Pin1 inhibition exerts potent activity against acute myeloid leukemia through blocking multiple cancer-driving pathways

Pin1 inhibition exerts potent activity against acute myeloid leukemia through blocking multiple... Background: The increasing genomic complexity of acute myeloid leukemia (AML), the most common form of acute leukemia, poses a major challenge to its therapy. To identify potent therapeutic targets with the ability to block multiple cancer-driving pathways is thus imperative. The unique peptidyl-prolyl cis-trans isomerase Pin1 has been reported to promote tumorigenesis through upregulation of numerous cancer-driving pathways. Although Pin1 is a key drug target for treating acute promyelocytic leukemia (APL) caused by a fusion oncogene, much less is known about the role of Pin1 in other heterogeneous leukemia. Methods: The mRNA and protein levels of Pin1 were detected in samples from de novo leukemia patients and healthy controls using real-time quantitative RT-PCR (qRT-PCR) and western blot. The establishment of the lentiviral stable-expressed short hairpin RNA (shRNA) system and the tetracycline-inducible shRNA system for targeting Pin1 were used to analyze the biological function of Pin1 in AML cells. The expression of cancer-related Pin1 downstream oncoproteins in shPin1 (Pin1 knockdown) and Pin1 inhibitor all-trans retinoic acid (ATRA) treated leukemia cells were examined by western blot, followed by evaluating the effects of genetic and chemical inhibition of Pin1 in leukemia cells on transformed phenotype, including cell proliferation and colony formation ability, using trypan blue, cell counting assay, and colony formation assay in vitro, as well as the tumorigenesis ability using in vivo xenograft mouse models. Results: First, we found that the expression of Pin1 mRNA and protein was significantly increased in both de novo leukemia clinical samples and multiple leukemia cell lines, compared with healthy controls. Furthermore, genetic or chemical inhibition of Pin1 in human multiple leukemia cell lines potently inhibited multiple Pin1 substrate oncoproteins and effectively suppressed leukemia cell proliferation and colony formation ability in cell culture models in vitro. Moreover, tetracycline-inducible Pin1 knockdown and slow-releasing ATRA potently inhibited tumorigenicity of U937 and HL-60 leukemia cells in xenograft mouse models. (Continued on next page) * Correspondence: xzhou@bidmc.harvard.edu; klu@bidmc.harvard.edu; chenyz@mail.fjmu.edu.cn Equal contributors Division of Translational Therapeutics, Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA Fujian Institute of Hematology, Fujian Provincial Key Laboratory on Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 2 of 14 (Continued from previous page) Conclusions: We demonstrate that Pin1 is highly overexpressed in human AML and is a promising therapeutic target to block multiple cancer-driving pathways in AML. Keywords: Acute myeloid leukemia (AML), Pin1 inhibitor, All-trans retinoic acid (ATRA), Oncogenic signaling, Leukemia treatment Background non-CSC tumor cells and CSCs, respectively, compared Acute myeloid leukemia (AML) is the most common form with normal breast epithelial cells [26]. Mechanically, Pin1 of acute leukemia and arises from a malignant transform- promotes breast cancer stem cell (BCSC)-proliferation ation of multipotent hematopoietic stem cells with a re- and tumorigenesis in vitro and in vivo by increasing markable genomic alteration [1]. AML development Rab2A transcription and thereby Erk activation, Zeb1 up- requires the collaboration of at least two classes of cyto- regulation, and β-catenin nuclear translocation [25]. genetic abnormalities [2]. This “two-hit model” [3], pre- Moreover, Pin1 can also stabilize NOTCH1 expression by sented by Gilliland and Griffin (2002), proposes that class reducing ubiquitin ligase FBW7 to promote self-renewal I mutations activate signaling transduction pathways to and metastasis in breast cancer CSCs [27, 29]. In acute promote cell proliferation and that class II mutations promyelocytic leukemia (APL), genetic or chemical inhib- affect transcription factors to block maturation of ition of Pin1 can induce the degradation of the disease- hematopoietic cells [4, 5]. The proteins involved in AML causing fusion oncogene PML-RARα, which causes APL processes include both fusion proteins (e.g., RUNX1/ETO, through blockage of promyelocyte differentiation and pro- CBFβ/MYH11, and PML/RARα) and mutated proteins (e. motion of self-renewal capacity [28]. In addition, Pin1 is g., NPM1, FLT3, and C/EPBα Eisfeld, 2017 #11467, [6–9]), required not only for the stability of the stem cell repro- as well as some molecular features that are a function of gramming factors Nanog, Oct4, and MYC but also for in- overexpression (e.g., BAALC, MN1, ERG-1, and AF1q) ducing and maintaining pluripotency [28, 30–33]. [10]. Such complexity of molecular and cytogenetic abnor- Therefore, Pin1 dysregulation has an important role in the malities poses a major challenge to formulating AML self-renewal and tumorigenic properties of CSCs. therapy [11, 12]. Chemotherapeutic approaches continue Given that Pin1 can promote tumorigenesis by activating to be the mainstay therapies used for AML treatments multiple signaling pathways and inducing and sustaining [13]. However, long-term survival using these therapies is self-renewal capability in a variety of solid tumors, Pin1 only obtained in 35 to 40% of younger patients [14, 15], may also play a critical role in leukemogenesis. Pin1 is a key and the long-term survival of elderly AML patients is even drug target for treating APL [28], and controls Notch3 pro- lower because only about one third of them are eligible tein expression and regulates T-ALL progression [34]. Si- for intensive chemotherapies [16]. Therefore, therapy still lencing Pin1 can delay the progression of lymphoma needs to come a long way to fully overcome AML. Hence, disease in Eμ-myc transgenic mice; however, much less is there is a pressing need to identify potent therapeutic tar- known about the role of Pin1 in the development and treat- gets to block multiple cancer-driving pathways for the ment of other more common and heterogeneous leukemia. treatment of AML. In this manuscript, our clinical data analyses demon- Pin1 is a unique peptidyl-prolyl isomerase (PPIase) that strated higher Pin1 expression in a variety of AML sub- catalyzes cis/trans isomerization of specific pSer/Thr-Pro types compared to healthy controls. Genetic or chemical motifs and central common phosphorylation motifs in cell inhibition of Pin1 downregulated multiple cancer- proliferation and transformation [17]. Hence, altered Pin1 promoting signaling pathways, leading to the inhibition function can play a profound role in pathogenesis of hu- of cell proliferation and colony formation capability in man disease, notably cancer. Pin1 is overexpressed and/or multiple human AML cell lines. Moreover, inducible over-activated in many human cancers, thereby disrupting downregulation or chemical inhibition of Pin1 inhibited the balance between oncoproteins and tumor suppressors, the tumorigenesis of AML in vivo. Taken together, these and by amplifying numerous cancer-driven pathways. results demonstrate that Pin1 is highly overexpressed in Moreover, overexpression and/or over-activation of Pin1 human AML and is a promising therapeutic target to frequently correlate with poor clinical prognosis [17–24]. block multiple cancer pathways in AML. Besides the well-known role of Pin1 in tumorigenesis, recent studies have revealed that Pin1 dysregulation plays Methods an important role in cancer stem cells (CSCs) in breast Sample source cancer and leukemia [25–28]. In primary human breast Bone marrow samples (n = 150) were collected from tumors, the expression of Pin1 is 5 and 30 times higher in healthy donors and patients treated in the Hematology Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 3 of 14 Department of the Fujian Medical University Union In line with the operation steps of the quantitative Hospital from September 2012 to February 2015. Of PCR kit (Roche), the reaction system contains SYBR these, 107 samples were taken from patients with acute Green Master (ROX) 10 μl, upstream primer (10 pmol/ leukemia (AL) and 43 were taken from healthy donors. μl) 0.15 μl, downstream primer (10 pmol/μl) 0.15 μl, AL patients after the first visit included 61 male and 46 cDNA 1 μl, and double distilled water 8.7 μl. The reac- female patients in age of 13~76 years old with median tion was conducted in ABI7500 fluorogenic quantitative age of 45 years old. According to FBA classification for PCR instrument (Applied Biosystem Company) under the diagnosis and classification of AML [35], there were conditions: at 50 °C for 2 min and 95 °C for 10 min, and 6 patients with M0, 9 with M1, 18 with M2, 7 with M3, then at 95 °C for 15 s and 60 °C for 1 min as 1 cycle for 1 with M4, 41 with M5, 3 with M6, 1 with M7, 21 cases 40 times in total. Then, melting curve reaction was con- with other types of AL, 43 healthy controls included 20 ducted under conditions: at 95 °C for 15 s, 60 °C for male, and 23 female donors in age of 20~45 years old 1 min, 95 °C for 15 s, 60 °C for 15 s, 1 cycle for once. with median 32 years old. All of the patients and healthy Three ventral orifices were set in every sample. With β- donors signed informed consent. actin as an internal reference, the relative expression quantity of mRNA was represented with RQ value by −ΔCT −ΔCT 2 method: RQ = 2 . The final results are Cell strain and reagents presented as the fold change of Pin1 expression in a All cells were cultured in RPMI1640 with 10 or 20% target sample relative to a reference sample, normalized FBS. HL60 (acute myelogenous leukemia cell line), U937 to β-actin. (histiocytic lymphoma cell line), KG-1a (acute myeloid leukemia cell line), NB4 (acute promyelocytic leukemia cell line), Nalm-6 (acute B acute lymphoblastic leukemia Lentiviral transduction and knockdown of Pin1 in AML cell line), Molt-4 (acute T acute lymphoblastic leukemia cells cell line), Kasumi-1 (acute myelogenous leukemia cell Recombinant lentiviral particles were produced in 293FT line), and K562 (chromic granulocytic leukemia cell line) cells by co-transfecting pRevTRE plasmid containing Pin1 were cultured at the Fujian Institution of Hematology. shRNA sequence, along with helper plasmids, including HL-60, U937, and KG-1a cell lines were cultured at the pCMV-VSVG and pCMV-dR8.91. The virus-containing BIDMC and were gifts from Dr. Daniel G Tenen. Anti- medium was harvested at 48 h after transfection and fil- bodies against various proteins were obtained from the tered using a 0.45-μm filter. For infection, the collected following sources: Pin1 was previously described [36]; β- virus medium was added to the rtTA-expressing cells with actin from Sigma; Cyclin D1 (DCS-6) from Biolegend; polybrene. The cells were selected with G418 and puro- NF-κB/p65 (D14E12, 8242) from Cell Signaling Technol- mycin. Pin1 shRNA were induced by doxycycline, and ogy; β-catenin from BD biosciences; and Rab-2A (15420- Pin1 protein level was analyzed by immunoblot. Pin1 1-AP) from Proteintech Group. All-trans retinoic acid shRNA sequence is CCACCGTCACACAGTATTTAT. (ATRA) were purchased from Sigma, 1,25-dihydroxyvi- tamin D3 were purchased from Cayman Chemicals, and Cell viability assay ATRA-releasing pellets were from Innovative Research Cells were seeded on 96-well plates at a density of 5000 of America. cells per well. Cells were stained with Trypan blue and counted every day. In two additional groups, cells were Quantitative real-time PCR treated with DMSO or ATRA. After 72 h, cell viability Total RNA from bone marrow mononuclear cell and cell was measured by CCK-8 assay (DOJINDO) and the line was extracted by TRIzol (Invitrogen), and cDNA number of cells was determined by CellTiter-Glo® 2.0 was transcribed with reverse transcription kit (Thermo Assay (Promega, Madison, WI) according to the manu- Fisher Scientific) in accordance with the manufacturer’s facturer’s instructions. protocols. PCR reactions were performed using quantita- tive PCR kit (Roche) and ABI7500 fluorogenic quantita- tive PCR instrument (Applied Biosystem Company). β- Colony formation assay actin was used as an internal control of samples. The Cells were seeded on 24-well plate at a density of 500 following primers were used: cells (KG-1a is 1000 cells). Methylcellulose (Sigma) in final concentration of 0.8% was added and mixed. After β-actin (F): AGTGTGACGTGGACATCCGCAA. 7 days (KG-1a is 14 days), cells were stained with 0.5 ml β-actin (R): ATCCACATCTGCTGGAAGGTGGAC. 1 mg/ml p-Iodonitrotetrazolium Violet. The number of Pin1 (F): GCTCAGGCCGAGTGTACTACTT. colonies and the total area were calculated by ImageJ Pin1(R): CGAGGCGTCTTCAAATGG. analysis. Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 4 of 14 PPIase assay using SPSS 23.0 and GraphPad Prism 5 software package. The PPIase activity on GST-Pin1 in response to ATRA and All data are presented as the means ± SD/SEM, followed by 1,25-(OH) vitamin D3 were determined using the determining significant differences using the two-tailed chymotrypsin-coupled PPIase activity assay with the sub- student’s t test or analysis of variance (ANOVA) test where strate Suc-Ala-pSer-ProPhe-pNA (50 nM) in buffer contain- *p <0.05, **p <0.01, and ***p < 0.001. ing 35 mM HEPES (pH 7.8) and 0.1 mg/ml BSA at 10 °C as described, with the exception that the compounds were pre- Results incubated with enzymes for 12 h at 4 °C [37]. Pin1 overexpression in leukemia patients To investigate the potential clinical significance of Pin1 FACS analysis in leukemia, we examined the relative expression of To assess cell surface expression of CD11b, cells were PIN1 mRNA in bone marrow mononuclear cells from washed with PBS, harvested by non-enzymatic cell dis- 107 newly diagnosed leukemia patients and 43 healthy sociation solution, and resuspended in blocking solution bone marrow donors. We found that PIN1 mRNA ex- (Ca2+, Mg2+-free PBS containing 2% FCS). Cells were pression was significantly higher in bone marrow cells of then incubated with CD11b-FITC (Biolegend) for 15 min acute leukemia (AL) patients compared to those of at 4 °C in the dark. Cells were washed and analyzed by healthy controls (Fig. 1a, lane 2 versus lane 1). Among using a Beckman Coulter’s Gallios flow cytometry. these samples, 86 samples were diagnosed with AML pa- tients, which also showed significantly higher PIN1 Western blot mRNA expression, as compared with healthy controls Cells from each group were collected, and cell lysis buf- (Fig. 1a, lane 3 versus lane 1). Further analysis of the re- fer (Roche Company) was added to extract total protein. lationship between the expression of PIN1 mRNA and The protein concentration was detected by the Bradford FAB (French–American–British classification system) method. The loading sample of equivalent protein was subtypes of AML showed that PIN1 mRNA was upregu- transferred to a nitrocellulose membrane (Bio-Rad, cat. lated in most subtypes of AML. In the case of M4 and No. 162-0115) through SDS-PAGE electrophoresis and M6, low sample sizes precluded sufficient power for stat- immunoblotting. istical analysis (Fig. 1b). Consistent with the results of PIN1 mRNA, Pin1 protein levels were higher in AML Animal studies patient samples compared with healthy controls (Fig. 1c, For xenograft experiments, 5 × 10 HL-60 and U937 cells d; Additional file 1: Figure S1a). A positive correlation stably expressing Tet-On shPin1 were injected subcutane- was found between PIN1 mRNA level and Pin1 protein ously into flank of 7-week-old BALB/c nude mice (Taco- level (Fig. 1e). In addition, we used different colors for nic Laboratories) and fed with normal or doxycycline- individual patients in western blot analysis. These results containing diet. For ATRA treatment, 5 × 10 U937 cells together demonstrate that Pin1 is highly overexpressed were injected subcutaneously into flank of 7-week-old in AML patients. BALB/c nude mice. After 5 days when tumor growth was We also examined Pin1 expression in human leukemia just about notable by sight, the mice were randomly di- cell lines, including six AML cell lines (Kasumi-1, U937, vided into placebo group or ATRA slow-releasing pellet K562, NB4, HL-60, and KG-1a) and two ALL cell lines group. Tumor growth was monitored twice a week until (Nalm-6 and Molt-4). Both PIN1 mRNA (Fig. 1f)and sacrifice criteria were met in the first mice. Tumor sizes Pin1 protein levels (Fig. 1g, h; Additional file 1:Figure were recorded twice a week by a caliper and tumor vol- S1b) were found to be significantly higher in human umes were calculated using formula L × W × 0.52, where leukemia cell lines as compared with bone marrow cells of L and W represent length and width, respectively. Tumors healthy control (Fig. 1f–h). A positive correlation of PIN1 were cut into small pieces and quickly stored in liquid mRNA and Pin1 protein levels was also found in leukemia nitrogen, partly for protein extraction. All BALB/c nude cell lines (Fig. 1i). These results further confirm that Pin1 mice were housed in laminar flow cabinets with free is overexpressed in leukemia cells including AML cells. access to food and water. Animal work was carried out in compliance with the ethical regulations approved by the Constitutive Pin1 knockdown inhibits cell proliferation Animal Care Committee, Beth Israel Deaconess Medical and clonogenicity of human AML cells in vitro Center, Boston, USA. Given the overexpression of Pin1 in AML, including bi- opsied bone morrow leukemia cells and established hu- Statistical analysis man AML cell lines, the question remains as to whether Experiments were routinely repeated at least three times, Pin1 plays any role in leukemogenesis. Based on the and the repeat number was increased according to effect “two-hit model,” the ability of a molecule to promote size or sample variation. Statistical analyses were performed cell proliferation and colony formation is required in Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 5 of 14 Fig. 1 Expression of PIN1 mRNA and protein in leukemia patients and cell lines. a The fold changes of PIN1 mRNA expression in bone marrow cells from normal controls, untreated acute leukemia patients (AL) and AML patients. b The fold changes of PIN1 mRNA expression in bone marrow cells from normal controls and FAB subtypes of untreated AML patients. c, d Pin1 protein levels in bone marrow cells from normal and AML patients were analyzed by immunoblotting (c). The quantitative results of Pin1 expression was analyzed from 1c (d). The individual patients used for immunoblot analysis were indicated by different colors. e The correlation between PIN1 mRNA and Pin1 protein levels in AML patients. Different colors were used to indicate different patients in 1c and 1d. f The fold changes of PIN1 mRNA expression in normal control bone marrow cells and several leukemia cell lines, including AML cells (Kasumi-1, U937, K562, NB4 and KG-1a) and ALL cells (Nalm-6 and Molt-4). g, h The protein levels (g) and quantitative results (h) of Pin1 in normal control bone marrow cells and leukemia cell lines. Different cell lines were indicated by corresponding colors. i The correlation between PIN1 mRNA and Pin1 protein levels in leukemia cell lines was analyzed based on 1f and 1 h. Individual cell lines were indicated by corresponding colors. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001) order to initiate leukemogenesis [38]. We investigated To test if Pin1 KD would impair the leukemogenic po- the effects of Pin1 on leukemogensis-related traits. To tential of AML cells to grow as clonogenic colonies, HL- this end, multiple AML cell lines, including HL-60, 60, U937, and KG-1a expressing Pin1 shRNA or control U937 and KG-1a, were infected with lentiviruses carry- shRNA were plated at a low density and observed for ing validated Pin1-specific shRNA [26] or control colony formation. A significant decrease both in the shRNA with Puro . Using puromycin selection, stable number (Fig. 2c, d) and size (Fig. 2c, e) of colonies was Pin1-knockdown (Pin1 KD) cell lines were established. observed in the Pin1 KD group compared with controls. Immunoblot analysis confirmed a dramatic decrease in Thus, our data demonstrates that Pin1 plays a positive endogenous Pin1 in cells carrying Pin1-shRNA com- role in leukemogenesis-relevant events in vitro. As a pared with cells carrying the control shRNA (Fig. 2a). major regulator of oncoproteins, Pin1 amplifies onco- The rates of cell growth of different pairs of AML genic pathways by activating more than 43 oncogenic Pin1-shRNA and control-shRNA cells were monitored molecules and suppressing at least 20 tumor-suppressing by counting cell number to demonstrate the effects of molecules, including many of which have well- Pin1 downregulation on cell proliferation. In all three established roles in CSCs [17, 21, 39]. To further AML cell lines examined, Pin1 KD resulted in a signifi- characterize the underlying mechanisms of Pin1- cant reduction in cell proliferation (Fig. 2b). mediated leukemogenesis, we analyzed the expression of Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 6 of 14 b c Fig. 2 Constitutive Pin1 downregulation suppresses oncogenic biological functions and signaling in vitro. a To establish stable-shPin1 cell lines, HL-60 or U937 or KG-1a cells were infected with lentivirus expressing scramble (Vec) or Pin1 shRNA (shPin1) with Puro . After puromycin selection for 1 week, Pin1 levels were validated by immunoblotting analysis. b Pin1 downregulation inhibits cell proliferation in indicated AML cell lines. Cell growth was monitored for 1 week by cell counting. p values were derived from the cell numbers for each group at the end point. c–e Cells were cultured in normal medium supplemented with methylcellulose for 1 or 2 weeks. When colonies became visible, cells were stained with p-iodonitrotetrazolium violet for counting. The number (d) and area (e) of colonies was measured and counted using ImageJ. Results present the mean ± SD of three independent experiments. f Cell lysates were subjected to western blot analysis with antibodies against the downstream oncogenic proteins of Pin1. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001) several leukemogeneis-related signaling molecules that have substrates in other cancer cells [26, 40–46]. AML Pin1 KD been reported to be Pin1 substrates. Wnt/β-catenin and cells also downregulated the expression of Pin1 oncopro- NF-κB pathways are well-known signaling pathways in tein substrates, including β-catenin and NF-κB(Fig. 2f), as leukemogenesis through regulating cell proliferation, differ- with solid tumors [26, 40–46]. Besides these key regulators entiation, or apoptosis and have been shown to be Pin1 in leukemogenesis, Pin1 also transcriptionally regulated Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 7 of 14 some stem cells-related molecules, including Rab2A [47] suppresses tumorigenesis through downregulation of and CyclinD1 [48, 49]. In AML Pin1 KD cells, these mole- oncogenic signaling pathways. cules were downregulated as well (Fig. 2f)(Additional file 2: Figure S2). Therefore, these biochemical analyses further The Pin1 inhibitor ATRA downregulates Pin1 oncoprotein support the role for Pin1 in the regulation of cell prolifera- substrates and inhibits cell growth and clonogenicity of tion and clonogenicity through deregulation of multiple human AML cells in vitro oncogenic pathways. Given the oncogenic role of Pin1 in AML, we wondered whether targeting Pin1 with chemical inhibitors would show any therapeutic benefit in the treatment of AML Doxycycline-inducible Pin1 knockdown inhibits [28]. ATRA has been reported to inhibit and degrade ac- clonogenicity of human AML cells in vitro tive Pin1, resulting in the blockade of multiple Pin1- To further confirm and evaluate the effects of Pin1 in- regulated oncogenic pathways in APL, breast cancer, and hibition on the tumorigenesis of AML, we generated liver cancer [28, 30, 52]. However, the effect of ATRA on stable tetracycline-inducible Pin1 knockdown HL-60 and AML cells is unknown. Therefore, we treated multiple U937 cells using a Tet-On system [28, 50]. We infected AML cells, HL-60, U937, and KG-1a, with different con- HL-60- and U937-rtTA cells with lentivirus containing centrations of ATRA. ATRA can degrade Pin1 and its either Pin1 shRNA or control shRNA with Puro . Stable downstream oncoproteins in a dose-dependent manner tetracycline-inducible Pin1 KD and control HL-60- and (Fig. 5a). Notably, in both cells, among these oncopro- U937-Tet ON cells were established using puromycin se- teins, while NF-κB was slightly upregulated in low con- lection. In the presence of doxycycline, Pin1 protein centrations of ATRA likely due to ATRA-induced levels were dramatically reduced in both cell lines (Fig. differentiation, they were downregulated once ATRA 3a, b). Furthermore, the clonogenic assay showed a sig- concentration was high enough to induce Pin1 degrad- nificant decrease in the number (Fig. 3c, d) and size of ation. This further supported that ATRA could down- colonies (Fig. 3e, f) after doxycycline treatment as com- regulate multiple oncoproteins through degrading Pin1 pared with the vehicle control. These results show that in AML. our Tet-On Pin1 KD system is highly inducible and Since ATRA induces differentiation in various could be used to confirm the role of Pin1 in leukemia cell lines, it is unclear whether differentiation leukemogenesis, as shown in constitutive Pin1 KD cells would affect Pin1 levels. To address this question, we (Fig. 2). treated cells with other differentiation-inducing reagent − 1-α, 25-(OH) vitamin D (1,25-D3), the active form of 2 3 Doxycycline-inducible Pin1 knockdown inhibits vitamin D , which has been shown to induce differenti- tumorigenesis of human AML cells in vivo. ation in various AML cell lines [53–56]. After 72 h of To examine whether inducible Pin1 inhibition would incubation of 1, 25-D3 or ATRA, differentiation was affect tumorigenesis of human AML cells in vivo, we evaluated by flow cytometry using a general myeloid subcutaneously implanted HL-60- and U937-Tet ON marker, CD11b. As shown in Figure S3a (Additional file 3; cells into the flanks of nude mice, then fed the mice with Figure S3a), the percentages of CD11b-positive cells a normal or a doxycycline-containing diet, respectively. were increased in a dose-dependent manner in 1,25-D3- Tumor growth was monitored twice a week until sacri- treated HL60 and U937, but not in KG-1a, likely due to fice criteria were met in the first mice. Our results low basal level of PKCβ [57]. The differentiation state of showed that doxycycline-induced Pin1 KD significantly 1,25-D3 was similar to that of ATRA in HL-60 and suppressed tumor growth in both HL-60 (Fig. 4a) and U937 (Additional file 3: Figure S3b). Using these models, U937 Pin1 KD cells (Fig. 4b) with reductions of both we further analyzed Pin1 stability and activity. The tumor weight and tumor volume exceeding 50% (Fig. results showed that 1,25-D3 neither reduced Pin1 levels 4c–f). Thus, inducible Pin1 downregulation suppressed in cells (Additional file 3: Figure S3c) nor inhibited Pin1 tumor growth in AML xenograft model in vivo. To as- PPIase activity in vitro (Additional file 3: Figure S3d). sess inactivation of Pin1-regulated cancer pathways in We also checked the stability of Pin1 downstream primary xenograft tumors in vivo, we extracted total targets in U937 (Additional file 3: Figure S3e). As shown proteins from xenograft tumor samples and then sub- in Additional file 3: Figure S3e, differentiation-unrelated jected to perform western blot analyses on Pin1 and its Rab2A did not show obvious changes. NF-κBand β- downstream oncoproteins, including β-catenin, NF-κB, catenin showed a slightly upregulated in high doses, and cyclinD1 and Rab-2A, whose protein levels have been cyclinD1 was slightly downregulated at high doses, shown to be upregulated by Pin1 [26, 40–46, 51]. The which could likely be due to proliferation inhibition. These protein levels of these oncoproteins were decreased with results show that differentiation-inducing drug per se could Pin1 downregulation (Fig. 4g, h), implying that Pin1 KD not downregulate Pin1 stability and activity, confirming Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 8 of 14 Fig. 3 Doxycycline-induced Pin1 downregulation in HL-60- and U937-Tet ON cells suppress clonogenicity in vitro. a, b Using Tet-On system, we generated inducible-shPin1 HL-60 (a) or U937 (b) cells. The effects of Pin1 downregulation were assayed by immunoblotting after Dox treatment (1 mg/ml) for 3 days. c–f Cells were cultured in normal medium supplemented with methylcellulose for 1 or 2 weeks. When colonies became visible, the morphology of cells were taken by transmission electron microscopy (c), followed by staining with p-iodonitrotetrazolium violet for counting (d). The number (e) and area (f) of colonies was measured and counted using ImageJ. Results present the mean ± SD of three independent experiments. Statistically significant differences using Student’s t test are indicated by p values. (*p <0.05, **p <0.01, ***p <0.001) that ATRA-induced Pin1 degradation and downregulation inhibition, chemical inhibition of Pin1 by ATRA can also of Pin1 substrates is differentiation-independent. Further- inhibit AML leukemogenesis through downregulation of more, ATRA treatment also significantly inhibited cell pro- multiple oncogenic pathways. liferation and colony formation of all three leukemia cells (Fig. 5b, c). To explore if ATRA could be effective for those Slow-releasing Pin1 inhibitor ATRA exerts potent normal cells that do not overexpress Pin1, we treated two anticancer activity against AML in vivo immortalized normal blood cells (N1 and N5 cells) with Given the effects of ATRA on cell growth, leukemogenesis, ATRA. First, in both normal cells, Pin1 protein levels were Pin1 protein levels, and the downstream in AML in vitro, a extremely low (Additional file 4: Figure S4a), as we have critical question is whether ATRA suppresses AML tumor shown in normal human bone marrow cells (Fig. 1c, g). Im- growth in vivo. We thus examined ATRA in mouse models portantly, these two normal blood cell lines were com- xenografted with U937 cells. ATRA is extremely light- pletely resistant to ATRA (Additional file 4:FigureS4b). sensitive and can be metabolized quickly in the liver, with a These results are consistent with our previous findings that 45-min half-life in humans. To improve the activity of drug ATRA selectively targets active Pin1 in breast cancer cells, and maintain a constant drug level in the blood, we but not normal breast cells [58]. Thus, besides genetic implanted slow-releasing ATRA pellets, as described in Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 9 of 14 Fig. 4 Pin1 knockdown suppresses tumor growth of human leukemia cells in vivo. After treating HL-60 and U937 Tet-On cells with or without Dox (1 mg/ml) for 3 days in vitro, cells were subcutaneously implanted into the flanks of nude mice fed with a normal or a doxycycline-containing diet,- respectively. a, b Tumor volumes were measured and the tumor growth curves were plotted over time. Error bars represent standard deviations. c, d Photographic illustration of tumors harvested from nude mice at the end point. Each scale of the ruler represents 1 mm. e, f Weightsoftumorsharvested from nude mice at the end point. Error bar represents SEM. g, h Total proteins from xenograft tumor samples were subjected to western blot analysis of the indicated proteins. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001) previous studies [28, 52]. As expected, ATRA blocked the Discussion tumor growth (P = 0.0037) (Fig. 5d)and tumorsizeand Since the heterogeneity of AML is a challenge to clinical weight (P = 0.0057) (Fig. 5e, f). Furthermore, the pro- therapy, combination therapy is increasingly being ex- tein level of Pin1 and Pin1’s oncogenic downstream plored for alternative means of overcoming biological (Fig. 5g) was downregulated in xenograft tumors from heterogeneity in AML. Combining one “targeted” agent nude mice treated with slow-releasing ATRA. These re- with other “targeted” agents or with conventional sults demonstrate that ATRA has potent anti-leukemia chemotherapy may enhance treatment efficacy. Our activity through targeting Pin1 and multi-cancer driving studies suggest that Pin1 could be a potent therapeutic pathway. target in AML. Not only is Pin1 highly overexpressed in Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 10 of 14 Fig. 5 ATRA inhibits the tumorigenesis of human leukemia cells and blocks multiple cancer-driving pathways in vitro and in vivo. a After 72 h incubation of different concentrations of ATRA, cell lysates were subjected to western blot analysis of the indicated proteins. b ATRA inhibits cell proliferation in indicated AML cell lines. After 3 days treatment of different concentrations of ATRA, cell growth was measured by CCK-8 analyses. c ATRA reduces colony formation in indicated AML cell lines. Cells were cultured in normal medium containing methylcellulose with or without ATRA (10 μM) for 1 or 2 weeks, followed by staining with p-iodonitrotetrazolium violet. d–f U937 cells were injected subcutaneously into flank of 7-week-old BALB/c nude mice, and the mice were randomly divided into placebo group and ATRA slow-releasing pellet group. Tumor volumes were measured and the tumor growth curves were plotted over time (d). Error bars represent standard deviations. Photographic illustration of tumors harvested from nude mice at the end point (e). Each scale of the ruler represents 1 mm. Weights of tumors harvested from nude mice at the end point (f). Error bar represents SEM. g Total proteins from xenograft tumor samples were subjected to western blot analysis of indicated Pin1 downstream oncoproteins. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p <0.01, ***p < 0.001) human primary AML cells and established AML cell this correlation due to insufficient numbers of C/EBPα- lines, but the chemical and genetic inhibition of Pin1 p30 positive samples. Despite the lack of sufficient C/ potently inhibits leukemogenesis in vitro and tumorigen- EBPα-p30 samples, significantly higher levels of Pin1 ex- esis in vivo. Thus, combining Pin1-targeted agents with pression were still detected in AML compared with other chemotherapy agents may provide a more efficient healthy controls, which implies that C/EBPα-p30 is not treatment to overcome the heterogeneity of AML. the only mechanism of Pin1 regulation. Elevated expression of Pin1 was detected in acute Several studies have shown that Pin1 can activate mul- leukemia patients in our study. The underlying mechan- tiple oncogenic signaling pathways in solid tumors [17]. ism of increased Pin1 expression likely arises from C/ We now show that Pin1 can promote tumorigenesis in EBPα-p30 which can increase Pin1 mRNA and protein AML via the activation of multiple oncogenes, including levels through E2F1, resulting in reduced C/EBPα func- β-catenin and NF-κB molecules, which belong to the tion, blocked cell differentiation and eventual AML [59]. Wnt/β-catenin and NF-κB pathways, respectively. All of From our own clinical data, we were unable to confirm these pathways are deregulated in AML leukemogenesis Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 11 of 14 [60–62]. In particular, The Wnt signaling pathway is es- maintain a constant drug concentration is used [52]. Here, sential for the maintenance of hematopoietic stem cells, we have shown an anti-leukemia activity against AML for and the fine-tuning regulation of β-catenin expression ATRA in vitro and in mice when slow-releasing ATRA levels is important for hematopoiesis progression [63, pellets are used. In contrast to free ATRA, these pellets 64]. The NF-κB signaling pathway also plays an import- are able to maintain ATRA serum concentrations in mice ant role in the development of AML [65]. Around 40% constant at the concentrations for Pin1 binding and inhib- of AML patients have high expression of constitutive ition as described previously [28]. The potent activity of NF-κB, the aberrant activity of which stimulates ATRA in inducing Pin1 degradation and inhibiting its leukemia cell proliferation and prevents leukemia cell oncogenic function in vitro and in vivo has been con- apoptosis, leading to leukemogenesis [66]. Given the firmed using another independent formulation of con- fundamental role of these signaling pathways in AML trolled release ATRA [79]. Similarly, while regular ATRA leukemogenesis, more and more therapeutic agents tar- needs to combine with others to treat APL, 13-year geting these molecules are being developed [67–70]. follow-up data show that liposomal ATRA with a longer Combining these targeted agents with Pin1 inhibitors, half-life has significant efficacy in APL patients as a single- for example ATRA, may result in even more efficacious agent front-line therapy [80]. These might be related to treatment of AML patients. the fact that ATRA, a vitamin A derivative, is metabolized In addition to its role in cell proliferation, the regula- rapidly in the liver with a very short half-life of 45 min tion of leukemic colony formation is another biological [81–84], possibly because it is an endogenous Pin1 inhibi- phenomenon affected by Pin1 in leukemogenesis. Silen- tor [28]. This idea is also consistent with the clinical find- cing Pin1 by either a stable-shRNA system or an ings that regular ATRA has some detectable but not inducible-shRNA system reduced colony numbers of striking results against AML [78] or solid tumors, but sec- AML cells in vitro. Furthermore, silencing Pin1 down- ond and third generations of much more stable and po- regulated β-catenin in AML cells both in vitro and in tent retinoid derivatives show little efficacy [85], likely vivo. Wnt/β-catenin signaling is known to be involved in because they potently target RARs or RXRs, but no longer the establishment of leukemia stem cells [71]. Leukemia bind to Pin1 [28, 86]. Thus, there is an urgent need to de- stem cells are hypothesized to be responsible for driving velop a longer half-life ATRA formulation or Pin1- leukemia development and disease relapse [72]. The ex- targeted ATRA derivatives or more specific Pin1 inhibitors pression of β-catenin is also correlated with the clono- for treating non-APL leukemia and other cancers. genic proliferation of AML cells and poor prognosis [73, 74]. This implies that Pin1 can maintain the leukemia Conclusion stem cell population and therefore represent an ideal Taken together, we have shown that Pin1 is overexpressed target for the effective treatment of AML. in most human AML patient tissues and cell lines, and In our studies, Pin1 degradation is not related to the that genetic and chemical inhibition of Pin1 inhibits cell effect of leukemia cell differentiation. When we treated proliferation and colony formation and leukemogenesis in AML cells with other differentiation-inducing reagent - AML in vitro and in mice through blocking multiple vitamin D3, Pin1 protein levels remain unchanged. oncogenic signaling pathways. Thus, our findings provide These results further support that ATRA-induced Pin1 evidence that Pin1 is a potential therapeutic target for degradation is through direct-binding [58]. On the other AML and suggest that the development of longer half-life hand, unlike the beneficial effects of differentiation in- ATRA or more potent and specific Pin1 inhibitors could duction in APL, it is difficult to predict the effects of dif- prove effective in the treatment of AML. ferentiation induction in individual AML patients [75– 77]. Therefore, to identify other agents targeting key Additional files molecules in AML, such as Pin1, in combination with differentiation induction therapy would be another strat- Additional file 1: Figure S1. The original whole blots of Pin1 expression in AML patients and leukemia cell lines. a The stronger signals of Pin1 egy to cure AML. protein levels of healthy controls and AML patients were detected with a ATRA has been identified as a Pin1 inhibitor, but it 5 min exposure of blot than with a 30 s exposure of blot. b The stronger had limited success in treatment of non-APL-AML and signals of Pin1 protein levels of healthy controls and leukemia cell lines were detected with a 5 min exposure of blot than with a 30 s exposure of the results of clinical trial have been overall disappoint- blot. (PDF 1205 kb) ing [78]. ATRA binds and induces degradation of Pin1 Additional file 2: Figure S2. The relative intensities of Pin1 downstream and its substrate PML-RARα and thereby exerts antican- oncoproteins in Vec and shPin1. The intensities of each protein signal cer activity against APL in cell and animal models and were determined by ImageJ. The semi-quantitative results were averaged from three independent experiments. (PDF 312 kb) human patients [28]. This anticancer activity of ATRA has Additional file 3: Figure S3. 1,25-(OH) VitaminD , does not affect Pin1 2 3 been confirmed and expanded to breast and liver cancer, stability and function. a, b The expression of CD11b were assayed by but only when slow-releasing ATRA formulations that Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 12 of 14 Consent for publication FACS in HL-60, U937 and KG-1a at 72 h after treatment (a). 1, 25-D3 All the patients that involved in the study have given their consent to publish induces HL-60 and U937 differentiation, but not KG-1a. The differentiation their individual data. state of each cell was assayed by the percentages of CD11b positive cells in indicated cell lines (b). c Pin1 protein levels were not changed after Competing interests 72 h incubation of 1, 25-D3 in HL-60 and U937. d 1,25-D3 does not inhibit Prof. Lu and Dr. Zhou are inventors of Pin1 technology, which was licensed PPIase activity of Pin1. Pin1 was incubated with different concentrations of by BIDMC to Pinteon Therapeutics. Both Prof. Lu and Dr. Zhou own equity 1, 25-D3, followed by chymotrypsin-coupled PPIase assay. e Pin1 down- stream oncoproteins were assayed after 72 h incubation of 1,25-D3 in U937. in, and consult for, Pinteon. Their interests were reviewed and managed by BIDMC in accordance with its conflict of interest policy. The other authors (PDF 2712 kb) declare that they have no competing interests. Additional file 4: Figure S4. Immortalized normal blood cells were resistant to ATRA. a Pin1 protein levels in two immortalized normal blood cells (N1 and N5 cells) were assayed by immunoblotting and compared Publisher’sNote with AML cell lines (HL-60, U937 and KG-1a). N was indicated normal Springer Nature remains neutral with regard to jurisdictional claims in published blood cells. b After 3 days treatment of different concentrations of ATRA, maps and institutional affiliations. cell growth rates were determined by CellTiter-Glo® 2.0 Assay. N1 and N5 cells were completely resistant to ATRA, compared with leukemia cell Author details lines. (PDF 222 kb) Fujian Institute of Hematology, Fujian Provincial Key Laboratory on Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian, China. Division of Translational Therapeutics, Department of Abbreviations Medicine and Cancer Research Institute, Beth Israel Deaconess Medical 1,25-D3: 1-α, 25-Dihydroxyvitamin D ; AL: Acute leukemia; ALL: Aacute Center, Harvard Medical School, Boston, MA 02215, USA. Fujian Key lymphoblastic leukemia; AML: Acute myeloid leukemia; APL: Aacute Laboratory for Translational Research in Cancer and Neurodegenerative promyelocytic leukemia; ATCC: American Type Culture Collection; ATRA: All- Diseases, Institute for Translational Medicine, Fujian Medical University, trans retinoic acid; BCSC: Breast cancer stem cell; CCK-8: Cell Counting Kit-8; Fuzhou 350108, Fujian, China. CSCs: Cancer stem cells; Dox: Doxycycline; FAB: French–American–British classification systems; Pin1 KD: Pin1-knockdown; Pin1: Peptidyl-prolyl cis-trans Received: 30 January 2018 Accepted: 29 April 2018 isomerase NIMA-interacting 1; PPIase: Peptidyl-prolyl isomerase; RT- qPCR: Real-time quantitative PCR; rtTA: Reverse tetracycline-controlled trans- activator; SDS–PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; shPin1: Pin1 shRNA; shRNA: Short hairpin RNA; References Tet: Tetracycline; Vec: Vector; WB: Western blot 1. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood. 2017;129:1577–85. 2. Falini B, Sportoletti P. A scale of “bad” co-mutations in NPM1-driven AML. Acknowledgements Blood. 2017;130:1877–9. Leukemia cell lines cultured in Harvard Medical School Beth Israel Deaconess 3. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Medical Center were gifts from Dr. Daniel G Tenen. 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Abstract

Background: The increasing genomic complexity of acute myeloid leukemia (AML), the most common form of acute leukemia, poses a major challenge to its therapy. To identify potent therapeutic targets with the ability to block multiple cancer-driving pathways is thus imperative. The unique peptidyl-prolyl cis-trans isomerase Pin1 has been reported to promote tumorigenesis through upregulation of numerous cancer-driving pathways. Although Pin1 is a key drug target for treating acute promyelocytic leukemia (APL) caused by a fusion oncogene, much less is known about the role of Pin1 in other heterogeneous leukemia. Methods: The mRNA and protein levels of Pin1 were detected in samples from de novo leukemia patients and healthy controls using real-time quantitative RT-PCR (qRT-PCR) and western blot. The establishment of the lentiviral stable-expressed short hairpin RNA (shRNA) system and the tetracycline-inducible shRNA system for targeting Pin1 were used to analyze the biological function of Pin1 in AML cells. The expression of cancer-related Pin1 downstream oncoproteins in shPin1 (Pin1 knockdown) and Pin1 inhibitor all-trans retinoic acid (ATRA) treated leukemia cells were examined by western blot, followed by evaluating the effects of genetic and chemical inhibition of Pin1 in leukemia cells on transformed phenotype, including cell proliferation and colony formation ability, using trypan blue, cell counting assay, and colony formation assay in vitro, as well as the tumorigenesis ability using in vivo xenograft mouse models. Results: First, we found that the expression of Pin1 mRNA and protein was significantly increased in both de novo leukemia clinical samples and multiple leukemia cell lines, compared with healthy controls. Furthermore, genetic or chemical inhibition of Pin1 in human multiple leukemia cell lines potently inhibited multiple Pin1 substrate oncoproteins and effectively suppressed leukemia cell proliferation and colony formation ability in cell culture models in vitro. Moreover, tetracycline-inducible Pin1 knockdown and slow-releasing ATRA potently inhibited tumorigenicity of U937 and HL-60 leukemia cells in xenograft mouse models. (Continued on next page) * Correspondence: xzhou@bidmc.harvard.edu; klu@bidmc.harvard.edu; chenyz@mail.fjmu.edu.cn Equal contributors Division of Translational Therapeutics, Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA Fujian Institute of Hematology, Fujian Provincial Key Laboratory on Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 2 of 14 (Continued from previous page) Conclusions: We demonstrate that Pin1 is highly overexpressed in human AML and is a promising therapeutic target to block multiple cancer-driving pathways in AML. Keywords: Acute myeloid leukemia (AML), Pin1 inhibitor, All-trans retinoic acid (ATRA), Oncogenic signaling, Leukemia treatment Background non-CSC tumor cells and CSCs, respectively, compared Acute myeloid leukemia (AML) is the most common form with normal breast epithelial cells [26]. Mechanically, Pin1 of acute leukemia and arises from a malignant transform- promotes breast cancer stem cell (BCSC)-proliferation ation of multipotent hematopoietic stem cells with a re- and tumorigenesis in vitro and in vivo by increasing markable genomic alteration [1]. AML development Rab2A transcription and thereby Erk activation, Zeb1 up- requires the collaboration of at least two classes of cyto- regulation, and β-catenin nuclear translocation [25]. genetic abnormalities [2]. This “two-hit model” [3], pre- Moreover, Pin1 can also stabilize NOTCH1 expression by sented by Gilliland and Griffin (2002), proposes that class reducing ubiquitin ligase FBW7 to promote self-renewal I mutations activate signaling transduction pathways to and metastasis in breast cancer CSCs [27, 29]. In acute promote cell proliferation and that class II mutations promyelocytic leukemia (APL), genetic or chemical inhib- affect transcription factors to block maturation of ition of Pin1 can induce the degradation of the disease- hematopoietic cells [4, 5]. The proteins involved in AML causing fusion oncogene PML-RARα, which causes APL processes include both fusion proteins (e.g., RUNX1/ETO, through blockage of promyelocyte differentiation and pro- CBFβ/MYH11, and PML/RARα) and mutated proteins (e. motion of self-renewal capacity [28]. In addition, Pin1 is g., NPM1, FLT3, and C/EPBα Eisfeld, 2017 #11467, [6–9]), required not only for the stability of the stem cell repro- as well as some molecular features that are a function of gramming factors Nanog, Oct4, and MYC but also for in- overexpression (e.g., BAALC, MN1, ERG-1, and AF1q) ducing and maintaining pluripotency [28, 30–33]. [10]. Such complexity of molecular and cytogenetic abnor- Therefore, Pin1 dysregulation has an important role in the malities poses a major challenge to formulating AML self-renewal and tumorigenic properties of CSCs. therapy [11, 12]. Chemotherapeutic approaches continue Given that Pin1 can promote tumorigenesis by activating to be the mainstay therapies used for AML treatments multiple signaling pathways and inducing and sustaining [13]. However, long-term survival using these therapies is self-renewal capability in a variety of solid tumors, Pin1 only obtained in 35 to 40% of younger patients [14, 15], may also play a critical role in leukemogenesis. Pin1 is a key and the long-term survival of elderly AML patients is even drug target for treating APL [28], and controls Notch3 pro- lower because only about one third of them are eligible tein expression and regulates T-ALL progression [34]. Si- for intensive chemotherapies [16]. Therefore, therapy still lencing Pin1 can delay the progression of lymphoma needs to come a long way to fully overcome AML. Hence, disease in Eμ-myc transgenic mice; however, much less is there is a pressing need to identify potent therapeutic tar- known about the role of Pin1 in the development and treat- gets to block multiple cancer-driving pathways for the ment of other more common and heterogeneous leukemia. treatment of AML. In this manuscript, our clinical data analyses demon- Pin1 is a unique peptidyl-prolyl isomerase (PPIase) that strated higher Pin1 expression in a variety of AML sub- catalyzes cis/trans isomerization of specific pSer/Thr-Pro types compared to healthy controls. Genetic or chemical motifs and central common phosphorylation motifs in cell inhibition of Pin1 downregulated multiple cancer- proliferation and transformation [17]. Hence, altered Pin1 promoting signaling pathways, leading to the inhibition function can play a profound role in pathogenesis of hu- of cell proliferation and colony formation capability in man disease, notably cancer. Pin1 is overexpressed and/or multiple human AML cell lines. Moreover, inducible over-activated in many human cancers, thereby disrupting downregulation or chemical inhibition of Pin1 inhibited the balance between oncoproteins and tumor suppressors, the tumorigenesis of AML in vivo. Taken together, these and by amplifying numerous cancer-driven pathways. results demonstrate that Pin1 is highly overexpressed in Moreover, overexpression and/or over-activation of Pin1 human AML and is a promising therapeutic target to frequently correlate with poor clinical prognosis [17–24]. block multiple cancer pathways in AML. Besides the well-known role of Pin1 in tumorigenesis, recent studies have revealed that Pin1 dysregulation plays Methods an important role in cancer stem cells (CSCs) in breast Sample source cancer and leukemia [25–28]. In primary human breast Bone marrow samples (n = 150) were collected from tumors, the expression of Pin1 is 5 and 30 times higher in healthy donors and patients treated in the Hematology Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 3 of 14 Department of the Fujian Medical University Union In line with the operation steps of the quantitative Hospital from September 2012 to February 2015. Of PCR kit (Roche), the reaction system contains SYBR these, 107 samples were taken from patients with acute Green Master (ROX) 10 μl, upstream primer (10 pmol/ leukemia (AL) and 43 were taken from healthy donors. μl) 0.15 μl, downstream primer (10 pmol/μl) 0.15 μl, AL patients after the first visit included 61 male and 46 cDNA 1 μl, and double distilled water 8.7 μl. The reac- female patients in age of 13~76 years old with median tion was conducted in ABI7500 fluorogenic quantitative age of 45 years old. According to FBA classification for PCR instrument (Applied Biosystem Company) under the diagnosis and classification of AML [35], there were conditions: at 50 °C for 2 min and 95 °C for 10 min, and 6 patients with M0, 9 with M1, 18 with M2, 7 with M3, then at 95 °C for 15 s and 60 °C for 1 min as 1 cycle for 1 with M4, 41 with M5, 3 with M6, 1 with M7, 21 cases 40 times in total. Then, melting curve reaction was con- with other types of AL, 43 healthy controls included 20 ducted under conditions: at 95 °C for 15 s, 60 °C for male, and 23 female donors in age of 20~45 years old 1 min, 95 °C for 15 s, 60 °C for 15 s, 1 cycle for once. with median 32 years old. All of the patients and healthy Three ventral orifices were set in every sample. With β- donors signed informed consent. actin as an internal reference, the relative expression quantity of mRNA was represented with RQ value by −ΔCT −ΔCT 2 method: RQ = 2 . The final results are Cell strain and reagents presented as the fold change of Pin1 expression in a All cells were cultured in RPMI1640 with 10 or 20% target sample relative to a reference sample, normalized FBS. HL60 (acute myelogenous leukemia cell line), U937 to β-actin. (histiocytic lymphoma cell line), KG-1a (acute myeloid leukemia cell line), NB4 (acute promyelocytic leukemia cell line), Nalm-6 (acute B acute lymphoblastic leukemia Lentiviral transduction and knockdown of Pin1 in AML cell line), Molt-4 (acute T acute lymphoblastic leukemia cells cell line), Kasumi-1 (acute myelogenous leukemia cell Recombinant lentiviral particles were produced in 293FT line), and K562 (chromic granulocytic leukemia cell line) cells by co-transfecting pRevTRE plasmid containing Pin1 were cultured at the Fujian Institution of Hematology. shRNA sequence, along with helper plasmids, including HL-60, U937, and KG-1a cell lines were cultured at the pCMV-VSVG and pCMV-dR8.91. The virus-containing BIDMC and were gifts from Dr. Daniel G Tenen. Anti- medium was harvested at 48 h after transfection and fil- bodies against various proteins were obtained from the tered using a 0.45-μm filter. For infection, the collected following sources: Pin1 was previously described [36]; β- virus medium was added to the rtTA-expressing cells with actin from Sigma; Cyclin D1 (DCS-6) from Biolegend; polybrene. The cells were selected with G418 and puro- NF-κB/p65 (D14E12, 8242) from Cell Signaling Technol- mycin. Pin1 shRNA were induced by doxycycline, and ogy; β-catenin from BD biosciences; and Rab-2A (15420- Pin1 protein level was analyzed by immunoblot. Pin1 1-AP) from Proteintech Group. All-trans retinoic acid shRNA sequence is CCACCGTCACACAGTATTTAT. (ATRA) were purchased from Sigma, 1,25-dihydroxyvi- tamin D3 were purchased from Cayman Chemicals, and Cell viability assay ATRA-releasing pellets were from Innovative Research Cells were seeded on 96-well plates at a density of 5000 of America. cells per well. Cells were stained with Trypan blue and counted every day. In two additional groups, cells were Quantitative real-time PCR treated with DMSO or ATRA. After 72 h, cell viability Total RNA from bone marrow mononuclear cell and cell was measured by CCK-8 assay (DOJINDO) and the line was extracted by TRIzol (Invitrogen), and cDNA number of cells was determined by CellTiter-Glo® 2.0 was transcribed with reverse transcription kit (Thermo Assay (Promega, Madison, WI) according to the manu- Fisher Scientific) in accordance with the manufacturer’s facturer’s instructions. protocols. PCR reactions were performed using quantita- tive PCR kit (Roche) and ABI7500 fluorogenic quantita- tive PCR instrument (Applied Biosystem Company). β- Colony formation assay actin was used as an internal control of samples. The Cells were seeded on 24-well plate at a density of 500 following primers were used: cells (KG-1a is 1000 cells). Methylcellulose (Sigma) in final concentration of 0.8% was added and mixed. After β-actin (F): AGTGTGACGTGGACATCCGCAA. 7 days (KG-1a is 14 days), cells were stained with 0.5 ml β-actin (R): ATCCACATCTGCTGGAAGGTGGAC. 1 mg/ml p-Iodonitrotetrazolium Violet. The number of Pin1 (F): GCTCAGGCCGAGTGTACTACTT. colonies and the total area were calculated by ImageJ Pin1(R): CGAGGCGTCTTCAAATGG. analysis. Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 4 of 14 PPIase assay using SPSS 23.0 and GraphPad Prism 5 software package. The PPIase activity on GST-Pin1 in response to ATRA and All data are presented as the means ± SD/SEM, followed by 1,25-(OH) vitamin D3 were determined using the determining significant differences using the two-tailed chymotrypsin-coupled PPIase activity assay with the sub- student’s t test or analysis of variance (ANOVA) test where strate Suc-Ala-pSer-ProPhe-pNA (50 nM) in buffer contain- *p <0.05, **p <0.01, and ***p < 0.001. ing 35 mM HEPES (pH 7.8) and 0.1 mg/ml BSA at 10 °C as described, with the exception that the compounds were pre- Results incubated with enzymes for 12 h at 4 °C [37]. Pin1 overexpression in leukemia patients To investigate the potential clinical significance of Pin1 FACS analysis in leukemia, we examined the relative expression of To assess cell surface expression of CD11b, cells were PIN1 mRNA in bone marrow mononuclear cells from washed with PBS, harvested by non-enzymatic cell dis- 107 newly diagnosed leukemia patients and 43 healthy sociation solution, and resuspended in blocking solution bone marrow donors. We found that PIN1 mRNA ex- (Ca2+, Mg2+-free PBS containing 2% FCS). Cells were pression was significantly higher in bone marrow cells of then incubated with CD11b-FITC (Biolegend) for 15 min acute leukemia (AL) patients compared to those of at 4 °C in the dark. Cells were washed and analyzed by healthy controls (Fig. 1a, lane 2 versus lane 1). Among using a Beckman Coulter’s Gallios flow cytometry. these samples, 86 samples were diagnosed with AML pa- tients, which also showed significantly higher PIN1 Western blot mRNA expression, as compared with healthy controls Cells from each group were collected, and cell lysis buf- (Fig. 1a, lane 3 versus lane 1). Further analysis of the re- fer (Roche Company) was added to extract total protein. lationship between the expression of PIN1 mRNA and The protein concentration was detected by the Bradford FAB (French–American–British classification system) method. The loading sample of equivalent protein was subtypes of AML showed that PIN1 mRNA was upregu- transferred to a nitrocellulose membrane (Bio-Rad, cat. lated in most subtypes of AML. In the case of M4 and No. 162-0115) through SDS-PAGE electrophoresis and M6, low sample sizes precluded sufficient power for stat- immunoblotting. istical analysis (Fig. 1b). Consistent with the results of PIN1 mRNA, Pin1 protein levels were higher in AML Animal studies patient samples compared with healthy controls (Fig. 1c, For xenograft experiments, 5 × 10 HL-60 and U937 cells d; Additional file 1: Figure S1a). A positive correlation stably expressing Tet-On shPin1 were injected subcutane- was found between PIN1 mRNA level and Pin1 protein ously into flank of 7-week-old BALB/c nude mice (Taco- level (Fig. 1e). In addition, we used different colors for nic Laboratories) and fed with normal or doxycycline- individual patients in western blot analysis. These results containing diet. For ATRA treatment, 5 × 10 U937 cells together demonstrate that Pin1 is highly overexpressed were injected subcutaneously into flank of 7-week-old in AML patients. BALB/c nude mice. After 5 days when tumor growth was We also examined Pin1 expression in human leukemia just about notable by sight, the mice were randomly di- cell lines, including six AML cell lines (Kasumi-1, U937, vided into placebo group or ATRA slow-releasing pellet K562, NB4, HL-60, and KG-1a) and two ALL cell lines group. Tumor growth was monitored twice a week until (Nalm-6 and Molt-4). Both PIN1 mRNA (Fig. 1f)and sacrifice criteria were met in the first mice. Tumor sizes Pin1 protein levels (Fig. 1g, h; Additional file 1:Figure were recorded twice a week by a caliper and tumor vol- S1b) were found to be significantly higher in human umes were calculated using formula L × W × 0.52, where leukemia cell lines as compared with bone marrow cells of L and W represent length and width, respectively. Tumors healthy control (Fig. 1f–h). A positive correlation of PIN1 were cut into small pieces and quickly stored in liquid mRNA and Pin1 protein levels was also found in leukemia nitrogen, partly for protein extraction. All BALB/c nude cell lines (Fig. 1i). These results further confirm that Pin1 mice were housed in laminar flow cabinets with free is overexpressed in leukemia cells including AML cells. access to food and water. Animal work was carried out in compliance with the ethical regulations approved by the Constitutive Pin1 knockdown inhibits cell proliferation Animal Care Committee, Beth Israel Deaconess Medical and clonogenicity of human AML cells in vitro Center, Boston, USA. Given the overexpression of Pin1 in AML, including bi- opsied bone morrow leukemia cells and established hu- Statistical analysis man AML cell lines, the question remains as to whether Experiments were routinely repeated at least three times, Pin1 plays any role in leukemogenesis. Based on the and the repeat number was increased according to effect “two-hit model,” the ability of a molecule to promote size or sample variation. Statistical analyses were performed cell proliferation and colony formation is required in Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 5 of 14 Fig. 1 Expression of PIN1 mRNA and protein in leukemia patients and cell lines. a The fold changes of PIN1 mRNA expression in bone marrow cells from normal controls, untreated acute leukemia patients (AL) and AML patients. b The fold changes of PIN1 mRNA expression in bone marrow cells from normal controls and FAB subtypes of untreated AML patients. c, d Pin1 protein levels in bone marrow cells from normal and AML patients were analyzed by immunoblotting (c). The quantitative results of Pin1 expression was analyzed from 1c (d). The individual patients used for immunoblot analysis were indicated by different colors. e The correlation between PIN1 mRNA and Pin1 protein levels in AML patients. Different colors were used to indicate different patients in 1c and 1d. f The fold changes of PIN1 mRNA expression in normal control bone marrow cells and several leukemia cell lines, including AML cells (Kasumi-1, U937, K562, NB4 and KG-1a) and ALL cells (Nalm-6 and Molt-4). g, h The protein levels (g) and quantitative results (h) of Pin1 in normal control bone marrow cells and leukemia cell lines. Different cell lines were indicated by corresponding colors. i The correlation between PIN1 mRNA and Pin1 protein levels in leukemia cell lines was analyzed based on 1f and 1 h. Individual cell lines were indicated by corresponding colors. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001) order to initiate leukemogenesis [38]. We investigated To test if Pin1 KD would impair the leukemogenic po- the effects of Pin1 on leukemogensis-related traits. To tential of AML cells to grow as clonogenic colonies, HL- this end, multiple AML cell lines, including HL-60, 60, U937, and KG-1a expressing Pin1 shRNA or control U937 and KG-1a, were infected with lentiviruses carry- shRNA were plated at a low density and observed for ing validated Pin1-specific shRNA [26] or control colony formation. A significant decrease both in the shRNA with Puro . Using puromycin selection, stable number (Fig. 2c, d) and size (Fig. 2c, e) of colonies was Pin1-knockdown (Pin1 KD) cell lines were established. observed in the Pin1 KD group compared with controls. Immunoblot analysis confirmed a dramatic decrease in Thus, our data demonstrates that Pin1 plays a positive endogenous Pin1 in cells carrying Pin1-shRNA com- role in leukemogenesis-relevant events in vitro. As a pared with cells carrying the control shRNA (Fig. 2a). major regulator of oncoproteins, Pin1 amplifies onco- The rates of cell growth of different pairs of AML genic pathways by activating more than 43 oncogenic Pin1-shRNA and control-shRNA cells were monitored molecules and suppressing at least 20 tumor-suppressing by counting cell number to demonstrate the effects of molecules, including many of which have well- Pin1 downregulation on cell proliferation. In all three established roles in CSCs [17, 21, 39]. To further AML cell lines examined, Pin1 KD resulted in a signifi- characterize the underlying mechanisms of Pin1- cant reduction in cell proliferation (Fig. 2b). mediated leukemogenesis, we analyzed the expression of Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 6 of 14 b c Fig. 2 Constitutive Pin1 downregulation suppresses oncogenic biological functions and signaling in vitro. a To establish stable-shPin1 cell lines, HL-60 or U937 or KG-1a cells were infected with lentivirus expressing scramble (Vec) or Pin1 shRNA (shPin1) with Puro . After puromycin selection for 1 week, Pin1 levels were validated by immunoblotting analysis. b Pin1 downregulation inhibits cell proliferation in indicated AML cell lines. Cell growth was monitored for 1 week by cell counting. p values were derived from the cell numbers for each group at the end point. c–e Cells were cultured in normal medium supplemented with methylcellulose for 1 or 2 weeks. When colonies became visible, cells were stained with p-iodonitrotetrazolium violet for counting. The number (d) and area (e) of colonies was measured and counted using ImageJ. Results present the mean ± SD of three independent experiments. f Cell lysates were subjected to western blot analysis with antibodies against the downstream oncogenic proteins of Pin1. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001) several leukemogeneis-related signaling molecules that have substrates in other cancer cells [26, 40–46]. AML Pin1 KD been reported to be Pin1 substrates. Wnt/β-catenin and cells also downregulated the expression of Pin1 oncopro- NF-κB pathways are well-known signaling pathways in tein substrates, including β-catenin and NF-κB(Fig. 2f), as leukemogenesis through regulating cell proliferation, differ- with solid tumors [26, 40–46]. Besides these key regulators entiation, or apoptosis and have been shown to be Pin1 in leukemogenesis, Pin1 also transcriptionally regulated Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 7 of 14 some stem cells-related molecules, including Rab2A [47] suppresses tumorigenesis through downregulation of and CyclinD1 [48, 49]. In AML Pin1 KD cells, these mole- oncogenic signaling pathways. cules were downregulated as well (Fig. 2f)(Additional file 2: Figure S2). Therefore, these biochemical analyses further The Pin1 inhibitor ATRA downregulates Pin1 oncoprotein support the role for Pin1 in the regulation of cell prolifera- substrates and inhibits cell growth and clonogenicity of tion and clonogenicity through deregulation of multiple human AML cells in vitro oncogenic pathways. Given the oncogenic role of Pin1 in AML, we wondered whether targeting Pin1 with chemical inhibitors would show any therapeutic benefit in the treatment of AML Doxycycline-inducible Pin1 knockdown inhibits [28]. ATRA has been reported to inhibit and degrade ac- clonogenicity of human AML cells in vitro tive Pin1, resulting in the blockade of multiple Pin1- To further confirm and evaluate the effects of Pin1 in- regulated oncogenic pathways in APL, breast cancer, and hibition on the tumorigenesis of AML, we generated liver cancer [28, 30, 52]. However, the effect of ATRA on stable tetracycline-inducible Pin1 knockdown HL-60 and AML cells is unknown. Therefore, we treated multiple U937 cells using a Tet-On system [28, 50]. We infected AML cells, HL-60, U937, and KG-1a, with different con- HL-60- and U937-rtTA cells with lentivirus containing centrations of ATRA. ATRA can degrade Pin1 and its either Pin1 shRNA or control shRNA with Puro . Stable downstream oncoproteins in a dose-dependent manner tetracycline-inducible Pin1 KD and control HL-60- and (Fig. 5a). Notably, in both cells, among these oncopro- U937-Tet ON cells were established using puromycin se- teins, while NF-κB was slightly upregulated in low con- lection. In the presence of doxycycline, Pin1 protein centrations of ATRA likely due to ATRA-induced levels were dramatically reduced in both cell lines (Fig. differentiation, they were downregulated once ATRA 3a, b). Furthermore, the clonogenic assay showed a sig- concentration was high enough to induce Pin1 degrad- nificant decrease in the number (Fig. 3c, d) and size of ation. This further supported that ATRA could down- colonies (Fig. 3e, f) after doxycycline treatment as com- regulate multiple oncoproteins through degrading Pin1 pared with the vehicle control. These results show that in AML. our Tet-On Pin1 KD system is highly inducible and Since ATRA induces differentiation in various could be used to confirm the role of Pin1 in leukemia cell lines, it is unclear whether differentiation leukemogenesis, as shown in constitutive Pin1 KD cells would affect Pin1 levels. To address this question, we (Fig. 2). treated cells with other differentiation-inducing reagent − 1-α, 25-(OH) vitamin D (1,25-D3), the active form of 2 3 Doxycycline-inducible Pin1 knockdown inhibits vitamin D , which has been shown to induce differenti- tumorigenesis of human AML cells in vivo. ation in various AML cell lines [53–56]. After 72 h of To examine whether inducible Pin1 inhibition would incubation of 1, 25-D3 or ATRA, differentiation was affect tumorigenesis of human AML cells in vivo, we evaluated by flow cytometry using a general myeloid subcutaneously implanted HL-60- and U937-Tet ON marker, CD11b. As shown in Figure S3a (Additional file 3; cells into the flanks of nude mice, then fed the mice with Figure S3a), the percentages of CD11b-positive cells a normal or a doxycycline-containing diet, respectively. were increased in a dose-dependent manner in 1,25-D3- Tumor growth was monitored twice a week until sacri- treated HL60 and U937, but not in KG-1a, likely due to fice criteria were met in the first mice. Our results low basal level of PKCβ [57]. The differentiation state of showed that doxycycline-induced Pin1 KD significantly 1,25-D3 was similar to that of ATRA in HL-60 and suppressed tumor growth in both HL-60 (Fig. 4a) and U937 (Additional file 3: Figure S3b). Using these models, U937 Pin1 KD cells (Fig. 4b) with reductions of both we further analyzed Pin1 stability and activity. The tumor weight and tumor volume exceeding 50% (Fig. results showed that 1,25-D3 neither reduced Pin1 levels 4c–f). Thus, inducible Pin1 downregulation suppressed in cells (Additional file 3: Figure S3c) nor inhibited Pin1 tumor growth in AML xenograft model in vivo. To as- PPIase activity in vitro (Additional file 3: Figure S3d). sess inactivation of Pin1-regulated cancer pathways in We also checked the stability of Pin1 downstream primary xenograft tumors in vivo, we extracted total targets in U937 (Additional file 3: Figure S3e). As shown proteins from xenograft tumor samples and then sub- in Additional file 3: Figure S3e, differentiation-unrelated jected to perform western blot analyses on Pin1 and its Rab2A did not show obvious changes. NF-κBand β- downstream oncoproteins, including β-catenin, NF-κB, catenin showed a slightly upregulated in high doses, and cyclinD1 and Rab-2A, whose protein levels have been cyclinD1 was slightly downregulated at high doses, shown to be upregulated by Pin1 [26, 40–46, 51]. The which could likely be due to proliferation inhibition. These protein levels of these oncoproteins were decreased with results show that differentiation-inducing drug per se could Pin1 downregulation (Fig. 4g, h), implying that Pin1 KD not downregulate Pin1 stability and activity, confirming Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 8 of 14 Fig. 3 Doxycycline-induced Pin1 downregulation in HL-60- and U937-Tet ON cells suppress clonogenicity in vitro. a, b Using Tet-On system, we generated inducible-shPin1 HL-60 (a) or U937 (b) cells. The effects of Pin1 downregulation were assayed by immunoblotting after Dox treatment (1 mg/ml) for 3 days. c–f Cells were cultured in normal medium supplemented with methylcellulose for 1 or 2 weeks. When colonies became visible, the morphology of cells were taken by transmission electron microscopy (c), followed by staining with p-iodonitrotetrazolium violet for counting (d). The number (e) and area (f) of colonies was measured and counted using ImageJ. Results present the mean ± SD of three independent experiments. Statistically significant differences using Student’s t test are indicated by p values. (*p <0.05, **p <0.01, ***p <0.001) that ATRA-induced Pin1 degradation and downregulation inhibition, chemical inhibition of Pin1 by ATRA can also of Pin1 substrates is differentiation-independent. Further- inhibit AML leukemogenesis through downregulation of more, ATRA treatment also significantly inhibited cell pro- multiple oncogenic pathways. liferation and colony formation of all three leukemia cells (Fig. 5b, c). To explore if ATRA could be effective for those Slow-releasing Pin1 inhibitor ATRA exerts potent normal cells that do not overexpress Pin1, we treated two anticancer activity against AML in vivo immortalized normal blood cells (N1 and N5 cells) with Given the effects of ATRA on cell growth, leukemogenesis, ATRA. First, in both normal cells, Pin1 protein levels were Pin1 protein levels, and the downstream in AML in vitro, a extremely low (Additional file 4: Figure S4a), as we have critical question is whether ATRA suppresses AML tumor shown in normal human bone marrow cells (Fig. 1c, g). Im- growth in vivo. We thus examined ATRA in mouse models portantly, these two normal blood cell lines were com- xenografted with U937 cells. ATRA is extremely light- pletely resistant to ATRA (Additional file 4:FigureS4b). sensitive and can be metabolized quickly in the liver, with a These results are consistent with our previous findings that 45-min half-life in humans. To improve the activity of drug ATRA selectively targets active Pin1 in breast cancer cells, and maintain a constant drug level in the blood, we but not normal breast cells [58]. Thus, besides genetic implanted slow-releasing ATRA pellets, as described in Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 9 of 14 Fig. 4 Pin1 knockdown suppresses tumor growth of human leukemia cells in vivo. After treating HL-60 and U937 Tet-On cells with or without Dox (1 mg/ml) for 3 days in vitro, cells were subcutaneously implanted into the flanks of nude mice fed with a normal or a doxycycline-containing diet,- respectively. a, b Tumor volumes were measured and the tumor growth curves were plotted over time. Error bars represent standard deviations. c, d Photographic illustration of tumors harvested from nude mice at the end point. Each scale of the ruler represents 1 mm. e, f Weightsoftumorsharvested from nude mice at the end point. Error bar represents SEM. g, h Total proteins from xenograft tumor samples were subjected to western blot analysis of the indicated proteins. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001) previous studies [28, 52]. As expected, ATRA blocked the Discussion tumor growth (P = 0.0037) (Fig. 5d)and tumorsizeand Since the heterogeneity of AML is a challenge to clinical weight (P = 0.0057) (Fig. 5e, f). Furthermore, the pro- therapy, combination therapy is increasingly being ex- tein level of Pin1 and Pin1’s oncogenic downstream plored for alternative means of overcoming biological (Fig. 5g) was downregulated in xenograft tumors from heterogeneity in AML. Combining one “targeted” agent nude mice treated with slow-releasing ATRA. These re- with other “targeted” agents or with conventional sults demonstrate that ATRA has potent anti-leukemia chemotherapy may enhance treatment efficacy. Our activity through targeting Pin1 and multi-cancer driving studies suggest that Pin1 could be a potent therapeutic pathway. target in AML. Not only is Pin1 highly overexpressed in Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 10 of 14 Fig. 5 ATRA inhibits the tumorigenesis of human leukemia cells and blocks multiple cancer-driving pathways in vitro and in vivo. a After 72 h incubation of different concentrations of ATRA, cell lysates were subjected to western blot analysis of the indicated proteins. b ATRA inhibits cell proliferation in indicated AML cell lines. After 3 days treatment of different concentrations of ATRA, cell growth was measured by CCK-8 analyses. c ATRA reduces colony formation in indicated AML cell lines. Cells were cultured in normal medium containing methylcellulose with or without ATRA (10 μM) for 1 or 2 weeks, followed by staining with p-iodonitrotetrazolium violet. d–f U937 cells were injected subcutaneously into flank of 7-week-old BALB/c nude mice, and the mice were randomly divided into placebo group and ATRA slow-releasing pellet group. Tumor volumes were measured and the tumor growth curves were plotted over time (d). Error bars represent standard deviations. Photographic illustration of tumors harvested from nude mice at the end point (e). Each scale of the ruler represents 1 mm. Weights of tumors harvested from nude mice at the end point (f). Error bar represents SEM. g Total proteins from xenograft tumor samples were subjected to western blot analysis of indicated Pin1 downstream oncoproteins. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p <0.01, ***p < 0.001) human primary AML cells and established AML cell this correlation due to insufficient numbers of C/EBPα- lines, but the chemical and genetic inhibition of Pin1 p30 positive samples. Despite the lack of sufficient C/ potently inhibits leukemogenesis in vitro and tumorigen- EBPα-p30 samples, significantly higher levels of Pin1 ex- esis in vivo. Thus, combining Pin1-targeted agents with pression were still detected in AML compared with other chemotherapy agents may provide a more efficient healthy controls, which implies that C/EBPα-p30 is not treatment to overcome the heterogeneity of AML. the only mechanism of Pin1 regulation. Elevated expression of Pin1 was detected in acute Several studies have shown that Pin1 can activate mul- leukemia patients in our study. The underlying mechan- tiple oncogenic signaling pathways in solid tumors [17]. ism of increased Pin1 expression likely arises from C/ We now show that Pin1 can promote tumorigenesis in EBPα-p30 which can increase Pin1 mRNA and protein AML via the activation of multiple oncogenes, including levels through E2F1, resulting in reduced C/EBPα func- β-catenin and NF-κB molecules, which belong to the tion, blocked cell differentiation and eventual AML [59]. Wnt/β-catenin and NF-κB pathways, respectively. All of From our own clinical data, we were unable to confirm these pathways are deregulated in AML leukemogenesis Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 11 of 14 [60–62]. In particular, The Wnt signaling pathway is es- maintain a constant drug concentration is used [52]. Here, sential for the maintenance of hematopoietic stem cells, we have shown an anti-leukemia activity against AML for and the fine-tuning regulation of β-catenin expression ATRA in vitro and in mice when slow-releasing ATRA levels is important for hematopoiesis progression [63, pellets are used. In contrast to free ATRA, these pellets 64]. The NF-κB signaling pathway also plays an import- are able to maintain ATRA serum concentrations in mice ant role in the development of AML [65]. Around 40% constant at the concentrations for Pin1 binding and inhib- of AML patients have high expression of constitutive ition as described previously [28]. The potent activity of NF-κB, the aberrant activity of which stimulates ATRA in inducing Pin1 degradation and inhibiting its leukemia cell proliferation and prevents leukemia cell oncogenic function in vitro and in vivo has been con- apoptosis, leading to leukemogenesis [66]. Given the firmed using another independent formulation of con- fundamental role of these signaling pathways in AML trolled release ATRA [79]. Similarly, while regular ATRA leukemogenesis, more and more therapeutic agents tar- needs to combine with others to treat APL, 13-year geting these molecules are being developed [67–70]. follow-up data show that liposomal ATRA with a longer Combining these targeted agents with Pin1 inhibitors, half-life has significant efficacy in APL patients as a single- for example ATRA, may result in even more efficacious agent front-line therapy [80]. These might be related to treatment of AML patients. the fact that ATRA, a vitamin A derivative, is metabolized In addition to its role in cell proliferation, the regula- rapidly in the liver with a very short half-life of 45 min tion of leukemic colony formation is another biological [81–84], possibly because it is an endogenous Pin1 inhibi- phenomenon affected by Pin1 in leukemogenesis. Silen- tor [28]. This idea is also consistent with the clinical find- cing Pin1 by either a stable-shRNA system or an ings that regular ATRA has some detectable but not inducible-shRNA system reduced colony numbers of striking results against AML [78] or solid tumors, but sec- AML cells in vitro. Furthermore, silencing Pin1 down- ond and third generations of much more stable and po- regulated β-catenin in AML cells both in vitro and in tent retinoid derivatives show little efficacy [85], likely vivo. Wnt/β-catenin signaling is known to be involved in because they potently target RARs or RXRs, but no longer the establishment of leukemia stem cells [71]. Leukemia bind to Pin1 [28, 86]. Thus, there is an urgent need to de- stem cells are hypothesized to be responsible for driving velop a longer half-life ATRA formulation or Pin1- leukemia development and disease relapse [72]. The ex- targeted ATRA derivatives or more specific Pin1 inhibitors pression of β-catenin is also correlated with the clono- for treating non-APL leukemia and other cancers. genic proliferation of AML cells and poor prognosis [73, 74]. This implies that Pin1 can maintain the leukemia Conclusion stem cell population and therefore represent an ideal Taken together, we have shown that Pin1 is overexpressed target for the effective treatment of AML. in most human AML patient tissues and cell lines, and In our studies, Pin1 degradation is not related to the that genetic and chemical inhibition of Pin1 inhibits cell effect of leukemia cell differentiation. When we treated proliferation and colony formation and leukemogenesis in AML cells with other differentiation-inducing reagent - AML in vitro and in mice through blocking multiple vitamin D3, Pin1 protein levels remain unchanged. oncogenic signaling pathways. Thus, our findings provide These results further support that ATRA-induced Pin1 evidence that Pin1 is a potential therapeutic target for degradation is through direct-binding [58]. On the other AML and suggest that the development of longer half-life hand, unlike the beneficial effects of differentiation in- ATRA or more potent and specific Pin1 inhibitors could duction in APL, it is difficult to predict the effects of dif- prove effective in the treatment of AML. ferentiation induction in individual AML patients [75– 77]. Therefore, to identify other agents targeting key Additional files molecules in AML, such as Pin1, in combination with differentiation induction therapy would be another strat- Additional file 1: Figure S1. The original whole blots of Pin1 expression in AML patients and leukemia cell lines. a The stronger signals of Pin1 egy to cure AML. protein levels of healthy controls and AML patients were detected with a ATRA has been identified as a Pin1 inhibitor, but it 5 min exposure of blot than with a 30 s exposure of blot. b The stronger had limited success in treatment of non-APL-AML and signals of Pin1 protein levels of healthy controls and leukemia cell lines were detected with a 5 min exposure of blot than with a 30 s exposure of the results of clinical trial have been overall disappoint- blot. (PDF 1205 kb) ing [78]. ATRA binds and induces degradation of Pin1 Additional file 2: Figure S2. The relative intensities of Pin1 downstream and its substrate PML-RARα and thereby exerts antican- oncoproteins in Vec and shPin1. The intensities of each protein signal cer activity against APL in cell and animal models and were determined by ImageJ. The semi-quantitative results were averaged from three independent experiments. (PDF 312 kb) human patients [28]. This anticancer activity of ATRA has Additional file 3: Figure S3. 1,25-(OH) VitaminD , does not affect Pin1 2 3 been confirmed and expanded to breast and liver cancer, stability and function. a, b The expression of CD11b were assayed by but only when slow-releasing ATRA formulations that Lian et al. Journal of Hematology & Oncology (2018) 11:73 Page 12 of 14 Consent for publication FACS in HL-60, U937 and KG-1a at 72 h after treatment (a). 1, 25-D3 All the patients that involved in the study have given their consent to publish induces HL-60 and U937 differentiation, but not KG-1a. The differentiation their individual data. state of each cell was assayed by the percentages of CD11b positive cells in indicated cell lines (b). c Pin1 protein levels were not changed after Competing interests 72 h incubation of 1, 25-D3 in HL-60 and U937. d 1,25-D3 does not inhibit Prof. Lu and Dr. Zhou are inventors of Pin1 technology, which was licensed PPIase activity of Pin1. Pin1 was incubated with different concentrations of by BIDMC to Pinteon Therapeutics. Both Prof. Lu and Dr. Zhou own equity 1, 25-D3, followed by chymotrypsin-coupled PPIase assay. e Pin1 down- stream oncoproteins were assayed after 72 h incubation of 1,25-D3 in U937. in, and consult for, Pinteon. Their interests were reviewed and managed by BIDMC in accordance with its conflict of interest policy. The other authors (PDF 2712 kb) declare that they have no competing interests. Additional file 4: Figure S4. Immortalized normal blood cells were resistant to ATRA. a Pin1 protein levels in two immortalized normal blood cells (N1 and N5 cells) were assayed by immunoblotting and compared Publisher’sNote with AML cell lines (HL-60, U937 and KG-1a). N was indicated normal Springer Nature remains neutral with regard to jurisdictional claims in published blood cells. b After 3 days treatment of different concentrations of ATRA, maps and institutional affiliations. cell growth rates were determined by CellTiter-Glo® 2.0 Assay. N1 and N5 cells were completely resistant to ATRA, compared with leukemia cell Author details lines. (PDF 222 kb) Fujian Institute of Hematology, Fujian Provincial Key Laboratory on Hematology, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian, China. Division of Translational Therapeutics, Department of Abbreviations Medicine and Cancer Research Institute, Beth Israel Deaconess Medical 1,25-D3: 1-α, 25-Dihydroxyvitamin D ; AL: Acute leukemia; ALL: Aacute Center, Harvard Medical School, Boston, MA 02215, USA. Fujian Key lymphoblastic leukemia; AML: Acute myeloid leukemia; APL: Aacute Laboratory for Translational Research in Cancer and Neurodegenerative promyelocytic leukemia; ATCC: American Type Culture Collection; ATRA: All- Diseases, Institute for Translational Medicine, Fujian Medical University, trans retinoic acid; BCSC: Breast cancer stem cell; CCK-8: Cell Counting Kit-8; Fuzhou 350108, Fujian, China. CSCs: Cancer stem cells; Dox: Doxycycline; FAB: French–American–British classification systems; Pin1 KD: Pin1-knockdown; Pin1: Peptidyl-prolyl cis-trans Received: 30 January 2018 Accepted: 29 April 2018 isomerase NIMA-interacting 1; PPIase: Peptidyl-prolyl isomerase; RT- qPCR: Real-time quantitative PCR; rtTA: Reverse tetracycline-controlled trans- activator; SDS–PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; shPin1: Pin1 shRNA; shRNA: Short hairpin RNA; References Tet: Tetracycline; Vec: Vector; WB: Western blot 1. Thomas D, Majeti R. Biology and relevance of human acute myeloid leukemia stem cells. Blood. 2017;129:1577–85. 2. Falini B, Sportoletti P. A scale of “bad” co-mutations in NPM1-driven AML. Acknowledgements Blood. 2017;130:1877–9. Leukemia cell lines cultured in Harvard Medical School Beth Israel Deaconess 3. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Medical Center were gifts from Dr. Daniel G Tenen. 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Journal of Hematology & OncologySpringer Journals

Published: May 30, 2018

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