Gas6/AXL Signaling Regulates Self-Renewal of Chronic Myelogenous Leukemia Stem Cells by Stabilizing Β-Catenin

Published OnlineFirst November 16, 2016; DOI: 10.1158/1078-0432.CCR-16-1298

Cancer Therapy: Preclinical Clinical Cancer Research Gas6/AXL Signaling Regulates Self-Renewal of Chronic Myelogenous Leukemia Stem Cells by Stabilizing b-Catenin Yanli Jin1, Danian Nie2, Juan Li3, Xin Du4, Yuhong Lu5, Yangqiu Li5, Chang Liu1, Jingfeng Zhou1, and Jingxuan Pan1,6

Abstract

þ Purpose: Quiescent leukemia stem cells (LSC) are important renewal capacity of human CML CD34 cells. Pharmacologic resources of resistance and relapse in chronic myelogenous leu- inhibition of AXL reduced the survival and self-renewal capacity of kemia (CML). Thus, strategies eradicating CML LSCs are required human CML LSCs in vitro and in long-term grafts in NSI mice. þ for cure. In this study, we discovered that AXL tyrosine kinase was Human CML CD34 cells conscripted bone marrow–derived þ selectively overexpressed in primary CML CD34 cells. However, stromal cells (BMDSC) and primary mesenchymal stem cells the role of AXL and its ligand Gas6 secreted by stromal cells in the (MSC) to secrete Gas6 to form a paracrine loop that promoted regulation of self-renewal capacity of LSCs has not been well self-renewal of LSCs. Suppression of AXL by shRNA and inhibitor investigated. prolonged survival of CML mice and reduced the growth of LSCs þ Experimental Design: The function of CML CD34 cells was in mice. Gas6/AXL ligation stabilizes b-catenin in an AKT-depen- þ evaluated by ﬂow cytometer, CFC/replating, long-term culture- dent fashion in human CML CD34 cells. initiating cells (LTC-IC), CML mouse model driven by human Conclusions: Our ﬁndings improve the understanding BCR-ABL gene and NOD-scid-IL2Rg / (NSI) mice. of LSC regulation and validate Gas6/AXL as a pair of thera- Results: AXL was selectively overexpressed in primary CML peutic targets to eliminate CML LSCs. Clin Cancer Res; 1–14. þ CD34 cells. AXL knockdown reduced the survival and self- 2016 AACR.

Introduction mesylate (IM; ref. 3). However, acquired resistance to IM conferred by multiple mechanisms [e.g., BCR-ABL mutations (4), Chronic myelogenous leukemia (CML) is a type of myelopro- leukemia stem cells (LSC; ref. 5)] is an emerging problem. In liferative disorder characterized by uncontrolled clonal expansion addition, BCR-ABL–positive pediatric acute lymphoblastic leuke- of immature granulocytes and their precursors (1). CML is mia and adult accelerated phase- or blast-crisis-CML usually believed to stem from the malignant transformation of bone respond poor to TKIs (6). marrow hematopoietic stem cells (HSC) triggered by a fusion LSCs in CML show common characteristics of cancer stem cells gene BCR-ABL because of a reciprocal chromosomal translocation (CSC; refs. 7–9). These traits endow LSCs with resistance to IM. t(9,22)(q34;q11) (2). Most adult patients with chronic-phase CML patients with complete molecular response, still showed CML respond well to tyrosine kinase inhibitor (TKI) imatinib long-term BCR-ABL–negative LSCs (10). Only approaches to effectively eliminate LSCs when combined with IM may help cure CML (7, 8, 11). Therefore, development of rational 1Jinan University Institute of Tumor Pharmacology; State Key Laboratory of approaches to eliminate LSCs is imperative. Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 2 The self-renewal capacity of LSCs is regulated by intrinsic CSC Guangzhou, China. Department of Hematology, Sun Yat-sen Memorial Hospital, regulation pathways (e.g., WNT/b-catenin, Hedgehog; refs. 12, 13), Sun Yat-sen University, Guangzhou, China. 3Department of Hematology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 4Department metabolism regulators (e.g., ALOX5, SCD; refs. 14, 15), tran- of Hematology, Guangdong General Hospital/Guangdong Academy of Medical scription factors (e.g., Foxo3a, HIF1a, g-catenin; refs. 16–18) Sciences, Guangzhou, China. 5Department of Hematology, The First Affiliated and epigenetic regulators (e.g., PRMT5; refs. 19, 20). However, Hospital, Jinan University, Guangzhou, China. 6Collaborative Innovation Center the whole regulation network of LSCs is not fully realized. The for Cancer Medicine, State Key Laboratory of Oncology in South China, Sun Yat- complexity is enhanced by the LSC local microenvironment Sen University Cancer Center, Guangzhou, China. in bone marrow (21). Stromal cells tightly regulate the self- Note: Supplementary data for this article are available at Clinical Cancer renewal of LSCs via direct contact and by secreting soluble Research Online (http://clincancerres.aacrjournals.org/). cytokines (22, 23). Corresponding Author: Jingxuan Pan, Jinan University Institute of Tumor AXL is a member of the TAM receptor tyrosine kinases Pharmacology; State Key Laboratory of Ophthalmology, Zhongshan Ophthal- (TAMR), which also includes Mer and Tyro3 (Sky) (24, 25). mic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060, Two highly homologous ligands for TAMRs are growth-arrest– P.R. China. Phone/Fax: 8620-3762-8262; E-mail: [email protected] specific gene 6 (Gas6) and protein S (25). Gas6 has the highest doi: 10.1158/1078-0432.CCR-16-1298 affinity for AXL, whereas protein S predominantly binds to 2016 American Association for Cancer Research. Tyro3 and Mer (26). AXL was originally identified in 1991 from

www.aacrjournals.org OF1

Jin et al.

in a humidiﬁed incubator with 5% CO for 48 hours. After Translational Relevance 2 discarding the nonadherent cells, the adherent cells were cultured Quiescent leukemia stem cells (LSC) that are intrinsically for 2 to 4 weeks and used as mesenchymal stromal cells (MSC) in insensitive to BCR-ABL tyrosine kinase inhibitors is a root subsequent experiments (34). cause of relapse for chronic myelogenous leukemia (CML) treated with imatinib mesylate. Treatments to eliminate LSCs Western blot analysis are urgently needed to cure CML. In this study, we discovered Whole cell lysates were prepared in RIPA buffer (35). The AXL receptor tyrosine kinase was overexpressed in CML, and membranes were scanned by using the Odyssey infrared imaging was an oncogenic protein required for survival and self-renew- system (LI-COR). Detailed information for antibodies and their þ al of CML LSCs. Human CML CD34 cells conscripted bone sources is described in Supplementary Materials and Methods. marrow–derived stromal cells (BMDSC) to secrete extra Gas6 to form a paracrine loop that promoted self-renewal of LSCs. Enzyme-linked immunosorbent assay AXL knockdown and pharmacologic inhibition of AXL pro- Human and mouse Gas6 immunoassay involved use of the longed survival of BCR-ABL–driven CML mice and eliminated Quantikine ELISA Kit (R&D Systems; ref. 36). Cell culture super- LSCs in mice. Gas6/AXL ligation stabilizes b-catenin in an AKT- natants or plasma from CML patients or healthy individuals were þ dependent manner in human CML CD34 cells. Disruption of used for detecting the secreted Gas6.

Gas6/AXL axis is an attractive approach to selectively eliminate þ LSCs in CML. Lentivirus transduction in CML CD34 cells Lentivirus were produced by transfection in 293T cells with control shRNA (Scramble) or specificshRNAtargetingAXL together with the pCMV-dR8.2 packing construct and the þ pCMV-VSVG envelope construct. CML or NBM CD34 cells patients with CML (27), then found to be overexpressed (1 106 cells/mL) were transduced by spinoculation (1,500 g, in multiple human cancers (24). AXL is involved in acquired 90 minutes, 32C) with virus-containing supernatants two resistance to endothelial growth factor receptor inhibitors in rounds. Cells were harvested 48 hours later for further analysis lung cancer (28), FLT3-targeted therapy in acute myelogenous (16, 20). leukemia (29) and IM through a bypass mechanism (30). – þ AXL overexpression was found to induce the epithelial Analysis of cell apoptosis in primary CD34 cells þ mesenchymal transition (EMT) and increase tumorigenicity in Apoptosis of primary CD34 cells in CML patients or NBM was breast cancer cells (31). The underlying mechanism may determined as described previously (35). involve upregulating b-catenin, snail, slug, and NF-kB triggered fi by Gas6/AXL activation (31). Recently, AXL was identi ed as a key CFSE assay. CFSE-labeled cells were cultured with XL880 and regulator for mesenchymal glioblastoma stem-like cells (32). We R428 for 96 hours, cells were harvested and stained with discovered that among the three TAMR family members, AXL was þ max þ Annexin V-PE, and quiescent cell apoptosis (CD34 CFSE þ selectively expressed in primary CML CD34 cells and hypoth- Annexin V ) was analyzed by using with BD FACS Aria II flow esized that overexpression of AXL in LSCs and Gas6 by bone cytometer (8, 16, 20). marrow stromal cells might increase the self-renewal capacity of LSCs and confer resistance to IM. In this study, we tested Colony-formation cell/replating and long-term culture- this hypothesis and investigated the impact of forced expression initiating cell (LTC-IC) assay of AXL, silencing it via shRNA or inactivating it by small-molecule Colony-formation cell (CFC)/replating and LTC-IC assay were inhibitors on survival and self-renewal capacity of LSCs in vitro performed as reported previously (16, 20, 37). Detailed informa- and in CML mice. tion is described in Supplementary Materials and Methods.

Materials and Methods CML mouse model driven by human BCR–ABL fusion gene Primary leukemia cells The retrovirus was produced by transient transfection with Peripheral blood or bone marrow samples (Supplementary the MSCV-BCR-ABL-IRES-EGFP construct in Plat-E cells (9). Table S1) were obtained from patients with CML and from Bone marrow cells from 6- to 8-week-old donor male C57BL/6 healthy adult donors in Sun Yat-sen Memorial Hospital, The First mice primed with 5-fluorouracil (5-FU) were stimulated with Affiliated Hospital of Sun Yat-sen University, Guangdong General cytokines in vitro, then transplanted by tail-vein injection into Hospital, and The First Affiliated Hospital of Jinan University after irradiated (550 cGy) recipient female C57BL/6 mice after informed consent according to the institutional guidelines and transduction two rounds with the MSCV-BCR-ABL-IRES-EGFP the Declaration of Helsinki principles. The isolation and culture of retrovirus (16, 20). All the mice developed CML in approxi- þ human CD34 cells were performed as described previously (16, mately 2 to 3 weeks. 19, 20, 33). The detailed information for the treatment of CML mice and detection of LSK, LT-HSC and ST-HSC is described in Supple- Mesenchymal stromal cells isolation and culture mentary Materials and Methods. Peripheral blood samples were obtained from patients with CML and from healthy adult donors. Mononuclear cells isolated Engraftment of human cells in immunodeficient mice þ by histopaque gradient centrifugation were resuspended in Primary CD34 cells from 2 CML patients (1 CP-CML patient DMEM-low glucose medium (Gibco) supplemented with 10% and 1 BP-CML patient) were cultured with XL880 (1.0 mmol/L) for FBS and 1% penicillin–streptomycin. The cells were kept at 37C 72 hours, then cells (1 106 cells/mouse) were transplanted by

OF2 Clin Cancer Res; 2017 Clinical Cancer Research

Disruption of Gas6/AXL Axis Eliminates LSCs in CML

www.aacrjournals.org Clin Cancer Res; 2017 OF3

Jin et al.

tail-vein injection into sublethally irradiated (300 cGy) 8-week- control (Supplementary Fig. S1C and S1D). In addition, AXL old NOD-scid-IL2Rg / (NSI) which were generated by TALEN- overexpression upregulated the expression of stemness-associated mediated Il2rg gene targeting in NOD/SCID mice (Guangzhou genes ALDHA1, OCT4, SOX2 and NOTCH1 but not LIN28A and Institutes of Biomedicine and Health, Chinese Academy of NANOG (Supplementary Fig. S1E). Sciences; refs. 20, 38). Mice were euthanized after 12 weeks, then bone marrow cells and spleen cells were harvested; the engraft- Silencing AXL reduces survival and self-renewal capacity of fl þ ment of human cells was analyzed by using BD FACS Aria II ow human primary CML CD34 cells þ cytometer. To further define the role of AXL in CML CD34 cells, we þ transduced the primary CD34 cells purified from CML patients Statistical analysis or NBM with scramble shRNA or lentivirus AXL shRNA for All experiments were performed 3 times, and results are pre- 48 hours, then treated them with IM for 24 hours. The knock- þ þ sented as mean SD. GraphPad Prism 5.0 (GraphPad Software) down effect of AXL in CML CD34 cells and NBM CD34 cells was used for statistical analysis. Data for 2 groups were compared was confirmed by qRT-PCR (Fig. 1F and Supplementary by Student t test and for multiple groups by one-way ANOVA, post Fig. S2A). Given the gene homology of AXL with Mer and Tyro3, hoc intergroup comparisons, Tukey test. A P value of <0.05 was we examined the levels of these receptors in cells treated with considered statistically significant. shRNA against AXL by Western blot analysis. The results showed that the effect of AXL knockdown was of selective (Fig. 1G). AXL þ silencing significantly increased apoptosis in CML CD34 cells Results þ þ (Fig. 1H and I) but not in NBM CD34 cells (Supplementary Selective overexpression of AXL in primary CML CD34 cells Fig. S2B and S2C). The effect of AXL knockdown on CML fi þ We rst examined the expression of AXL and other TAMRs CD34 cells was augmented with combined IM treatment (Fig. þ fi members in primary CD34 cells puri ed with immunomagnetic 1I and Supplementary Fig. S2C), in which the BCR-ABL activity columns from CML patients and NBM from healthy donors. The as reflected by its phosphorylation (Y245) was completely mRNA level of AXL, but not other TAMR members (e.g., Mer and inhibited by IM (Fig. 1J). Because the self-renewal capacity is fi þ Tyro3), was signi cantly higher in the primary CD34 cells from an important trait of CSCs, we determined whether silencing CML patients than from NBM (Fig. 1A). The levels of phospho- AXL by its specific lentivirus shRNA impaired the CFC/replating þ þ þ AXL and AXL were higher in CML CD34 cells than NBM CD34 ability of CML stem cells. The primary CD34 cells of human cells as determined by Western blot analysis (Fig. 1B). Flow CML and NBM were transduced with lentiviral shAXL for cytometry results showed that there was surface expression of þ 72 hours and underwent three rounds of 14-day serially replat- AXL in CML CD34 cells (Fig. 1C). We next examined whether þ ing of CFC culture in methylcellulose medium. When AXL was BCR-ABL regulated AXL expression in CML CD34 cells. The silenced, the CFC/replating ability was significantly decreased þ þ levels of phospho-AXL and AXL were not changed after treatment in human CML CD34 but not in NBM CD34 cells (Fig. 1K with IM (Fig. 1D). However, BCR-ABL knockdown by lentiviral and Supplementary Fig. S2D). Thus, AXL knockdown reduced þ þ shRNA in the primary CML CD34 cells dramatically decreased CML CD34 cell survival and growth, and IM treatment further þ the AXL mRNA levels (Fig. 1E). These data suggest that AXL suppressed CML CD34 cell growth. expression is dependent on BCR-ABL protein level rather than its tyrosine kinase activity. AXL knockdown eliminates LSCs and prolongs survival of BCR-ABL–driven CML mice Forced overexpressing AXL in CML cells increases side We next used a widely used human BCR-ABL gene-driven CML þ population and ALDH cells and contributes to IM resistance mouse model (Fig. 2A) to examine the in vivo effect of AXL Forced overexpression of human AXL in K562 cells reduced knockdown on CML LSCs (14, 16, 20, 33). We knocked down þ sensitivity to IM, suggesting that increased AXL confers resistance AXL with shRNA in spleen GFP cells from the first generation of to IM (Supplementary Fig. S1A and S1B). The percentage of side CML mice (Fig. 2B), then transplanted these spleen cells into þ population (SP) cells and Aldefluor cells were also significantly sublethally irradiated recipient C57BL/6 mice. The results showed increased in K562 overexpressing AXL as compared with the that AXL knockdown alone or combination with IM potently

Figure 1. AXL is overexpressed in CML CD34þ cells and knockdown AXL reduces survival and CFC/replating ability in human CML CD34þ cells. A, Selective overexpression of AXL in primary CD34þ cells from CML patients. The mRNA level of AXL (left), Mer (middle) and Tyro3 (right) in CML patients (n ¼ 6) and normal bone marrow (NBM; n ¼ 4) CD34þ cells analyzed by qRT-PCR. , P < 0.0001, Student t test. B, The protein levels of phospho-AXL and AXL were detected by Western blot analysis in CML CD34þ cells (n ¼ 5) and NBM CD34þ cells (n ¼ 3). C, The surface expression of AXL in CML CD34þ cells was examined by flow cytometer. D, CML CD34þ cells were treated with IM for 24 hours, the expression of phospho-AXL and AXL were detected by Western blot analysis (left); the mRNA levels of AXL were detected by qRT-PCR (right). E, BCR-ABL knockdown decreased AXL mRNA levels. Human CML CD34þ (n ¼ 3) were transduced with control shRNA (Scramble), shBCR-ABL #1, or shBCR-ABL #2 lentivirus for 48 hours. The knockdown effect of BCR-ABL was confirmed by Western blot analysis (left). AXL mRNA level was determined by qRT-PCR (right). F and J, Human CML CD34þ (n ¼ 3) were transduced with control shRNA (Scramble), shAXL #1, or shAXL #2 for 48 hours, then treated with IM (2.5 mmol/L) for 24 hours. The knockdown effect of AXL was confirmed by qRT-PCR (F)andWesternblot(G)analysis.H, Representative flow cytometry plots for apoptosis in CML CD34þ cells. I, Apoptosis in CD34þ cells in CML analyzed by Annexin V-FITC and anti–CD38-PE labeling. J, BCR-ABL phosphorylation in CML CD34þ cells was inhibited after IM treatment. K, Knockdown of AXL suppressed the serially replating capacity of CML CD34þ cells. The same number of Scramble, shAXL #1 or shAXL #2 cells (5,000 cells/ well) were seeded in methylcellulose medium (H4434) for three rounds, and colonies were counted on day 14 after each round of culture. , P < 0.05; , P < 0.01; , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test.

OF4 Clin Cancer Res; 2017 Clinical Cancer Research

Disruption of Gas6/AXL Axis Eliminates LSCs in CML

Figure 2. AXL knockdown eliminates LSCs and prolongs survival in CML mice. A, A schema to generate retroviral human BCR-ABL–driven CML mouse model. B, AXL knockdown in spleen cells from CML mice was confirmed by Western blot analysis. C, A representative photograph of spleens from each group. D–I, AXL knockdown eliminated LSCs andsensitizedCMLLSCstoIMin vivo.GFPþ (leukemia) cells (D)andGFPþ myeloid cells (Gr-1þ Mac-1þ; E)inbone marrow were analyzed by flow cytometer. F, Representative flow cytometry plots of LSKs, LT-HSCs, and ST-HSCs in bone marrow from CML mice. Results for the GFPþ populations in bone marrow are shown: LSK cells (G), LT-HSCs (H), and ST-HSCs (I). J, Representative flow cytometry plots of progenitor cells in the bone marrow of mice. GMP cells (K) and CMP cells (L)inbonemarrow., P < 0.0001, compared with control; #, P < 0.05; ##, P < 0.01, shAXL compared with shAXLþIM; ns: not significant, one-way ANOVA, post hoc intergroup comparisons, Tukey test. M, AXL knockdown significantly prolonged survival of CML mice. Kaplan–Meier survival curves were plotted. Control (n ¼ 10), IM (n ¼ 12), shAXL (n ¼ 10), shAXLþIM (n ¼ 10), Control versus IM: , P ¼ 0.0003; Control versus shAXL: , P < 0.0001; Control versus shAXLþIM: , P < 0.0001. Log-rank test.

www.aacrjournals.org Clin Cancer Res; 2017 OF5

Jin et al.

Figure 3. Pharmacologic inhibition of AXL activity suppresses survival and self-renewal of human primary CML CD34þ cells. A, Representative ﬂow cytometry plots for apoptosis in human CML CD34þ cells treated with IM and AXL inhibitors XL880 (top) or R428 (bottom). CD34þ cells from CML bone marrow were treated with XL880 (top) or R428 (bottom) with IM (2.5 mmol/L) for 24 hours; apoptotic cells were examined by Annexin V-FITC and anti–CD38-PE labeling. B, XL880 or R428 treatment inhibited AXL phosphorylation in CML CD34þ cells. C, Pharmacologic inhibition of AXL by XL880 selectively þ induced apoptosis in quiescent CML LSCs. Results for CFSEmax and Annexin V cells are shown. , P < 0.01, Student t test. D, Pharmacologic inhibition of AXL by XL880 and R428 induced apoptosis in quiescent CML LSCs. Results for CFSEmax and Annexin Vþ cells are shown. E and F, Pharmacologic inhibition of AXL selectively suppressed the serially replating capacity of CML LSCs. G, Pharmacologic inhibition of AXL selectively lessened the long-term culture- initiating cell (LTC-IC) capacity of CML LSCs. , P < 0.01; , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test.

OF6 Clin Cancer Res; 2017 Clinical Cancer Research

Disruption of Gas6/AXL Axis Eliminates LSCs in CML

www.aacrjournals.org Clin Cancer Res; 2017 OF7

Jin et al.

blocked splenomegaly (Fig. 2C) and markedly decreased the replating and LTC-IC assay. In the presence of XL880 and þ proportion of BCR-ABL–expressing (i.e., GFP ) leukemia cells, R428, the CFC/replating ability was greatly decreased in the þ þ þ þ myeloid cells (Gr-1 Mac-1 ) in bone marrow cells (Fig. 2D primary CML CD34 cells but not in NBM CD34 cells and E) and splenic cells (Supplementary Fig. S3A and S3B) of (Fig. 3E and F and Supplementary Fig. S4E). Similarly, XL880 CML mice. greatly decreased the number of LTC-IC–derived colony-forming AXL knockdown or combination with IM greatly decreased the units for CML bone marrow cells as compared with NBM cells þ þ proportions of LSK cells (Lin Sca-1 c-Kit ), LT-HSCs (LSK (Fig. 3G and Supplementary Fig. S4F). Combined treatment þ Flt3 CD150 CD48 ) and ST-HSCs (LSK Flt3 CD150 CD48 ) with XL880 and IM enhanced the decreased CML LTC-IC fre- in bone marrow cells (Fig. 2F–I) and splenic cells (Supple- quency as compared with either agent alone (Fig. 3G). Thus, mentary Fig. S3C–S3E) of CML mice. Similarly, the proportions pharmacologic inhibition of AXL may reduce the survival and of granulocyte-macrophage progenitors (GMPs, Lin Sca- self-renewal ability of human CML LSCs. þ þ 1 c-Kit CD34 FcgRII/IIIhig) and common myeloid progenitors þ þ (CMPs, Lin Sca-1 c-Kit CD34 FcgRII/IIIlow) in bone marrow Pharmacologic inhibition of AXL prolongs survival of CML cells (Fig. 2J–L) and splenic cells (Supplementary Fig. S3F and mice and reduces the percentage of CML stem cells in mice S3G) were significantly reduced in AXL knockdown mice. IM To examine the in vivo effect of pharmacologic inhibition of treatment alone did not significantly reduce the proportions of AXL on CML LSCs, we used the CML mouse model again men- LSKs, LT-HSCs, ST-HSCs, GMPs and CMPs in bone marrow cells tioned before. Mice were treated with XL880, IM or both (Fig. 4A). and splenic cells of CML mice (Fig. 2F–L and Supplementary Splenomegaly was ameliorated in mice that received XL880 or Fig. S3C–S3G), which agreed with previous reports (7, 8). AXL both XL880 and IM (Fig. 4B). XL880-treated CML mice showed knockdown significantly prolonged survival of CML mice significantly prolonged survival, and XL880 with IM extended (Fig. 2M). In conclusion, silencing AXL eliminated LSCs in vivo survival as compared with each alone (Fig. 4C). and prolonged survival of CML mice. XL880 alone or with IM markedly decreased the propor- þ tion of BCR-ABL–expressing (GFP ) leukemia cells, myeloid þ þ Pharmacologic inhibition of AXL reduces survival and self- cells (Gr-1 Mac-1 ) in bone marrow cells (Fig. 4D and E) þ renewal capacity of human CML CD34 cells and splenic cells (Supplementary Fig. S5A and S5B) of CML Next, we determined the effect of the small-molecule AXL mice. XL880 alone or with IM greatly decreased the pro- þ þ inhibitors XL880 (39) and R428 (40) on survival of primary CML portions of LSK cells (Lin Sca-1 c-Kit ), LT-HSCs (LSK þ þ CD34 cells. The data showed that XL880 treatment increased Flt3 CD150 CD48 ), and ST-HSCs (LSK Flt3 CD150 CD48 ) þ the apoptosis in CML CD34 cells (Fig. 3A, top). Combined in bone marrow cells (Fig. 4F–I) and splenic cells (Supplementary treatment with XL880 and IM enhanced the apoptosis in CML Fig. S5C–S5E) of CML mice. þ þ CD34 cells, suggesting that XL880 sensitized CML CD34 cells The proportions of granulocyte-macrophage progenitors þ þ to apoptosis in response to IM. XL880 alone or with IM did not (GMPs, Lin Sca-1 c-Kit CD34 FcgRII/IIIhig) and common þ þ þ induce apoptosis in CD34 cells from NBM (Supplementary Fig. myeloid progenitors (CMPs, Lin Sca-1 c-Kit CD34 FcgRII/ S4A and S4B). Similar results were obtained when the primary IIIlow) in bone marrow cells (Fig. 4J–L) and splenic cells þ CML CD34 cells were exposed to R428, a more selective AXL (Supplementary Fig. S5F and S5G) were significantly reduced inhibitor (Fig. 3A, bottom). In these settings, both XL880 and in XL880-treated mice. Taken together, pharmacologic inhibi- R428 treatment blocked the AXL tyrosine phosphorylation in tion of AXL eliminated LSCs and sensitized CML LSCs to IM þ CML CD34 cells (Fig. 3B). in vivo. To further identify the effectiveness of AXL inhibition on þ quiescent LSCs, we labeled primary CD34 cells of human CML AXL inhibition reduces long-term engraftment of human CML þ patients or NBM with carboxyfluorescein diacetate succinimidyl CD34 cells in NSI mice ester (CFSE), then incubated with XL880 (1.0 mmol/L) for 96 We investigated the effect of ex vivo treatment with XL880 in þ hours. XL880 treatment markedly increased apoptosis in undi- human CML CD34 cells on their ability to be engrafted in NOD- þ vided CML CD34 cells but not undivided NBM HSCs (Fig. 3C scid-IL2Rg / (NSI) mice (Supplementary Fig. S6A). XL880 treat- þ and Supplementary Fig. S4C and S4D). R428 treatment exhibited ment reduced the engraftment of human CML CD45 cells in þ similar effect compared with XL880 in undivided CML CD34 bone marrow (Supplementary Fig. S6B) and spleens (Supple- cells (Fig. 3D). mentary Fig. S6C) at 12 weeks after transplantation. The engrafted þ We next evaluated whether inhibiting AXL by XL880 and R428 human CML CD45 cells sorted by flow cytometry from NSI impaired the self-renewal ability of CML stem cells by CFC/ murine bone marrow cells expressed BCR-ABL, and XL880

Figure 4. Pharmacologic inhibition of AXL reduces in vivo growth of CML stem cells and prolongs survival of CML mice. A, A schema of drug treatment in CML mice. B, A representative photograph of spleens from each group. C, Treatment with XL880 alone or with IM significantly prolonged the survival of CML mice. Kaplan-Meier survival curves of CML mice treated with IM, XL880 or their combination. Control (n ¼ 9), IM (n ¼ 9), XL880 (n ¼ 9), XL880þIM (n ¼ 9), Control versus IM: , P ¼ 0.0327; Control versus XL880: , P ¼ 0.0210; Control versus XL880þIM: , P ¼ 0.0001; XL880 versus XL880þIM: , P ¼ 0.0298. Log-rank test. D–I, XL880 eliminated LSCs and sensitized CML LSCs to IM in vivo.GFPþ (leukemia) cells (D)andGFPþ myeloid cells (Gr-1þ Mac-1þ; E) in bone marrow were analyzed by flow cytometry. F, Representative flow cytometry plots of LSKs, LT-HSCs, and ST-HSCs in bone marrow from CML mice. Results for the GFPþ populations in the bone marrow are shown: LSK cells (G), LT-HSCs (H), and ST-HSCs (I). J, Representative flow cytometry plots of progenitor cells in the bone marrow of mice. GMP cells (K) and CMP cells (L)inbonemarrow., P < 0.05; , P < 0.0001, compared with control; #, P < 0.05; ##, P < 0.01; ###, P < 0.0001, XL880 compared with XL880þIM; ns: not significant, one-way ANOVA, post hoc intergroup comparisons, Tukey test.

OF8 Clin Cancer Res; 2017 Clinical Cancer Research

Disruption of Gas6/AXL Axis Eliminates LSCs in CML

www.aacrjournals.org Clin Cancer Res; 2017 OF9

Jin et al.

treatment markedly decreased BCR-ABL mRNA levels (Supple- increased, CML cells indeed educated OP9 cells to dose-depen- mentary Fig. S6D). The proportions of engrafted human CML dently secrete Gas6 (Supplementary Fig. S7B). These data þ þ CD33 and CD14 myeloid cells in NSI murine bone marrow suggest that BMDSCs secreted Gas6, which activated the AXL cells were decreased with XL880 treatment (Supplementary Fig. membrane receptor on leukemia cells to form a paracrine S6E and S6F). XL880 treatment may selectively target human regulation loop. primitive CML cells with in vivo engraftment capacity. To assess whether Gas6 regulates CML responses in vitro,we found that Gas6 stimulation induced AXL activation (Fig. 5G) and increased growth of CML cells (Fig. 5H). The protective role Gas6/AXL paracrine loop confers self-renewal capacity of þ of stromal cells or Gas6 on CML cells was then investigated in human CML CD34 cells the co-culture system with or without AXL-Fc chimeric protein Wild-type AXL executes its function dependently by binding its treatment. AXL-Fc contains the extracellular domain of the AXL ligand Gas6. Given that AXL was overexpressed in CML LSCs, we þ protein and the Fc portion of human IgG1. AXL-Fc binds Gas6 wondered whether CML CD34 cells produced extra Gas6 to form and prevents it from interacting with AXL (39). We counted the an autocrine regulation loop. We evaluated Gas6 mRNA level in þ number of K562 cells when incubated in the presence or primary CML and NBM CD34 cells and found that Gas6 gene þ absence of AXL-Fc chimeric proteins without or with co-culture transcription was similar in the primary CD34 cells from CML with human HS5 BMDSCs or Gas6 stimulation. AXL-Fc treat- patients (Fig. 5A). The data did not support a Gas6/AXL autocrine ment counteracted the protective role of BMDSCs (Fig. 5I) or regulation loop in CML LSCs. Gas6 (Fig. 5J). The stemness of LSCs is tightly regulated by the components of In addition, Gas6 stimulation increased the self-renewal its surrounding niches (22). We next examined the plasma con- þ ability of CML CD34 cells detected by CFC/replating assay centration of Gas6 in CML patients and found increased plasma (Fig. 5K). Next, we evaluated whether blocking the Gas6/AXL Gas6 level in CML patients as compared with healthy donors paracrine loop inhibited the self-renewal of human CML (Fig. 5B). We detected the cellular human Gas6 expression in þ CD34 cells. After transduced with human AXL lentiviral the primary MSCs from CML patients and healthy individuals by þ shRNA for 48 hours, the CML CD34 cells were co-cultured qRT-PCR, the results showed that the Gas6 mRNA levels were with human HS5 cells for another 72 hours for CFC/replating higher in CML MSCs than normal MSCs (Fig. 5C). Furthermore, assay. In parallel, HS5 cells were infected with human Gas6 the secreted Gas6 levels as determined by ELISA were also lentiviral shRNA for 48 hours, then co-cultured with the pri- increased in the cell culture supernatants of CML MSCs compared þ mary CML CD34 cells for 72 hours for CFC/replating assay. with normal MSCs (Fig. 5D). These data together suggest the þ Silencing AXL in primary CML CD34 cells or Gas6 in stromal cellular and secreted Gas6 levels appears increased in CML MSCs. cells markedly inhibited the CFC/replating ability of primary Co-cultured with CML MSCs for 10 days significantly promoted þ þ CML CD34 cells in the co-culture system (Fig. 5L–O). Sim- growth of CML CD34 cells (Supplementary Fig. S7A). The þ ilarly, blocking the Gas6/AXL paracrine loop diminished the number of CFCs for CML CD34 cells co-cultured with MSCs þ self-renewal of human CML CD34 cells co-cultured with was significantly elevated in comparison with those without co- human CML MSCs (Supplementary Fig. S7C–S7F). culture with MSCs (Fig. 5E). Collectively, the Gas6/AXL paracrine loop positively regulated To determine the reason for the increased Gas6 level in CML, the self-renewal capacity of human CML LSCs, hinting that we seeded human leukemia K562 cells on the feeder layer of targeting the Gas6/AXL paracrine loop may be of benefit for murine BMDSCs OP9 cells, then evaluated murine Gas6 secre- treatment of CML. tion in the culture medium by ELISA. The level of murine Gas6 was higher in co-culture than with OP9 or K562 cells alone. Of note, murine Gas6 but not human soluble Gas6 level was Gas6/AXL ligation stabilizes b-catenin in an AKT-dependent þ increased in the co-culture system, suggesting that CML cells manner in CML CD34 cells might educate OP9 cells to secrete Gas6 (Fig. 5F). Indeed, with The WNT/b-catenin pathway regulates the self-renewal capacity the number of K562 cells increased in the system of a constant of CML LSCs (12, 41). We examined whether AXL affected number of OP9 cells, the soluble murine Gas6 level was b-catenin level. Levels of b-catenin and its downstream target

Figure 5. Gas6/AXL paracrine regulation loop elevates self-renewal of human CML CD34þ cells. A, mRNA levels of Gas6 gene in CML (n ¼ 6) and NBM (n ¼ 4) CD34þ cells analyzed by qRT-PCR. B, Plasma levels of human Gas6 in CML patients versus healthy individuals detected by ELISA. , P < 0.05, Student t test. C, MSCs were isolated from CML patients (n ¼ 2) and healthy adult donors (n ¼ 3). Human Gas6 mRNA levels in the MSCs cells were detected by qRT-PCR. D, Gas6 levels in the MSCs cell culture supernatants were measured by ELISA. E, CD34þ cells from CML patients (n ¼ 2) were co-cultured with MSCs for 10 days, then the cells (10,000/35 mm dish) were seeded in the methylcellulose medium. Colonies were counted on day 14. , P < 0.0001, Student t test. F, Human and murine Gas6 level in the culture medium of human CML K562 cells, murine bone marrow–derived stromal cells (BMDSCs) OP9, or co-culture detected by ELISA. ND, not detectable. G, Gas6 stimulation induced AXL activation in CML CD34þ cells. H, Gas6 increased the growth of CML cells. Starved CML cells were stimulated with recombinant human Gas6 (500 ng/mL) for 48 h; cell number was examined by trypan blue exclusion assay. , P < 0.05; , P < 0.001, compared with control. I, Total cell numbers were plotted after incubation in the presence or absence of AXL-Fc chimeric proteins (1.0 mg/mL) without or with human BMDSCs HS5. J, Number of K562 cells counted after incubation in the presence or absence of AXL-Fc chimeric proteins (1.0 mg/mL) without or with Gas6 stimulation. K, Gas6 stimulation increased the self-renewal ability of CML CD34þ cells. L–O, Disturbed Gas6/AXL paracrine regulation loop inhibited the self-renewal of human CML stem cells. L and M, CML CD34þ cells were transduced with human AXL shRNA lentivirus for 48 hours, co-cultured with HS5 cells for 72 hours, then underwent CFC/replating assay. N and O, HS5 cells were infected with human Gas6 shRNA þ lentivirus 48 hours, co-cultured with CML CD34 cells for 72 hours, then underwent CFC/replating assay. , P < 0.05; , P < 0.01; , P < 0.0001, one-way ANOVA, post hoc intergroup comparisons, Tukey test.

OF10 Clin Cancer Res; 2017 Clinical Cancer Research

Disruption of Gas6/AXL Axis Eliminates LSCs in CML

www.aacrjournals.org Clin Cancer Res; 2017 OF11

Jin et al.

genes (e.g., c-MYC and LEF1) were greatly increased in AXL- mediated the phosphorylation of GSK3b S9 in primary CML þ overexpressing K562 cells (Fig. 6A, left and middle). Given CD34 cells. that DVL (Dishevelled) is a positive upstream regulator in the To examine the effect of Gas6/AXL on AKT activation in the WNT/b-catenin pathway, we set to examine DVL. Among the absence of BCR/ABL, we knocked down BCR-ABL in primary þ three isoforms of DVL, only DVL2 and DVL3 were detectable in CD34 cells with lentivirus BCR-ABL shRNA, and then the K562 cells. The AXL-overexpressing K562 cells showed an cells were stimulated with Gas6, the results showed that AKT increased DVL3 but not DVL2 (Fig. 6A, left and middle). Similar was activated with Gas6 stimulation in BCR-ABL knockdown results were obtained in Gas6-stimulated K562 cells (Fig. 6A, cells (Fig. 6G), which is indicative of the critical role of Gas6/ þ right). Conversely, AXL knockdown in the primary CML CD34 AXL on AKT. cells dramatically inhibited WNT/b-catenin pathway (Fig. 6B). Finally, we determined whether AXL exerted its function þ However, the level of BCR-ABL was unaltered, suggesting that through b-catenin. The CML CD34 cells were transduced with BCR-ABL was not likely to be regulated by AXL in the primary CML Scramble (Scr) or AXL lentivirus for 48 hours, then the cells þ CD34 cells (Fig. 6B). Similarly, AXL inhibition by XL880 reduced underwent transfection with pcDNA3 (Vector) or pcDNA3-b-cate- levels of b-catenin and downstream genes in primary human CML nin (b-catenin) for another 48 hours. Ectopic expression of þ CD34 cells (Supplementary Fig. S8A). b-catenin rescued the AXL knockdown-mediated decrease in CFC þ Next, we ascertained whether AXL overexpression activates formation in CML CD34 cells (Fig. 6H). TCF4/LEF1-dependent gene transcription. The data indicated that Taken together, Gas6/AXL ligation leads to AKT-dependent þ AXL overexpression increased TCF4/LEF1-dependent luciferase stabilization of b-catenin protein in CML CD34 cells. activity (Supplementary Fig. S8B). AXL overexpression also increased the levels of b-catenin target genes (e.g., AXIN2, c-MYC, LEF1 and CCND1; Supplementary Fig. S8C). In a separate Discussion approach, the addition of Gas6 increased TCF4/LEF1-dependent LSCs are important resources of TKI resistance and CML relapse. luciferase activity (Supplementary Fig. S8D). Conversely, treat- In the present study, we discovered that among three of the TAMR ment with AXL inhibitor XL880 in K562 cells decreased luciferase family members, AXL was selectively overexpressed in primary þ activity (Supplementary Fig. S8E), as well as the transcription of CML CD34 cells. Inhibiting AXL by shRNA or small-molecule þ these b-catenin target genes in human primary CML CD34 cells inhibitors reduced the survival and self-renewal capacity of (Supplementary Fig. S8F). human CML LSCs. AXL knockdown and pharmacologic inhibi- To explore the mechanism by which AXL regulates b-catenin, tion of AXL prolonged the survival of CML mice and eliminated we performed time–chase experiments. AXL overexpression decel- LSCs in mice. AXL inhibition reduced long-term engraftment of þ erated the turnover of b-catenin protein (Fig. 6C). Similar results human CML CD34 cells in NSI mice. A Gas6/AXL paracrine loop þ were obtained with Gas6 stimulation (Fig. 6D). K562 cells stably conferred the self-renewal capacity of human CML CD34 cells. overexpressing AXL and Gas6-stimulated K562 cells showed Gas6/AXL ligation stabilizes b-catenin in an AKT-dependent þ increased phospho-(S9)-GSK3b, phospho-AKT and phospho- manner in CML CD34 cells. Our findings improve the under- ERK1/2 levels (Fig. 6E, left and middle). Conversely, pharmaco- standing of LSC regulation and validate a pharmacologic target for logic inhibition of AXL decreased the levels in human primary eliminating LSCs. Our study represents the first description of þ CML CD34 cells (Fig. 6E, right). Because phospho-(S9)-GSK3b Gas6/AXL as a critical regulator of the self-renewal capacity of inhibited the activity of GSK3b and decreased the degradation of CML LSCs conferring IM resistance. þ b-catenin in primary CML CD34 cells (Fig. 6E, right), we Among all members of the TAMR family, only AXL has been hypothesized that AXL activation might phosphorylate GSK3b reported to be involved in the EMT in glioblastoma and breast at S9. To identify the regulator responsible for GSK3b S9 phos- cancer (31, 32, 42). Consistently, we identified AXL overexpres- phorylation, we used specific inhibitors against MEK/ERK1/2 or sion in CML LSCs. AXL-overexpressing in CML cells increased the þ PI3K/AKT pathway. The PI3K inhibitor LY294002 but not MEK proportions of SP and Aldefluor cells and stemness-related inhibitor U0126 abrogated the Gas6-induced accumulation of genes including ALDHA1. In agreement with our findings, erlo- b-catenin (Fig. 6F), which suggests that AKT but not ERK1/2 tinib-resistant non–small cell lung carcinoma cell populations

Figure 6. Gas6/AXL stabilized b-catenin in an AKT-dependent manner in human CML CD34þ cells. A, AXL overexpression or ligation by Gas6 activates WNT/b-catenin signaling in K562 cells. Cells stably expressing AXL (C1 and C6; left), (C2 and B3; middle) or starved K562 cells were stimulated with Gas6 (400 ng/mL; right) for 24 hours; the activation of WNT/b-catenin pathway was examined by Western blot analysis. B, Knockdown of AXL by lentiviral shRNA þ inhibited b-catenin pathway but not BCR-ABL expression and activity in primary human CML CD34 cells detected by Western blot analysis. C and D, AXL overexpression or Gas6 stimulation decelerated the turnover rate of b-catenin protein. E, Gas6/AXL signaling increased b-catenin levels in an AKT-dependent fashion. The phosphorylation of AKT and ERK1/2 were examined by Western blot analysis in K562 cells stably overexpressing AXL (C1 and C6; left) or starved K562 cells were stimulated with Gas6 for 24 hours (middle). Pharmacologic inhibition of AXL inhibited phosphorylation of AKT and ERK1/2 þ þ in primary human CML CD34 cells (right). F, Gas6/AXL ligation enables b-catenin accumulation via AKT but not ERK1/2. CML CD34 cells were pretreated with MEK inhibitor U0126 (10.0 mmol/L) or PI3K inhibitor LY294002 (10.0 mmol/L) for 2 hours, then stimulated with Gas6 (400 ng/mL) for 24 hours; the protein level of b-catenin and phosphorylation of AKT and ERK1/2 were examined by Western blot analysis. , P < 0.05; , P < 0.01; , P < 0.0001, versus the corresponding control. G, Primary CML CD34þ cells were transduced with lentivirus BCR-ABL shRNA for 48 hours, then the cells were stimulated with Gas6 for 24 hours, BCR-ABL knockdown and AKT activation were conﬁrmed by Western blot analysis. H, Ectopic expression of b-catenin rescued the AXL knockdown-mediated decrease in CFC formation in CML CD34þ cells. CML CD34þ cells were transduced with Scramble (Scr) or AXL lentivirus for 48 hours, then the cells underwent electrotransfection with pcDNA3 (Vector) or pcDNA3-b-catenin (b-catenin) for another 48 hours. AXL knockdown and b-catenin overexpression were conﬁrmed by Western blot analysis (left). The cells were seeded in the methylcellulose medium. Colonies were counted on day 14 (right). , P < 0.05, Student t test. I, Proposed model by which self-renewal capacity of CML LSCs is positively regulated by Gas6/AXL paracrine loop.

OF12 Clin Cancer Res; 2017 Clinical Cancer Research

Disruption of Gas6/AXL Axis Eliminates LSCs in CML

þ contained a greater proportion of Aldefluor cells than parental direct manner. Therefore, we turned our attention to the canonical erlotinib-responsive cells with concurrent increase in AXL expres- cascade of WNT/b-catenin. After inhibiting AXL by shRNA or þ sion (43). small-molecule inhibitor XL880 in CML CD34 cells, phospho- Overexpressed AXL may be abundantly accessed by its ligand (S9)-GSK3b was markedly decreased, which was helpful to Gas6. However, identifying any gain-of-function mutations explain the decreased b-catenin levels. Regarding the mediator or genomic amplification in AXL in CML LSCs needs further to render the decreased phospho-(S9)-GSK3b, our results pin- investigation. pointed its upstream regulators—AKT but not ERK1/2. AKT is able Little is known about the underlying mechanism of AXL over- to phosphorylate b-catenin at S552 to facilitate its nucleus accu- expression in LSCs. Our results showed that AXL mRNA and mulation and transcriptional activity (48). Consistently, AKT is þ protein levels were higher in human primary CML CD34 cells required for Gas6/AXL to protect cells from apoptosis in ovarian as compared with NBM counterparts. Furthermore, AXL transcrip- cancer (49). Because LY294002 is a PI3K inhibitor with toxicity, tional expression appears dependent on BCR-ABL protein level future studies should define the potential application value of rather than its tyrosine kinase activity. It was previously reported more specific inhibitors of the PI3K/AKT pathway in eliminating that transcription factors (e.g., AP1) and miRNAs (e.g., miR-34a) LSCs. lead to AXL overexpression (40). TIG1 in breast cancer cells can In conclusion, overexpression of AXL or ligation of Gas6/AXL increase AXL protein stabilization by physical interaction (44). increases the self-renewal capacity of CML LSCs in vitro and in vivo. Further study is needed to identify the mechanism of increased LSCs may educate BMDSCs to secrete Gas6, assumably forming a AXL expression in LSCs. vicious circle (proposed model, Fig. 6I). Our findings may provide Multiple types of cells, including fibroblasts, endothelial cells, a rationale for a clinical trial of IM combined with AXL inhibitor osteoblasts, liver cells, and cancer cells can secrete Gas6 (45). In foretinib, which is tolerant during a phase II clinical trial, in bone marrow, Gas6 is believed to be secreted by fibroblasts and resistant and refractory CML (50). other stromal cells to maintain hematopoietic homeostasis (46). Our results support that Gas6 ligation to AXL to promote survival Disclosure of Potential Conflicts of Interest and self-renewal capacity of LSCs. Conversely, Gas6 knockdown No potential conflicts of interest were disclosed. in different stromal cells (e.g., HS5, primary CML MSCs) atten- uated the self-renewal of LSCs. Similarly, the addition of soluble Authors' Contributions AXL-Fc into the co-culture of LSCs and HS5 BMDSCs blocked the Conception and design: Y. Jin, J. Pan pro-survival and pro-self-renewal effect of Gas6 on CML LSCs. Development of methodology: Y. Jin, C. Liu, J. Zhou Our data did not support that LSCs generated Gas6 for autocrine Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Jin, D. Nie, Y. Lu, Y. Li, C. Liu, J. Zhou, J. Pan regulation. This finding disagrees with a previous report that bulk Analysis and interpretation of data (e.g., statistical analysis, biostatistics, AML cells enhanced Gas6 secretion to form an autocrine loop computational analysis): Y. Jin, J. Zhou, J. Pan (45). The difference may be due to variant cell types. Of interest, Writing, review, and/or revision of the manuscript: Y. Jin, J. Pan CML cells educated BMDSCs to secrete Gas6, which can increase Administrative, technical, or material support (i.e., reporting or organizing þ the self-renewal ability of CML CD34 cells. data, constructing databases): Y. Jin, J. Pan Overexpression of AXL in CML cells increased the protein Study supervision: J. Pan Other (provided specimens of CML patients): J. Li, X. Du level of b-catenin and facilitated its transcriptional activity, as reflected by the expression of its downstream target genes. Grant Support Stimulation of Gas6 in CML cells provoked similar effects. This study was supported by grants from National Natural Science funds Conversely, silencing AXL decreased b-catenin protein and its (grant nos. U1301226, 81373434, 81025021, and 91213304 to J. Pan; transcriptional activity. In function, the critical role of b-catenin 81473247 and 81673451; to Y. Jin), Guangdong Natural Science Funds for in CML LSCs was revealed as forced expression of b-catenin Distinguished Young Scholars (Grant no. 2016A030306036 to Y. Jin the rescued the AXL knockdown-mediated decrease in CFC forma- Research Foundation of Education Bureau of Guangdong Province, China þ tion in CD34 cells. (grant no. cxzd1103; to J. Pan). BCR-ABL tyrosine kinase can directly phosphorylate b-catenin The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked at Y86 and Y654 residues to increase b-catenin protein stability advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate (47). However, the phosphorylation at Y654 of b-catenin was not this fact. altered in AXL-overexpressing CML cells or when starved K562 cells were stimulated with Gas6 (data not shown), which suggests Received May 21, 2016; revised October 14, 2016; accepted November 7, that AXL might not increase b-catenin protein stability in such a 2016; published OnlineFirst November 16, 2016.

References 1. Druker BJ. Translation of the Philadelphia chromosome into therapy for phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002;2: CML. Blood 2008;112:4808–17. 117–25. 2. Perrotti D, Jamieson C, Goldman J, Skorski T. Chronic myeloid leukemia: 5. Jiang X, Zhao Y, Smith C, Gasparetto M, Turhan A, Eaves A, et al. Chronic mechanisms of blastic transformation. J Clin Invest 2010;120:2254–64. myeloid leukemia stem cells possess multiple unique features of resistance 3. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, et al. Efﬁcacy to BCR-ABL targeted therapies. Leukemia 2007;21:926–35. and safety of a speciﬁc inhibitor of the BCR-ABL tyrosine kinase in chronic 6. Quintas-Cardama A, Kantarjian H, Cortes J. Flying under the radar: the new myeloid leukemia. N Engl J Med 2001;344:1031–7. wave of BCR-ABL inhibitors. Nat Rev Drug Discov 2007;6:834–48. 4. Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J, et al. 7. Li L, Wang L, Wang Z, Ho Y, McDonald T, Holyoake TL, et al. Activation of Multiple BCR-ABL kinase domain mutations confer polyclonal resis- p53 by SIRT1 inhibition enhances elimination of CML leukemia stem cells tance to the tyrosine kinase inhibitor imatinib (STI571) in chronic in combination with imatinib. Cancer Cell 2012;21:266–81.

www.aacrjournals.org Clin Cancer Res; 2017 OF13

Jin et al.

8. Zhang B, Strauss AC, Chu S, Li M, Ho Y, Shiang KD, et al. Effective targeting 31. Asiedu MK, Beauchamp-Perez FD, Ingle JN, Behrens MD, Radisky DC, of quiescent chronic myelogenous leukemia stem cells by histone deace- Knutson KL. AXL induces epithelial-to-mesenchymal transition and tylase inhibitors in combination with imatinib mesylate. Cancer Cell regulates the function of breast cancer stem cells. Oncogene 2014;33: 2010;17:427–42. 1316–24. 9. Zhang H, Peng C, Hu Y, Li H, Sheng Z, Chen Y, et al. The Blk pathway 32. Cheng P, Phillips E, Kim SH, Taylor D, Hielscher T, Puccio L, et al. Kinome- functions as a tumor suppressor in chronic myeloid leukemia stem cells. wide shRNA screen identifies the receptor tyrosine kinase AXL as a key Nat Genet 2012;44:861–71. regulator for mesenchymal glioblastoma stem-like cells. Stem Cell Rep 10. Chu S, McDonald T, Lin A, Chakraborty S, Huang Q, Snyder DS, et al. 2015;4:899–913. Persistence of leukemia stem cells in chronic myelogenous leukemia 33. Jin B, Wang C, Li J, Du X, Ding K, Pan J. Anthelmintic niclosamide disrupts patients in prolonged remission with imatinib treatment. Blood 2011; the interplay of p65 and FOXM1/b-catenin and eradicates leukemia stem 118:5565–72. cells in chronic myelogenous leukemia. Clin Cancer Res 2016 Aug 4. [Epub 11. Prost S, Relouzat F, Spentchian M, Ouzegdouh Y, Saliba J, Massonnet G, ahead of print]. PMID:27492973. et al. Erosion of the chronic myeloid leukaemia stem cell pool by PPARg 34. Schmal O, Seifert J, Schaffer TE, Walter CB, Aicher WK, Klein G. Hemato- agonists. Nature 2015;525:380–3. poietic stem and progenitor cell expansion in contact with mesenchymal 12. Zhao C, Blum J, Chen A, Kwon HY, Jung SH, Cook JM, et al. Loss of stromal cells in a hanging drop model uncovers disadvantages of 3D b-catenin impairs the renewal of normal and CML stem cells in vivo. culture. Stem Cells Int 2016;2016:4148093. Cancer Cell 2007;12:528–41. 35. Jin Y, Lu Z, Ding K, Li J, Du X, Chen C, et al. Antineoplastic mechanisms of 13. Zhao C, Chen A, Jamieson CH, Fereshteh M, Abrahamsson A, Blum J, et al. niclosamide in acute myelogenous leukemia stem cells: inactivation of the Hedgehog signalling is essential for maintenance of cancer stem cells in NF-kB pathway and generation of reactive oxygen species. Cancer Res myeloid leukaemia. Nature 2009;458:776–9. 2010;70:2516–27. 14. Chen Y, Hu Y, Zhang H, Peng C, Li S. Loss of the Alox5 gene impairs 36. Waizenegger JS, Ben-Batalla I, Weinhold N, Meissner T, Wroblewski M, leukemia stem cells and prevents chronic myeloid leukemia. Nat Genet Janning M, et al. Role of Growth arrest-specific gene 6-Mer axis in multiple 2009;41:783–92. myeloma. Leukemia 2015;29:696–704. 15. Zhang H, Li H, Ho N, Li D, Li S. Scd1 plays a tumor-suppressive role in 37. Neviani P, Harb JG, Oaks JJ, Santhanam R, Walker CJ, Ellis JJ, et al. PP2A- survival of leukemia stem cells and the development of chronic myeloid activating drugs selectively eradicate TKI-resistant chronic myeloid leuke- leukemia. Mol Cell Biol 2012;32:1776–87. mic stem cells. J Clin Invest 2013;123:4144–57. 16. Jin Y, Yao Y, Chen L, Zhu X, Jin B, Shen Y, et al. Depletion of g-catenin by 38. Xiao Y, Jiang Z, Li Y, Ye W, Jia B, Zhang M, et al. ANGPTL7 regulates the histone deacetylase inhibition confers elimination of CML stem cells in expansion and repopulation of human hematopoietic stem and progenitor combination with imatinib. Theranostics 2016;6:1947–62. cells. Haematologica 2015;100:585–94. 17. Naka K, Hoshii T, Muraguchi T, Tadokoro Y, Ooshio T, Kondo Y, et al. 39. Park IK, Mishra A, Chandler J, Whitman SP, Marcucci G, Caligiuri MA. TGF-b-FOXO signalling maintains leukaemia-initiating cells in chronic Inhibition of the receptor tyrosine kinase Axl impedes activation of the myeloid leukaemia. Nature 2010;463:676–80. FLT3 internal tandem duplication in human acute myeloid leukemia: 18. Ng KP, Manjeri A, Lee KL, Huang W, Tan SY, Chuah CT, et al. Physiologic implications for Axl as a potential therapeutic target. Blood 2013;121: hypoxia promotes maintenance of CML stem cells despite effective BCR- 2064–73. ABL1 inhibition. Blood 2014;123:3316–26. 40. Scaltriti M, Elkabets M, Baselga J. Molecular pathways: AXL, a mem- 19. Chen Y, Li S. Molecular signatures of chronic myeloid leukemia stem cells. brane receptor mediator of resistance to therapy. Clin Cancer Res 2016; Biomark Res 2013;1:21. 22:1313–7. 20. Jin Y, Zhou J, Xu F, Jin B, Cui L, Wang Y, et al. Targeting methyltransferase 41. Heidel FH, Bullinger L, Feng Z, Wang Z, Neff TA, Stein L, et al. Genetic and PRMT5 eliminates leukemia stem cells in chronic myelogenous leukemia. pharmacologic inhibition of b-catenin targets imatinib-resistant leukemia J Clin Invest 2016;126:3961–80. stem cells in CML. Cell Stem Cell 2012;10:412–24. 21. Goff DJ, Court Recart A, Sadarangani A, Chun HJ, Barrett CL, Krajewska M, 42. Bosurgi L, Bernink JH, Delgado Cuevas V, Gagliani N, Joannas L, et al. A Pan-BCL2 inhibitor renders bone-marrow-resident human leuke- Schmid ET, et al. Paradoxical role of the proto-oncogene Axl and Mer mia stem cells sensitive to tyrosine kinase inhibition. Cell Stem Cell receptor tyrosine kinases in colon cancer. Proc Natl Acad Sci U S A 2013;12:316–28. 2013;110:13091–6. 22. Crews LA, Jamieson CH. Selective elimination of leukemia stem cells: 43. Corominas-Faja B, Oliveras-Ferraros C, Cuyas E, Segura-Carretero A, Joven hitting a moving target. Cancer Lett 2013;338:15–22. J, Martin-Castillo B, et al. Stem cell-like ALDH(bright) cellular states in 23. Zhang B, Ho YW, Huang Q, Maeda T, Lin A, Lee SU, et al. Altered EGFR-mutant non-small cell lung cancer: a novel mechanism of acquired microenvironmental regulation of leukemic and normal stem cells in resistance to erlotinib targetable with the natural polyphenol silibinin. Cell chronic myelogenous leukemia. Cancer Cell 2012;21:577–92. Cycle 2013;12:3390–404. 24. Graham DK, DeRyckere D, Davies KD, Earp HS. The TAM family: phos- 44. Wang X, Saso H, Iwamoto T, Xia W, Gong Y, Pusztai L, et al. TIG1 promotes phatidylserine sensing receptor tyrosine kinases gone awry in cancer. Nat the development and progression of inflammatory breast cancer through Rev Cancer 2014;14:769–85. activation of Axl kinase. Cancer Res 2013;73:6516–25. 25. Hafizi S, Dahlback B. Gas6 and protein S. Vitamin K-dependent ligands for 45. Ben-Batalla I, Schultze A, Wroblewski M, Erdmann R, Heuser M, Waize- the Axl receptor tyrosine kinase subfamily. FEBS J 2006;273:5231–44. negger JS, et al. Axl, a prognostic and therapeutic target in acute myeloid 26. Nagata K, Ohashi K, Nakano T, Arita H, Zong C, Hanafusa H, et al. leukemia mediates paracrine crosstalk of leukemia cells with bone marrow Identification of the product of growth arrest-specific gene 6 as a common stroma. Blood 2013;122:2443–52. ligand for Axl, Sky, and Mer receptor tyrosine kinases. J Biol Chem 1996; 46. Dormady SP, Zhang XM, Basch RS. Hematopoietic progenitor cells grow on 271:30022–7. 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 27. O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, et al. axl, (GAS6). Proc Natl Acad Sci U S A 2000;97:12260–5. a transforming gene isolated from primary human myeloid leukemia cells, 47. Coluccia AM, Vacca A, Dunach M, Mologni L, Redaelli S, Bustos VH, et al. encodes a novel receptor tyrosine kinase. Mol Cell Biol 1991;11:5016–31. Bcr-Abl stabilizes b-catenin in chronic myeloid leukemia through its 28. Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activation of the tyrosine phosphorylation. EMBO J 2007;26:1456–66. AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat 48. Fang D, Hawke D, Zheng Y, Xia Y, Meisenhelder J, Nika H, et al. Phos- Genet 2012;44:852–60. phorylation of b-catenin by AKT promotes b-catenin transcriptional activ- 29. Park IK, Mundy-Bosse B, Whitman SP, Zhang X, Warner SL, Bearss DJ, et al. ity. J Biol Chem 2007;282:11221–9. Receptor tyrosine kinase Axl is required for resistance of leukemic cells to 49. Lee WP, Wen Y, Varnum B, Hung MC. Akt is required for Axl-Gas6 signaling FLT3-targeted therapy in acute myeloid leukemia. Leukemia 2015;29: to protect cells from E1A-mediated apoptosis. Oncogene 2002;21:329–36. 2382–9. 50. Choueiri TK, Vaishampayan U, Rosenberg JE, Logan TF, Harzstark AL, 30. Dufies M, Jacquel A, Belhacene N, Robert G, Cluzeau T, Luciano F, et al. Bukowski RM, et al. Phase II and biomarker study of the dual MET/VEGFR2 Mechanisms of AXL overexpression and function in Imatinib-resistant inhibitor foretinib in patients with papillary renal cell carcinoma. J Clin chronic myeloid leukemia cells. Oncotarget 2011;2:874–85. Oncol 2013;31:181–6.

OF14 Clin Cancer Res; 2017 Clinical Cancer Research

Gas6/AXL Signaling Regulates Self-Renewal of Chronic Myelogenous Leukemia Stem Cells by Stabilizing β-Catenin

Yanli Jin, Danian Nie, Juan Li, et al.

Clin Cancer Res Published OnlineFirst November 16, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-1298

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2016/11/16/1078-0432.CCR-16-1298.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2017/03/17/1078-0432.CCR-16-1298. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.