Apatinib exhibits anti-leukemia activity in preclinical models of acute lymphoblastic leukemia

Abstract

Background: Acute lymphoblastic leukemia (ALL) is a clonal malignant disorder characterized by an uncontrolled proliferation of immature B or T lymphocytes. Extensive studies have suggested an involvement of angiogenesis signaling in ALL progression and resistance to treatment. Thus, targeting angiogenesis with anti-angiogenic drugs may be a promising approach for ALL treatment. In this study, we investigated the effectiveness of Apatinib, a novel receptor tyrosine kinase inhibitor selectively targeting VEGFR-2 in ALL cells.

Method: ALL cell lines were treated with different concentration of Apatinib and then CCK8 assay, flow cytometry were used to determine the IC₅₀ value and cell apoptosis, respectively. The effect of Apatinib against primary ALL cells from 11 adult patients and normal counterparts were also analyzed by apoptosis with flow cytometry. Next, we used western bolting and mass cytometry (CyTOF) assay to explore the underlying mechanism of the cytotoxicity of Apatinib. Finally, the anti-leukemia activity was further evaluated in an in vivo xenograft model of ALL.

Results: Our results showed that Apatinib significantly inhibited cell growth and promoted apoptosis in both B and T lineage ALL cell lines in a dose- and time-dependent manner. The IC₅₀ values of Apatinib against Nalm6, Reh, Jurkat and Molt4 for 48 h were 55.76 ± 13.19, 51.53 ± 10.74, 32.43 ± 5.58, 39.91 ± 9.88 μmol/L, and for 72 h were 30.34 ± 2.65, 31.96 ± 3.92, 17.62 ± 5.90, and 17.65 ± 2.17 μmol/L respectively. Similarly, Apatinib shows cytotoxic activity against primary adult ALL cells while sparing their normal counterparts in vitro. Moreover, Apatinib suppressed ALL growth and progression in an in vivo xenograft model. Mechanistically, Apatinib-induced cytotoxicity was closely associated with inhibition of VEGFR2 and its downstream signaling cascades, including the PI3 K, MAPK and STAT3 pathways.

Conclusion: Our study indicates that Apatinib exerts its anti-leukemia effect by inducing apoptosis through suppressing the VEGFR2 signaling pathway, supporting a potential role for Apatinib in the treatment of ALL.

Keywords: Acute lymphoblastic leukemia; Anti-angiogenic agent; Apatinib; Leukemia therapy; VEGFR2.

Fig. 1

Apatinib exhibits a dose- and time-dependent inhibition of proliferation of B and T-lineage ALL cell lines. B-lineage (a, b) and T-lineage ALL cells (c, d) were exposed to indicated concentrations of Apatinib for 48 or 72 h, and cell viability was subsequently determined by a CCK-8 kit

Fig. 2

Apatinib induces apoptosis of both B and T lineage ALL cells in a dose and time-dependent manner. B-lineage (a, b) and T-lineage ALL cells (c, d) were treated with indicated doses of Apatinib for 48 or 72 h, and the percentage of apoptotic cells was examined by Annexin V/PI double staining. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Apatinib shows preferential cytotoxic activity against primary adult ALL cells while sparing normal PBMCs. The percentage of apoptotic cells (Annexin V+) of primary ALL patients (a) and peripheral blood or bone marrow of healthy donors (b) after exposure to indicated doses of Apatinib for 48 h. *P < 0.05; **P < 0.01

Fig. 4

Apatinib down-regulates the phosphorylation of VEGFR2 and its downstream signaling pathway in ALL cells. Jurkat (a), Nalm6 (b) cells were treated with 0, 10, 20 and 40 μM Apatinib for 48 h, respectively. The protein level of VEGFR2, p-VEGFR2 and its downstream signaling pathways were examined by western blotting. β-actin and GAPDH was used as a loading control in this experiment

Fig. 5

The effect of Apatinib on intracellular signaling pathways was examined by CyTOF at the single-cell level in primary ALL. The primary ALL cells were exposed to 20 μM Apatinib for 48 h, and subjected to CyTOF analysis of intracellular signaling pathways downstream of VEGFR2

Fig. 6

Apatinib suppresses leukemia cell growth and leukemia progression in a xenograft model. Nalm6 cells were intravenously injected into NSI mice. 7 days later, mice were randomized (n = 6 per group) and treated with vehicle (control group) or Apatinib (administered by oral gavage at the dose of 100 mg/kg for 2 consecutive weeks). Percentage of human CD45+ (hCD45) cells in peripheral blood (a), bone marrow (b) and spleen (c) were determined by flow cytometry. d Spleens of mice were weighted at the end of the experiment. e Spleen and bone marrow of mice were embedded with paraffin and stained with H&E. Scale bar, 25 μm.*P < 0.05; **P < 0.01