Anlotinib Induced Apoptosis and Regulated the Chemosensitivity and Immune-Related Properties of Leukemia Stem Cells By Inhibiting JAK2-STAT3/5 Signaling

Acute myeloid leukemia (AML) is derived from small populations of leukemia stem cells (LSCs) characterized by the self-renewal and chemoresistant properties. Residual LSCs after chemotherapy remain as the critical barriers to cure. Clearance of LSCs might rationally lead to an improvement of clinical outcome. Recently studies showed that JAK/STAT signaling play an important role in the self-renewal of AML-LSCs due to increased growth factor (GF) receptor expression such as c-kit, FLT3, CD123 and altered GF signaling by activating tyrosine kinases. Therefore, targeting such tyrosine kinases might be a strategy to eliminate LSCs. Anlotinib displayed its anti-tumor activity in lung cancer by targeting tyrosine kinase of VEGFR, FGFR, PDGFR and c-kit. However, whether anlotinib could inhibit the GF receptor-related tyrosine kinase overactivation and its downstream JAK-STAT signaling, and subsequently kill LSCs or regulate LSCs biology remains largely unknown.

To explore whether anlotinib could exert effective ani-LSCs activity, we treated LSC like cell lines (CD34+CD38-KG-1 and Kasumi-1) with anlotinib, and found anlotinib could effectively induce apoptosis of LSC-like cells in a dose- and time-dependent manner. Similar results were observed in primary CD34+CD38-AML LSCs; notably, anlotinib did not significantly kill normal CD34+ cells in vitro. Additionally, the anti-LSC activity of anlotinib was further confirmed in the xenograft mouse model by injection of Kasumi cells (LSC-like cell line) into irradiated female BALB/c nude mice.

To determine whether anlotinib could inhibit the over activation of the GF receptor-related tyrosine kinase, we performed western blot at 12h after anlotinib treatment when LSC-like cells did not showed significant apoptosis. As a result, anlotinib inhibit c-kit phosphorylation and JAK2 activation. Intriguingly, unlike JAK2 inhibitors, anlotinib could not only the inhibit phosphorylation of STAT3 and STAT5 but also downregulate their expression.

Chemoresistance and immune evasion were the key features of LSCs, JAK2-STAT3/5 signaling was reported to involved in chemoresistance by upregulating anti-apoptotic proteins such as Bcl-2 ,Mcl-1 and also involved in immune escape by inducing immune suppressive molecules such as PD-L1 ,TGF-β.Thus we evaluated Bcl-2 expression and found a significant decrease in LSC-likes cells after anlotinib treatment. Similarly, PD-L1 and TGF-β were also significantly downregulated after anlotinib treatment.

In conclusion, anlotinib not only displayed the effective anti-LSCs activity but also might regulate the chemoresistance and immune evasion of LSC by downregulating the anti-apoptotic proteins and suppressive molecules such as PD-L1, TGF-β respectively. Consequently, anlotinib might has the potential to contribute to a deeper clearance of LSCs by combining with chemotherapy or immunotherapy.