Preferential Eradication of Acute Myelogenous Leukemia Stem Cells by Fenretinide
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Preferential eradication of acute myelogenous leukemia stem cells by fenretinide Hui Zhanga,1, Jian-Qing Mia,1, Hai Fanga,b,1, Zhao Wanga,1, Chun Wangc, Lin Wua,c, Bin Zhanga, Mark Mindend, Wen-Tao Yanga, Huan-Wei Wanga, Jun-Min Lia, Xiao-Dong Xia, Sai-Juan Chena, Ji Zhanga,b,e,2, Zhu Chena,e,2, and Kan-Kan Wanga,e,2 aState Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU- SM), Shanghai 200025, China; bInstitute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and SJTU-SM, Shanghai 200025, China; cThe First People Hospital, Shanghai Jiao Tong University, Shanghai 200080, China; dPrincess Margaret Hospital/Ontario Cancer Institute, University Health Network, Toronto, ON, Canada M5G 2M9; and eSamuel Waxman Cancer Research Foundation Laboratory, Shanghai Ruijin Hospital, Shanghai 200025, China Contributed by Zhu Chen, February 8, 2013 (sent for review November 14, 2012) Leukemia stem cells (LSCs) play important roles in leukemia activation of nuclear factor κB (NF-κB), active Wnt/β-catenin initiation, progression, and relapse, and thus represent a critical signaling, and elevated levels of interferon regulatory factor-1 target for therapeutic intervention. However, relatively few (IRF-1) and death-associated protein kinase (DAPK) (8–12). agents have been shown to target LSCs, slowing progress in the Most recently, emerging evidence points to oxidative signaling as treatment of acute myelogenous leukemia (AML). Based on in being a two-edged sword in AML: moderate levels of reactive vitro and in vivo evidence, we report here that fenretinide, a well- oxygen species (ROS) are important for driving disease, whereas tolerated vitamin A derivative, is capable of eradicating LSCs higher levels result in cell death (13–15). The dual roles of oxi- but not normal hematopoietic progenitor/stem cells at physiolog- dative signaling suggest that LSCs, in comparison with normal ically achievable concentrations. Fenretinide exerted a selective HSCs, are more vulnerable to ROS-generating agents. Accord- cytotoxic effect on primary AML CD34+ cells, especially the LSC- ingly, pharmacological agents favoring the generation of ROS are enriched CD34+CD38− subpopulation, whereas no significant ef- worth exploring in LSC-targeted therapy. Indeed, ROS induction fect was observed on normal counterparts. Methylcellulose colony has been shown as a critical mechanism for the selective eradication formation assays further showed that fenretinide significantly of LSCs by several compounds, such as parthenolide (PTL), di- suppressed the formation of colonies derived from AML CD34+ methyl-aminoparthenolide (DMAPT), 4-benzyl-2-methyl-1,2,4- + thiadiazolidine-3,5-dione (TDZD-8), and 4-hydroxynonenal cells but not those from normal CD34 cells. Moreover, fenretinide – significantly reduced the in vivo engraftment of AML stem cells (HNE) (16 19). but not normal hematopoietic stem cells in a nonobese diabetic/ Another promising agent that could be used in this regard is SCID mouse xenotransplantation model. Mechanistic studies fenretinide, a synthetic retinoid that lacks a carboxyl functional revealed that fenretinide-induced cell death was linked to a series group likely necessary for retinoid receptor activity (20). We and others have previously demonstrated that fenretinide, unlike of characteristic events, including the rapid generation of reactive classical retinoids that often induce differentiation, triggers ap- oxygen species, induction of genes associated with stress responses optotic effects; it is largely achieved through the generation of and apoptosis, and repression of genes involved in NF-κBandWnt ROS (21–24), enhanced cellular ceramide, and/or ganglioside signaling. Further bioinformatic analysis revealed that the fenreti- – fi D3 (25). Moreover, several key stem cell survival-associated sig- nide down-regulated genes were signi cantly correlated with the naling pathways, such as NF-κB, c-Jun N-terminal kinase (JNK), existing poor-prognosis signatures in AML patients. Based on these fi and extracellular signal-regulated kinase (ERK), have been ndings, we propose that fenretinide is a potent agent that selec- reported to be inactivated in the fenretinide-induced apoptosis in tively targets LSCs, and may be of value in the treatment of AML. different cancer cell types (25, 26); this further suggests the therapeutic value of fenretinide in targeting cancer stem cells. cute myelogenous leukemia (AML) represents a group of Fenretinide has been used clinically for some time as an ef- Aclonal hematopoietic stem cell disorders, in which a small fective chemopreventive agent for various cancers (27). It can subpopulation of leukemia stem cells (LSCs) are responsible for significantly reduce the risk of breast cancer and small cell lung the accumulation of large numbers of immature myeloblasts in cancer (28, 29), suggesting an ability to prevent the development the bone marrow of AML patients. In addition to their crucial of cancer and/or eliminate early-stage malignant cells (likely roles in leukemia initiation and progression, LSCs are also re- cancer-initiating cells). Furthermore, long-term clinical trials have sponsible for the high frequency of relapse that is characteristic demonstrated only minimal side effects in patients receiving fen- of current AML therapies. Of patients receiving treatment with retinide (28, 30–32). In particular, no significant hematopoietic curative intent, less than one-half will achieve long-term survival toxicity has been observed in patients treated with fenretinide (28). (1). Similar to normal hematopoietic stem cells (HSCs), LSCs To illustrate the potential value of fenretinide in AML therapies, in exhibit stem cell-like characteristics such as the capacity for self- renewal, differentiation potential, and relative quiescence (2, 3). The quiescent feature renders LSCs resistant to conventional Author contributions: J.Z., Z.C., and K.-K.W. designed research; J.-Q.M., C.W., and J.-M.L. chemotherapeutic agents that predominantly target proliferating collected samples; H.Z., Z.W., L.W., B.Z., and K.-K.W. performed research; H.Z., J.-Q.M., rather than quiescent cells (1). For this reason, it is not surprising H.F., C.W., M.M., W.-T.Y., H.-W.W., J.-M.L., X.-D.X., S.-J.C., and K.-K.W. analyzed data; and that relapse occurs in the majority of cases; this is further sup- H.Z. and K.-K.W. wrote the paper. ported by recent studies showing that AML patients with LSCs The authors declare no conflict of interest. – enrichment have worse clinical outcomes (4 7). It is therefore Data deposition: The microarray expression data reported in this paper have been de- crucial that therapies be developed targeting the quiescent and posited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (ac- drug-resistant LSCs. cession no. GSE33243). Despite the similarities shared by LSCs and HSCs, LSCs often 1H.Z., J.-Q.M., H.F., and Z.W. contributed equally to this work. possess several unique features as well, which may provide im- 2To whom correspondence may be addressed. E-mail: [email protected], [email protected]. portant hints for designing LSC-targeted therapy. For instance, cn, or [email protected]. LSCs are usually associated with the abnormal expression of CD This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. markers (e.g., CD44, CD47, CD96, and CD123), constitutive 1073/pnas.1302352110/-/DCSupplemental. 5606–5611 | PNAS | April 2, 2013 | vol. 110 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1302352110 Downloaded by guest on September 27, 2021 + this study, we defined the fenretinide effects on primary AML Table 1. Fenretinide on AML and normal CD34 cell viability + + − CD34 cells and LSC-enriched AML CD34 CD38 cells. Viable cells, % Results Specimens Untreated 2.5 μM5μM 7.5 μM + Fenretinide Preferentially Induces Death of Primary AML CD34 Cells + but Not Normal CD34 Hematopoietic Cells. Our initial study was AML specimens + performed to compare fenretinide effects on AML CD34 and AML1 87.3 48.9 14.3 0.3 + normal CD34 cells using 32 primary AML and 6 normal sam- AML2 67.1 55.9 19.0 3.8 ples. The AML specimens were classified into different French– AML3 83.9 68.7 58.3 29.0 American–British (FAB) subtypes (including M0, M1, M2, M4, AML4 90.1 77.5 24.2 1.1 M5, and M6), thus representing the spectrum of AML subtypes. AML5 86.8 61.2 19.5 8.9 The clinical characteristics of these patients are described in AML6 97.3 66.2 12.0 0.4 Table S1. In the clinic, it is possible to achieve plasma concen- AML7 86.9 80.3 77.2 60.2 trations of 10 μM while maintaining minimal side effects in pa- AML8 92.8 73.3 58.9 7.6 tients receiving standard doses of fenretinide (28, 32). We + AML9 90.2 81.5 54.3 7.2 therefore treated CD34 cells with concentrations of fenretinide AML10 93.1 75.2 19.5 0.8 ranging from 2.5 to 7.5 μM to evaluate its effect. We determined AML11 91.7 97.2 63.9 13.6 cell viability and apoptosis by Annexin V and 7-aminoactinomycin AML12 91.3 81.5 46.1 ND D (7-AAD) staining after 18 h of treatment. As shown in Table 1, AML13 80.3 98.3 69.3 1.8 fenretinide exerted a dose-dependent cytotoxic effect on AML + AML14 92.9 93.4 72.3 5.5 CD34 cells (75.3%, 49.0%, and 26.5% viable cells for 2.5, 5, and AML15 73.6 69.7 47.2 17.8 7.5 μM, respectively). Moreover, we found that fenretinide killed + AML16 67.6 44.1 36.2 24.2 AML CD34 cells mainly through apoptosis, as indicated by a significant increase in the number of Annexin V-positive apoptotic AML17 88.2 89.4 65.3 42.5 cells (Table S2 and Fig.