Anlotinib Potentially Eliminates Leukemia Stem Cells and Modulates the Chemosensitivity via Inhibiting JAK2-STAT3/5 Signaling

Leukemia stem cells (LSCs) remain as the critical barrier to cure of acute myeloid leukemia (AML) due to its chemoresistance. Here, we explore the role anlotinib -a multiple tyrosine kinases inhibitor in killing LSCs and regulating the chemoresistance. We found anlotinib could effectively induce apoptosis of LSC-like cells as well as primary CD34+ AML LSCs while sparing the normal mononuclear cells in vitro. The anti-leukemia activity of anlotinib was also conrmed in the mice model with Kasumi-1 cells; we further found that anlotinib could impair the regeneration capacity of LSCs in the patient-derived leukemia xenograft mouse model. Mechanistically, anlotinib could not only inhibit phosphorylation of c-kit and JAK2 /STAT3 and STAT5 but also downregulate STAT3 and STAT5 expression. In addition, anlotinib downregulated the anti-apoptotic protein Bcl-2 and Bcl-xl and upregulated Bax, thereby enhancing the sensitivity of LSCs to idarubicin in vitro. In conclusion, our results demonstrated anlotinib showed anti-LSCs activity and enhanced the chemosensitivity via inhibiting JAK2/STAT signaling in preclinical study and provided a rational basis for combinatory strategies that invoving anlotinib and idarubicin.


Introduction
The treatment of acute myeloid leukemia (AML) has been rapidly evolved from conventional chemotherapy to target therapeutics such as FLT3-ITD IDH2 inhibitors and subsequently contribute to an improvement in complete remission rates over the past decade 10 . However, novel therapeutics did not signi cantly reduce the relapse of AML, which led to the failure to converting complete remission to long term survival of patients 14 . Consequently, preventing relapse remains to be the key point during the management of AML.
Leukemia stem cells are a small population at the ape of AML differentiation hierarchy and characterized by its self-renewal and chemoresistance property 16 . LSCs are considered as the source of relapse after chemotherapy 6, 17 . Therefore, targeting LSC is a rationally promising strategy to prevent AML relapse. As previously reported, deprivation of cytokines such IL-3, G-CSF, SCF could induce gradual loss of stemness of LSC 16 . Accordingly, the phenotypic features such IL-3 receptor (CD123) and IL-2 receptor (CD25) high expression confer its capacity to receive cytokines signaling 4,9,11 . Recently, et al reported LSC displayed high expression of multiple growth factor receptors such c-kit, FMS-related tyrosine kinase 3(FLT3), which incurred the overactivation of their downstream tyrosine kinases such as Janus kinase 2 (JAK2) 4 . The JAK family of nonreceptor tyrosine kinases are critical mediators of cytokines and growth factor signaling 12 and represent an important signaling mechanism supporting AML LSC growth and survival 2 . JAK/STAT signaling could also regulate anti-apoptotic protein such as Bcl-2 and Bcl-xl as well as pro-apoptotic proteins 18 . Inhibition of JAK2 signaling could contribute to the blockade of LSC proliferation 4 , which implicated that targeting growth factor receptors and their downstream signaling might be effective to eliminate LSC. However, most tyrosine kinases inhibitor displayed their speci city on the target, which might not be suitable in multiple tyrosine kinase activation settings of LSC rationally.
Anlotinib as multiple kinase inhibitor could target c-kit, platelet derived growth factor receptor (PDGF-R), broblast growth factor receptor (FGF-R), vascular endothelial growth factor receptor (VEGF-R) has displayed its e cacy in non-small cell lung cancer and other cancers with mild adverse complications 13 .
Additionally, Liang L et al reported anlotinib could exert its effect by inhibiting JAK2 signaling 7 . Our previous data showed that other tyrosine inhibitor such as apatinib displayed its e cacy in elimination of AML cells 5 . Therefore, our study aim to exploring the effect of anlotinib in elimination of LSC and its potential mechanism.

Patients and donor samples
Primary samples from bone marrow of AML patients (n = 9) and peripheral blood of healthy donors (n = 8) were collected in Department of Hematology, First A liated Hospital of Xiamen University after patients were informed consent. This study was carried out in accordance with the Declaration of Helsinki and approved by the Ethics Review Board of First A liated Hospital of Xiamen University, Clinical characteristics of patients with AML are summarized in Table 1. Mononuclear cells were isolated by density gradient centrifugation using Ficol (BD, Franklin Lakes, NJ, USA) and supplemented with RPMI1640 and 10% FBS for short culture.
Cell viability assay Flow cytometry detection of apoptosis Apoptosis of LSC or LSC-like cells was detected by ow cytometry after staining of Annexin V and PI following the manufacturer's instruction. In brief, cells exposed to different treatments were harvested and washed with ice-cold PBS (Gibco, USA) and 1 × Annexin V binding buffer and then subjected to Annexin V-FITC/PI staining. Flow cytometry analysis was performed within 1h after staining. Primary samples with spontaneous apoptosis over 30% without treatment were excluded. The speci c apoptosis was adopted to adjust the variation in basal levels of spontaneous cell death following the formula: (% apoptosis in treated cells − % apoptosis in untreated cells)/(1 − % apoptosis in untreated cells) to analysis of the potential relationship between patients' characteristics and ex vivo e cacy of anlotinib, Western blot analysis Cells with 2 × 10 5 /well were treated with anlotinib for 12 h. Apoptosis of cells were tested to control apoptotic rates no more than 10% before immunoblot assays. Hereafter cells mentioned above were lysed in RIPA solution containing protease inhibitor cocktails. The protein level of each sample was

Mice models
All animal experiments were approved by the Animal Center of Xiamen University (Xiamen, China).
Kasumi-1 cells (6×10 6 ) were subcutaneously injected into 6-weeks-old BALB/c male nude mice (Animal Center of Xiamen University, Xiamen, China). The tumor volume (V) was calculated using V=L*W 2 /2 (L: tumor length, and W: tumor width) twice a week. After tumor volume reached 50 mm 3 , mice were randomly divided into 2 groups (n =5/group) by oral gavage of vehicle or anlotinib (6mg/kg/d) for 1 week respectively. All mice were sacri ced after anlotinib exposure for 7 days.

Human AML xenograft experiments
Pre-treated bone marrow mononuclear cells of AML patients with anlotinib (2.5μM) or vechicle for 12h respectively in vitro. To exclude the impact of apoptotic cells on engraftment of LSCs, we detected apoptosis of the two groups. The equal accounts of alive cells were injected into tail vein of NOD-Prkdc-/-IL2rg -/-(NSG) mice (purchased from DMO ltd., Beijing, China) after 1Gy irradiation. In order to con rm the engraftment, the peripheral blood will be stained with human CD45.At the end of this experiment, the NSG mice will be culled to analyze the tumor burden with human CD45-FITC (clone HI30, Biolegend) and murine CD45-BV421 antibodies (clone 30-F11, Biolegend) as well as CD34-APC( clone 581,Biolegend ).

Statistical analysis
The value was expressed as the mean ± standard deviation (S.E.M) for at least three independent experiments. The Student t-test or Wilcoxon and Mann-Whitney tests were applied in two-group comparison according to distribution of the data. Multiple-group comparisons were conducted using the One-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. P<0.05 was considered statistically signi cant. All statistical analyses were performed using SPSS 22.0 software (La Jolla, CA). The results showed that anlotinib treatment signi cantly inhibited Kasumi-1 and KG-1α proliferation in a dose-and time-dependent manner (Fig.1A, B). To investigate whether cell proliferation blockade were caused by apoptosis or cell cell cycle arrest. As a result, signi cantly higher percentages of apoptosis after anlotinib treatment in a dose-and time dependent manner were observed (Fig.1C, D). In addition, cell cycle arrest in G2/M phase were also observed after anlotinib treatment (Fig.1E, F). Therefore, these data indicated that the anti-LSC potential of anlotinib might be dependent on its cytotoxicity and the capacity of inducing cell cycle arrest in G2/M phase.

Anlotinib targets primary CD34+ AML cells while sparing normal hematopoietic cells ex vivo
Despite anlotinib could effectively kill LSC-like cells, the exact effect of anlotinib on primary LSCs remains unclear. Therefore, we treated the isolated mononuclear cells from the bone marrow of 9 AML patients with anlotinib with escalating doses. Clinical characteristics of AML patients were summarized in Table 1. Consistent with the anti-leukemia effect in LSC-like cell lines, anlotinib could also signi cantly induce the apoptosis of CD34+ AML cells in a dose-dependent manner ( Fig.2A). In addition, we further evaluated the adverse effects of anlotinib on normal blood cells. Mononuclear cells from the peripheral blood of healthy donors were treated with anlotinib for 48h. Intriguingly, even the highest dose (10μM) did not show signi cant cytotoxicity of mononuclear cells from peripheral blood the donors (Fig.2B).

Anlotinib exerted anti-leukemia effects and impaired the regeneration capacity of LSCs in vivo
To further investigate the anti-LSCs activity in vivo, we establish a subcutaneous leukemia xenograft mice model with Kasumi-1 cell line. Anlotinib was administered after tumor volume reached 50 mm 3 . As shown in gure, the tumor volume were signi cantly lower in the group treated by anlotinib after 7 days (Fig.3A). Regeneration of leukemia is the feature of LSCs that distinguish leukemia cells. Whether anlotinib could impair the regeneration capacity of LSCs remains unknown. Mononuclear cells of bone marrow from AML patients were pretreated by anlotinib or vehicle in vitro for 12h, the same amounts of alive cells were injected into tail vein of NSG mice to establish the PDX model of AML. As a result, we found anlotinib pretreatment signi cantly decrease the engraftment of hCD45+cells and hCD45+CD34+ cells in bone marrow of mice at the 4 th week (Fig.3C, D); additionally, the spleen weight in anlotinib group was also signi cantly less than the vehicle group (Fig.3B). Taken together, anlotinib could not only exert effective anti-leukemia effects but also could suppress the regeneration capacity of LSCs derived from AML patients.
The anti-LSCs activity of anlotinib might be associated with the inhibition of JAK-STAT pathway.
Overactivation of JAK/STAT signaling is constitutively increased in AML stem and progenitor cells due to the mutations of RTKs or growth factors stimulation and responsible for the self-renewal of LSCs 3 . Whether anlotinib could inhibit JAK-STAT pathway remains to be elucidated. We found that anlotinib could signi cantly inhibit the activation of c-kit and JAK2. Moreover, the phosphorylation of downstream transcript factors of JAK2 including STAT3 and STAT5 were inhibited after anlotinib treatment. Notably, the expression of STAT3 AND STAT5 were decreased by anlotinib as well. These results indicated anlotinib not only inhibited the activation of JAK2/STAT signaling but also downregulated STAT3 and STAT5 (Fig.4).
Anlotinib enhanced the chemosensitivity of LSCs via regulating apoptotic related-proteins.
Chemoresistance is considered as the key feature of LSCs 9 . Overexpression of anti-apoptotic proteins such as BCL-2 and Bcl-xl were linked to chemoresistance. Pro-apoptotic such as BAX and anti-apoptotic proteins were reported as the targets of JAK2/STAT signaling in various tumor such as leukemia and lymphoma 18, 21 . Hence we evaluated the role of anlotinib in regulating those proteins of LSCs. As shown in the gure, upregulation of Bax and inversely downregulation of Bcl-2 and Bcl-xl in Kasumi-1 and KG-1α were observed in anlotinib-treated group (Fig.4A,B) Thereafter, we further evaluated the role of anlotinib in modulating chemosensitivity by combining with idarubicin (IDA). As a result, anlotinib could signi cantly enhance the sensitivity of Kasumi-1 and KG-1α to IDA (Fig.4C,D).

Discussion
The current study demonstrated anlotinib displayed the effective anti-LSC activity which might associated with the inhibition of JAK2-STAT3/5 signaling. Additionally, anlotinib could signi cantly enhanced the sensitivity of LSCs to IDA by downregulating Bcl-2. Our results provide a strong scienti c rationale and impetus for application of anlotinib in AML.
As reported by Cook, A. M. et al , JAK2-STAT signaling overactivation caused by various cytokines or growth factors or mutation of receptor tyrosine kinases such as c-kit, FLT3 or PDGFR was associated with self-renewal of LSC 19 . Anlotinib could not only directly induce the apoptosis of LSC but also could inhibit the regeneration of LSC in vivo. Compared with the JAK2 inhibitor, anlotinib showed the mechanistic advantages by the inhibition of JAK2-STAT3/STAT5 activation and STAT3 and STAT5 expression. Such advantages might be associated with multiple target inhibition by anlotinib such as c-kit and PDGFR which could activate other oncogenic signaling including Ras/MAPK PI3K/AKT and NF-ΚB signaling besides JAK2 signaling 2 . However, further study are warranted to investigate the detailed mechanism. Additionally, hematopoiesis toxicity was reported as one of main adverse effects of JAK2 inhibitor in clinical practice 1 , which limited their widely application. However, both clinical data in non-small cell lung cancer 13 and our laboratory data showed that anlotinib spared no adverse effect on normal mononuclear cells.
IDA combined with cytarabine regimen is the frontline therapy for AML patients 3 . BCL-2 could interact with pro-apoptotic BH3 only proteins such as Bax, BIM, BID and subsequently blockade intrinsic apoptotic pathway 15 . Bcl-2 upregulation in leukemia cells contributed predominantly to the resistance of IDA or cytarabine in AML, which is one of main targets of JAK2-STAT3/5 pathway 20 . Inhibition of JAK signaling could downregulate the expression of Bcl-2 was demonstrated in lymphoma and pulmonary brosis 8, 21 . Consistent with the inhibition of JAK2-STAT3/5, anlotinib could also signi cantly decrease Bcl-2 expression and upregulate Bax expression in LSC-like cell lines. Combinatory experiments suggested anlotinib could enhance the sensitivity of LSCs to IDA.
In conclusion, our preclinical ndings provide the evidence for clinical implementation of anlotinib as a novel approach to eliminating LSCs and further displayed the synergistic potential of combination of anlotinib with IDA. Considering the limited toxicity of hematopoiesis and its synergic effect with IDA, anlotibib combined with IA regimen might be a promising strategy to deeper clearance of LSCs without causing further myelosuppression.