitumor efficacy in all 8 HCC models tested (Supplementary Table 2). In contrast, infigratinib, FGF401, and combination infigratinib/FGF401 exhibited no significant antitumor activity in HCC10-0505 model, which expressed undetectable levels of FGF19, FGFR1/2 and low levels of FGFR3 (Supplementary Table 2; Supplementary Data 1).
We next sought to determine if lower doses of both drugs could achieve antitumor efficacy similar to that of standard doses. Treatment of mice bearing HCC09-0913 tumors with 15:30, 10:20, and 7.5:15 mg/kg infigratinib:FGF401 for 9 days led to approximately 97.2%, 93.2%, and 88.5% reductions in tumor burden, respectively (Fig. 2A-C, p < 0.0001). Similarly, treatment of mice bearing HCC01-0909 xenografts with 15:30, 10:20, 7.5:15, and 5:10 mg/kg infigratinib:FGF401 for 15 days led to approximately 98.0%, 98.3%, 97.1%, and 96% reductions in tumor burden, respectively (Fig. 2D-F, p < 0.0001). In these two models, 10 mg/kg infigratinib and 20 mg/kg FGF401 yielded efficacy similar to that as the standard dose of 15 mg/kg infigratinib and 30 mg/kg FGF401, suggesting that dose reduction is possible without compromising the efficacy. Throughout the course of treatment, no significant body weight loss or other signs of toxicity were observed in mice from treatment groups compared with those from the vehicle group, suggesting that the treatments are well-tolerated (Supplementary Data 2A). Moreover, combination infigratinib/FGF401 resulted in the increase in white blood cells, lymphocytes, monocytes, platelets, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST), and a significant decrease in serum creatinine (Supplementary Data 2B, p < 0.01).
Combination infigratinib/FGF401 induced cell cycle arrest and apoptosis in HCC cells in vitro
Infigratinib and FGF401 significantly induced G1 cell cycle arrest (Supplementary Data 1A). Cells treated with combination infigratinib/FGF401 showed a higher proportion of sub-G1 phase in a dose-dependent manner, compared with those treated with infigratinib or FGF401 alone, suggesting that the combination is more potent than a single agent in inducing apoptosis.
Combination infigratinib/FGF401 induced apoptosis, inhibited cell proliferation, switched off the angiogenic rescue program and reduced tumor hypoxia via blood vessel normalization
In high FGF19 expressing HCC09-0913 model, FGF401 was more potent than infigratinib in inhibiting cell proliferation but had similar potency to induce apoptosis. FGF401 was more potent than infigratinib to induce apoptosis but had similar potency to inhibit cell proliferation in HCC13-0212 (Supplementary Data 3A). In contrast, infigratinib, but not FGF401, significantly inhibited cell proliferation in high FGFR2/3 expressing HCC01-0909 model (Fig. 3A, p < 0.01). Both Infigratinib and FGF401 had minimal or no effect on apoptosis in HCC01-0909 model (Fig. 3B). Neither FGF401 nor infigratinib had any significant effect on proliferation or apoptosis of low FGFR2/3 HCC10-0505 tumors (Supplementary Data 3B). In high FGFR2/3- or FGF19-expressing tumors, the infigratinib/FGF401-treated group showed a lower number of p-Histone 3-positive cells and more cleaved PARP-positive cells than treatment with infigratinib or FGF401 alone (Fig. 3A-B, Supplementary Data 3A-B, p < 0.001), suggesting a combination effect in promoting apoptosis and inhibition of cell proliferation.
Blood vessels in the infigratinib- and to a lesser extent, FGF401-treated HCC09-913 and HCC01-0909 tumors were slim, resembling capillary-like vessels (Fig. 3A-B). Furthermore, the majority of capillary-like blood vessels induced by infigratinib and combination infigratinib/FGF401 were positive for lectin, suggesting productive blood vessels. Hypoxyprobe staining was negative across large sections of these tumors, indicating that the regions were well-oxygenated. In HCC09-0913, combination FGF401/infigratinib did not induce any significant alteration in total blood vessels, number of lectin-positive blood vessels, and tumor hypoxia compared with infigratinib or FGF401 monotherapy (Fig. 3, Supplementary Data 4). In HCC01-0909, significant increase in total blood vessels and number of lectin-positive blood vessels were observed (Fig. 3B, Supplementary Data 4, p < 0.01). Neither FGF401 nor infigratinib had any significant effect on total blood vessels of low FGFR2/3 HCC10-0505 tumors (Supplementary Data 3B).
We next analyzed the mRNA levels of proangiogenic factors in vehicle- and drug-treated tumors using qRT-PCR. In the high FGFR2/3 HCC21-0208 model, infigratinib- and FGF401-treated tumors had two-fold lower levels of VEGF and HIF-1α than vehicle-treated tumors (Supplementary Data 3C, p < 0.05). bFGF expression was undetected in infigratinib-, FGF401-, and combination infigratinib/FGF401-treated samples. Combination infigratinib/FGF401 further decreased the levels of VEGF, CYR61, PDGF-AA, and HIF-1α mRNAs. Infigratinib-treated high FGFR2/3-expressing HCC01-0909 tumors showed a four-fold lower level of VEGF mRNAs and a two-fold lower level of CYR61 mRNAs compared to the vehicle-treated tumors in the model (Supplementary Data 3D, p < 0.05). Further reduction of VEGF, CYR61, and PDGF-AA mRNA levels were observed with combination infigratinib/FGF401. Similar data were obtained when mice bearing high FGF19-expressing HCC13-0212 tumors were treated with combination infigratinib/FGF401 (Supplementary Data 3E). There were no significant changes in the expression of proangiogenic factors in low FGFR-expressing HCC10-0505 tumors treated with infigratinib, FGF401, or combination infigratinib/FGF401 (Supplementary Data 3F).
Infigratinib, but not FGF401, inhibited p-FRS2α and p-ERK1/2 in HCC01-0909 and HCC06-0606 (high FGFR2/3 and undetectable FGF19). Both FGF401 and infigratinib potently reduced p-FRS2α and p-ERK1/2 in HCC09-0913 (high FGF19 and FGFR2/3 expression). In the three models analyzed, treatment with combination infigratinib/FGF401 further reduced the levels of FGFR2/3, p-FRS2α, p-p70S6K/4EBP1/S6R, p-ERK1/2, p-Cdk2, p-Cdc2, CDC25C, p-Rb, p-Cdc2, c-Myc, cyclin D1, and survivin compared with infigratinib or FGF401 treatment alone. Significant upregulation of p27, dephosphorylated form of Bim (fast-migrated form) and cleaved caspase 3 were detected in infigratinib/FGF401-treated tumors (p < 0.05; Fig. 4). However, changes in p-ERK1/2, p-AKT, survivin, p-Cdk2 and cyclin D1 were not detected in the infigratinib/FGF401-treated HCC10-0505 tumors, which expressed low FGFRs and null-FGF19 (Supplementary Data 5).
As shown in Fig. 5A, infigratinib but not FGF401 significantly suppressed the growth of high FGFR2/3-expressing HCC01-0909 tumors (p < 0.01). However, combination infigratinib/FGF401 showed further inhibition of tumor growth compared to infigratinib or FGF401 alone (p < 0.01). In this model, lung metastasis was detected in 90% (9/10), 20% (2/10), 80% (8/10), and 0% (0/10) of vehicle-, infigratinib-, FGF401-, and infigratinib/FGF401-treated mice, respectively. The survival of mice bearing HCC01-0909 tumors treated with combination infigratinib/FGF401 was significantly longer than that of mice treated with single agents (Fig. 5B; p < 0.001, log-rank test). For the high FGF19- and FGFR2/3-expressing HCC09-0913 model, more than 60% of the mice in the combination group were still alive on day 250 (Fig. 5B; p < 0.001, log-rank test). Thus, combination infigratinib/FGF401 inhibited lung metastasis and prolonged the survival of mice bearing FGF19- or FGFR2/3-dependent tumors.
We next investigated whether inhibition of the FGF19/FGFR signaling pathway improves intratumoral immune cell infiltration. Compared with infigratinib or FGF401 alone, combination infigratinib/FGF401 caused insignificant changes on body weight (Fig. 6A) but significantly reduced tumor growth (Fig. 6B) and tumor weight (Fig. 6C). The tumor regression in Infigratinib/FGF401 was associated with significant increase in the infiltration of CD8 + T-cells, CD4 + T-cells, CD11c + dendritic cells, B-cells, and macrophages concomitant with granzyme-B mediated apoptosis (Supplementary Data 7A-B, p < 0.01). In contrast, infigratinib or FGF401 monotherapy caused an insignificant increase in immune cell infiltration (Supplementary Data 7B).