E35 ablates acute leukemia stem and progenitor cells in vitro and in vivo

Abstract Leukemia stem cells (LSCs) have critical functions in acute leukemia (AL) pathogenesis, participating in its initiation and relapse. Thus, identifying new molecules to eradicate LSCs represents a high priority for AL management. This work identified E35, a novel Emodin derivative, which strongly inhibited growth and enhanced apoptosis of AL stem cell lines, and primary stem and progenitor cells from AL cases, while sparing normal hematopoietic cells. Furthermore, functional assays in cultured cells and animals suggested that E35 preferentially ablated primitive leukemia cell populations without impairing their normal counterparts. Moreover, molecular studies showed that E35 remarkably downregulated drug‐resistant gene and dramatically inhibited the Akt/mammalian target of rapamycin signaling pathway. Notably, the in vivo anti‐LSC activity of E35 was further confirmed in murine xenotransplantation models. Collectively, these findings indicate E35 constitutes a novel therapeutic candidate for AL, potentially targeting leukemia stem and progenitor cells.

Foundation of China, Grant/Award Numbers: 81370630, 81470326, 81500158 1 | INTRODUCTION Acute leukemia (AL) represents a clonal hematopoietic stem cell (HSC) dysregulation marked by arrested differentiation, unsuitable growth of immature progenitor cells. Persistence of leukemia stem cells (LSCs) constitutes an important factor that contributes to both initiation and relapse in patients with AL. Meanwhile, LSCs typically are quiescent and not susceptible to most conventional chemotherapy regimens. In addition, currently available regimens might not efficiently distinguish normal from malignant cells (Akbarzadeh et al., 2019;Bonnet & Dick, 1997;Guan, Gerhard, & Hogge, 2003;Guzman et al., 2005;Lapidot et al., 1994;van Rhenen et al., 2005). Thus, identifying novel molecules that could specifically target LSCs represents a high priority for leukemia therapy.
Emodin, a plant-derived anthraquinone, has recently attracted increasing attention because of its overt pharmacological features in cancer therapy (Fu et al., 2012;Liu et al., 2016;Subramaniam et al., 2013). Brown, Bellon, and Nicot (2007) and our previous studies demonstrated the promising anti-leukemic activities of Emodin in different types of hematological malignancies (Chen et al., 2014;Chen, Mei et al., 2018). We showed that Emodin can enhance all-trans retinoic acid (ATRA)-associated differentiation and induce apoptotic death in acute myeloid leukemia (AML) cells (Chen et al., 2014). In addition, our team found that Emodin may significantly increase the chemosensitivity of resistant AML cells to cytarabine (Ara-C; . Our recent study further demonstrated that TP53 and PI3K are the targets by which Emodin may function in diffuse large B cell lymphoma (Chen, Mei et al., 2018). However, Emodin is poorly soluble, which limits its potential application in clinic.
Among these compounds, E35 (C 34 H 50 BrNO 5 ·H 2 O, MW: 631.29), which was obtained by introducing a long quaternary ammonium salt containing a long carbon chain to Emodin, exhibited potent antileukemia effects with low half maximal inhibitory concentration (IC50) values and markedly enhanced aqueous solubility. The positive charge of quaternary ammonium salt makes it easy for E35 to enter the mitochondria of cancer cells preferentially. Meanwhile, the long carbon chain renders E35 lipophilic and allows it to pass through the mitochondrial membrane easily (Hu et al., 2019;Li et al., 2015). E35 was interestingly found to have significant inhibitory effects on chronic myeloid leukemia (CML) 32Dp210-T315I cells harboring T315I mutation in our previous report . It is well-accepted that the T315I gatekeeper mutation promotes resistance to both imatinib and second-generation tyrosine kinase inhibitors, such as nilotinib and dasatinib in patients with CML (O'Hare et al., 2005;Tamai et al., 2018 Collection, China). Cell culture followed the procedures described in our previous study (Chen et al., 2014). Human leukemic stem-like KG1a cells (>95% CD34 + CD38 − , American Type Culture Collection, ATCC) were maintained in Iscove's Modified Dulbecco's medium containing 10% fetal bovine serum (Gibco-BRL) at 37℃ in a humid incubator containing 5% CO 2 . CD34 + cells enriched from HL-60/ADR cells were termed HARs. E35 stock solution was prepared as reported in our previous study .

| Primary cell isolation
Peripheral blood was collected from 33 primary patients with AL. The specimens contained ≥70% blasts before any manipulation. All patients had AL, as described by standard French-American-

| Statistical analysis
Data are mean ± standard deviation, and were evaluated by t test.
GraphPad Prism 6.0 was employed for statistical analysis.

| E35 decreases leukemic but not normal stem and progenitor cell activity
The in vitro colony formation assay was employed to assess whether E35 altered colony formation in primitive cells. Figure 2a, (Figure 2a,c).
3.4 | E35 reduces NOD/SCID repopulating ability in AL cells but not normal cells NOD/SCID mouse xenograft assays were performed to assess whether E35 targets functionally defined leukemia progenitor/stem cells. Primary AL and normal cells after an 18-hr treatment with E35 were administered to immunodeficient NOD/SCID mice. Bone marrow cells were collected and examined for human-derived CD45 cells in recipients at 8 weeks after transplantation. Figure 3a shows that E35 treatment markedly reduced human leukemic cell engraftment in NOD/SCID mice. The engraftment levels of seven independent AL specimens in the recipients were decreased by 85% compared with the untreated group (p = .005, n = 7). In contrast, hCD45 cell assessment in animals treated versus untreated healthy cells showed no statistically significant difference (Figure 3b; p = .742, n = 4), indicating that E35 specifically targets AL stem/ progenitor cells with no effect on the engraftment potential of normal primitive cells.

| E35 specifically induces apoptotic response in primitive AL cells
To explore the underlying mechanism by which E35 affects AL stem and progenitor cells, we investigated whether treatment with E35 was associated with apoptotic induction in leukemic cells. As shown in Figure 4a Figure 5a,b).

| E35 downregulates drug resistance genes and inhibits the Akt/mammalian target of rapamycin signaling pathway
To evaluate the molecular consequences of E35 treatment in primitive leukemia cells, we examined the expression changes of drug-resistant genes by qRT-PCR and immunoblot, respectively. As depicted in  Figure 6b). Flow cytometric analysis was also performed to track human KG1a-R cells in the bone marrow from individual mice. As shown in Figure 6c, the percentages of CD34 + CD38 − KG1a-R cells in recipients were markedly reduced following E35 treatment in comparison with control values (p < .0001). Of note, all treated animals appeared healthy; none of them appeared to succumb to therapeutic toxicity, and all survived to the end of observation. In contrast, one mouse in the saline control group died due to rapid disease progression (data not shown).
Hence, the in vivo study further confirmed the potential of E35 for the eradication of leukemic stem/progenitor cells.

| DISCUSSION
AL arises from immature cells in the bone marrow. It represents a severe and common hematologic malignancy. Because LSCs play a critical role in AL initiation, multidrug resistance and recurrence of leukemia (Bonnet & Dick, 1997;Lapidot et al., 1994;Ratajczak, Bujko, Mack, Kucia, & Ratajczak, 2018;van Rhenen et al., 2007), selectively targeting LSCs has been identified as a promising strategy for leukemia treatment (Baquero et al., 2018;Ding et al., 2016;Guzman et al., 2014;Liu et al., 2013;Tremblay et al., 2018). Here, we provide the first in vitro and in vivo evidence that E35, a novel Emodin derivative, preferentially eradicates AL stem and progenitor cells.
We recently demonstrated that Emodin has several properties, including inhibiting leukemic cell proliferation, sensitizing resistant leukemic cells to chemotherapeutic agents and suppressing diffuse large B cell lymphoma. In this study, we increased the antileukemic properties of Emodin by designing a more pharmacologically potent derivative, E35, which retained the key features of the parent molecule. The IC50 values of E35 in 10 different leukemia/lymphoma cell lines were between 6.07-and 25.47-fold lower than those of Emodin. We then examined whether E35 may eliminate AL stem and progenitor cells. According to previous reports, KG1a cells with the similar LSC phenotype of CD34 + CD38 − are considered an ideal cell model for studying LSCs Liu et al., 2013;Weng, Zeng, Huang, Fan, & Guo, 2015 We further explored the underlying molecular mechanisms evoked by E35 treatment in KG1a cells. It is known that nuclear factor κB (NF-κB) modulates mesenchymal stem cell accumulation at tumors. Inhibition of NF-κB activity might contribute to apoptotic induction in LSCs (Guzman et al., 2001;Guzman et al., 2002;Guzman et al., 2014;Ji et al., 2016;Jin et al., 2017;Uchibori et al., 2013). Mohammadi et al. (2016) reported  (Chen, Lee, Kang, Minden, & Zhang, 2018;Ding et al., 2016;Fuchs, Daniel, Sadeghi, Opelz, & Naujokat, 2010;Rao et al., 2011).
In this study, we found that HARs and KG1a cells in response to E35 showed dramatic reductions in both mRNA and protein expression levels of MDR1, MRP1, GSTπ, TopⅡβ, and BCL-2. These molecules are associated with drug-resistance in AL (Lin et al., 2013;Wang et al., 2015). Therefore, we speculate that blockade of the AKT/mTOR pathway  Ding et al. (2016) showed that alantolactone dose-dependently induces apoptosis in KG1a cells via suppression of NF-κB and its downstream target proteins, while its prodrug DMA-alantolactone could greatly inhibit KG1a xenograft in vivo. Guzman et al. (2007) found that the anti-LSC activity mediated by F I G U R E 6 In vivo therapeutic effects of E35 in KG1a-R xenograft mice. Nude mice harboring KG1a-R xenografts were randomized into two groups and intraperitoneally administered 20 mg/kg E35 or vehicle once daily for 2 weeks. All animals were followed up for 10 weeks after the initial treatment with E35. (a)The leukemic burden was assessed on an IVIS LUMINA II Imaging System. (b) Harvested bone marrow (BM) cells were stained by the Wright-Giemsa method.
(c) The percentages of CD34 + CD38 − KG1a-R cells in BM were measured by flow cytometry. ****p < .0001 versus vehicle control the dimethyl-amino analog of parthenolide in canine models is strongly associated with induced oxidative stress responses and NF-κB suppression. The latter authors also reported that Histone Deacetylase Inhibitor AR-42 reduces Hsp90's ability to stabilize its oncogenic effectors, causing enhanced and specific cytotoxicity in LSCs (Guzman et al., 2014). Our findings demonstrated that the therapeutic response to E35 is mediated, at least in part, by differentiation induction in LSCs.
However, the underpinning molecular mechanisms still need to be explored under in vitro and in vivo settings.
In summary, we identified E35 as a novel agent that can ablate AL cells at the bulk, stem, and progenitor cell levels. Going forward, further investigation in large animal models may enable greater success in applying this compound to clinical cases.

ACKNOWLEDGMENTS
We appreciate the reviewers for critical comments of the manuscript. We