Abstract
The formation of the BCR-ABL fusion gene drives human chronic myeloid leukemia (CML). The last 2 decades have witnessed that specific tyrosine kinase inhibitors (TKIs, e.g., imatinib mesylate, IM) against ABL1 improve disease treatment, although some patients still suffer from relapse and TKI resistance. Therefore, a better understanding of the molecular pathology of CML is still urgently needed. miR-181a-5p (miR-181a) acts as a tumor suppressor in CML; however, the molecular mechanism of miR-181a in CML stem/progenitor cells remains elusive. Herein, we showed that miR-181a inhibited the growth of CML CD34+ cells, including the quiescent subset, and sensitized them to IM treatment, while miR-181a inhibition by a sponge sequence collaborated with BCR-ABL to enhance the growth of normal CD34+ cells. Transcriptome data and biochemical analysis revealed that SERPINE1 was a bona fide and critical target of miR-181a, which deepened the understanding of the regulatory mechanism of SERPINE1. Genetic and pharmacological inhibition of SERPINE1 led to apoptosis mainly mediated by caspase-9 activation. The dual inhibition of SERPINE1 and BCR-ABL exhibited a significantly stronger inhibitory effect than a single agent. Taken together, this study demonstrates that a novel miR-181a/SERPINE1 axis modulates CML stem/progenitor cells, which likely provides an important approach to override TKI resistance.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Chronic myeloid leukemia originates from hematopoietic stem cells acquiring the BCR-ABL fusion gene, which encodes a tyrosine kinase with consecutive activity. The development of tyrosine kinase inhibitors (TKIs, e.g., imatinib mesylate, IM) has witnessed the transition of CML from a fatal disease to a manageable ailment [1,2,3]. Nevertheless, up to 25% of patients in the chronic phase are confronted with drug resistance and relapse. For those who achieve long-term major molecular remission (MMR), the discontinuation of TKI results in frequent relapse [3]. Accumulating evidence indicates that CML stem cells are responsible for relapse and that the survival of these cells is not entirely dependent on BCR-ABL activity [4,5,6,7,8,9]. Therefore, the identification of other factors contributing to the survival of CML stem/progenitor cells will provide new clues to improve disease treatment.
microRNAs (miRNAs, or miRs) are 18 to 25-nucleotide long, non-coding single-stranded RNAs that regulate gene expression by degrading target transcripts or blocking their translation [10, 11]. It is well established that miRNAs play crucial roles in the growth and drug response of CML cells [12,13,14], including leukemic stem/progenitor cells [15,16,17,18,19,20,21,22,23,24]. Thus the study of miRNAs likely leads to the identification of novel predictive biomarkers or therapeutic targets [23, 25,26,27]. miR-181a-5p (hereinafter referred to as miR-181a) plays a tumor-suppressor role in CML [17, 28,29,30,31,32], and small activating (sa) RNAs targeting the promoter of miR-181a exhibits anti-leukemia activity through enhancing the expression of miR-181a [33]. We have reported the downregulation of miR-181a in CML CD34+ cells [17]; however, the direct target of miR-181a in these cells has not yet been elucidated.
Plasminogen activator inhibitor-1 (PAI-1) is one of the most important inhibitors of the plasminogen/plasmin system, which belongs to the conserved serine protease inhibitor (SERPIN) family and is designated as SERPIN family E member 1 (SERPINE1) [34, 35]. In general, SERPINE1 promotes tumor cell growth via its proangiogenic activity or antiapoptotic property [36,37,38]. A recent report shows that SERPINE1 facilitates the recruitment and polarization of macrophage in cancer, which provides a new mechanism explaining its pro-tumorigenic function [39]. SERPINE1 inhibitors exhibit antitumor activities in vitro and in vivo [40,41,42,43,44]. Recently, it has been reported that PAI-1 induced by TGF-β is responsible for retaining hematopoietic stem cells in the niche [45]. PAI-1 deficiency impairs BCR-ABL+ leukemic stem cells (LSCs), and a PAI-1 inhibitor (TM5614) plus IM shows a stronger effect to eradicate LSCs than a single agent in mice [46]. Importantly, TM5614 exhibits anti-leukemia activity in human CML cells in vitro [47]. Additionally, the combination therapy of TM5614 and TKIs is well tolerated in patients and achieves a higher molecular response (MR) rate than TKI treatment alone [48]. To date, the regulatory microRNA of SERPINE1 in human CML cells has not been reported and the effects of SERPINE1 silencing or SERPINE1 pharmacological inhibition on human CML stem/progenitor cells have not been well evaluated.
In the present study, we demonstrate that miR-181a plays an important role in the growth and TKI response of CML stem/progenitor cells. Moreover, SERPINE1 is identified as a bona fide target of miR-181a in CML cells. Genetic and pharmacological inhibition of SERPINE1 inhibits the in vitro and in vivo growth of BCR-ABL+ cells. Taken together, our data reveal a novel miR-181a/SERPINE1 axis modulating the growth and TKI response of CML stem/progenitor cells.
Materials and methods
Patients and cells
K562 cells, KU812 cells and 293 T cells were obtained from the Cell Bank of the Chinese Academy (www.cellbank.org.cn). K562 and KU812 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), and 293 T cells were maintained in DMEM medium plus 10% FBS. Murine BaF3 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 5 ng/mL mIL-3. BaF3/BCR-ABL cells were generated by BCR-ABL lentiviral transduction and maintained in RPMI 1640 medium plus 10% FBS. Bone marrow cells from human CML patients and healthy donors were obtained from the Hematological Biobank, Jiangsu Biobank of Clinical Resources, in accordance with the Declaration of Helsinki and with written informed consent approved by the Ethical Committee of Soochow University (SUDA20211224H03, Suzhou, China). A gradient centrifuge with Lymphocyte-H cell separation medium (Cedarlane Laboratories, Burlington, NC, USA) was used to obtain the nucleated cells, and an EasySep CD34 positive selection kit (STEMCELL Technologies, Vancouver, BC, Canada) was used to purify CD34+ cells. Quiescent CD34+ cells were isolated with Hoechst and Pyronin Y staining following Holyoake’s study [49]. The clinical characteristics of CML patients recruited in the present study are summarized in Supplementary Table S1.
RNA extraction and RT-qPCR
For the assessment of mRNA, the extraction of total RNA and RT-qPCR analysis were performed as previously described [50]. For the measurement of microRNA, the total RNA was prepared by TRIzol (Thermo Scientific, Waltham, MA USA). 500 ng extracted RNA was reversed-transcribed into cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche (China) Holding Ltd., Shanghai, China) and miRNA specific primers (GenePharma Co., Ltd., Shanghai, China) according to manufacturer’s protocol. Real-time quantification was carried out using 0.5 μL cDNA, 400 nM primers, 12.5 μL 2 × Power SYBR Green PCR Master Mix (ThermoFisher, Applied Biosystems, Foster City, CA, USA) on 7500 Real Time PCR System (Applied Biosystems). β-ACTIN or u6 were used as endogenous controls, to normalize mRNA and miRNA input. The gene-specific primers are detailed in Supplementary Table S2.
Lentiviral vectors and viral production
The shRNA sequences, mature miR-181a, and scramble sequence were synthesized and subcloned into a LV3 (H1/GFP&Puro) vector by GenePharma Co., Ltd. The sponge sequence against miR-181a were reported previously by others [51], and 3 consecutive sponge sequences were synthesized and subcoloned into LV3 vector by GenePharma Co., Ltd. All above sequences are shown in Supplementary Table S3. SERPINE1 cDNA was amplified with specific primers (Supplementary Table S2) and then subcloned into a lentiviral vector [52].
Lentiviral production was performed with polyethylenimine (PEI) transfection of 293 T cells. 6 × 106 293 T cells were plated into a 10 cm dish for 16 h and medium was replaced with fresh medium 2 h before transfection. 450 μL Opti-MEM containing 6 μg lentiviral vector and three packaging constructs was mixed with 50 μL PEI solution (1 mg/mL). After incubation at room temperature for 15 min, the mixture was added to 293 T cells. The lentiviruses were harvested 48 h and 72 h post transfection. The concentrated viruses were prepared by ultracentrifugation.
Colony-forming cell assay
Normal bone marrow (NBM) and CML CD34+ cells were transduced with concentrated lentivirus, and the transduced CD34+ cells were isolated using fluorescence-activated cell sorting (FACS) (BD FACSAria III, Becton Dickinson, Franklin Lakes, NJ, USA). The colony-forming cell (CFC) assay was performed as previously described [52, 53]. A total of 1000 FACS-isolated cells were plated in methylcellulose medium (MethoCult H4230, STEMCELL Technologies) supplemented with a cocktail of cytokines, including stem cell factor (SCF, 50 ng/mL), interleukin-3 (IL-3, 20 ng/mL), interleukin-6 (IL-6, 20 ng/mL), granulocyte macrophage colony-stimulating factor (GM-CSF, 20 ng/mL), granulocyte colony-stimulating factor (G-CSF, 20 ng/mL), and erythropoietin (EPO, 3 IU/mL). The colonies were classified and counted 14–16 days later. For CFC assays with imatinib mesylate (IM), SCF and EPO were not supplemented.
Western blotting
Protein samples were prepared using protein lysate buffer (Beyotime, Shanghai, China) and equal amounts of protein samples were separated with SDS-PAGE. These samples were transferred from the electrophoresed gel onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was subsequently incubated with specific antibodies and developed the films with an ECL detection system (GE Healthcare Life Sciences, Piscataway, NJ, USA). The antibodies used in this study are listed in Supplementary Table S4.
Microarray analysis
K562 cells were infected with scramble and miR-181a viruses, and after 72 h GFP+ cells were sorted for RNA extraction. RNA was prepared using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer's recommendations. Microarray analysis was performed using Agilent whole human genome oligo-chips (4 × 180 K) in Shanghai Biotechnology Corporation and analyzed as previously described [50].
Luciferase reporter assay
pEZX vector with the full-length 3′-UTR of SERPINE1 (NM_000602, from stop codon to the end, 1805 bp) and the blank control were purchased from GeneCopoeia, Inc. (Guangzhou, China, Cat#: HmiT054357-MT01). Two small constructs containing the predicted miR-181a recognition motifs (motif-1:1904–1926, motif-2:2899–2921) and their mutants (“ugaaugu” changed to “uacuagu”) were subcloned into pEZX blank vector between its EcoRI and XhoI sites using various primers (listed in Supplementary Table S2). The transfection was performed as previously described [53], 3 μg vector plasmid was transfected into 293 T cells using Lipofectamine 3000 (Thermo Scientific), or it was transfected into K562 cells using electrotransfection. 48 h later, the harvested cells were lysated to measure the luciferase and renilla activities using a Dual-luciferase Reporter Assay Kit (Promega, Madison, WI, USA) and presented as a Firefly/Renilla ratio. The relative reporter activities of individual samples in miR-181a-overexpressiong cells were normalized to control cells.
Assays of ROS, mitochondrial activity and membrane potential
The cells were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime), and DMSO was added as the control. Then, the cells were analyzed by flow cytometry.
Mitochondrial activity was analyzed using MitoTracker Red (CMXRos) staining (Beyotime). The cells were stained with the dye and then analyzed by flow cytometry. The mean fluorescence intensity (MFI) of tiplaxtinin-treated cells was compared with untreated control cells.
Mitochondrial membrane potential was analyzed with a mitochondrial membrane potential detection kit (Beyotime). The cells were stained with JC-1 and were analyzed by flow cytometry. The fluorescent emission of JC-1 aggregates (590 nm, red) and that of JC-1 monomers (520 nm, green) were measured, and the ratio of the fluorescent intensities of aggregate to monomer dyes was determined.
Activity assays of caspase-8 and caspase-9
Caspase-8 and 9 activities in cell lysates were determined using caspase-8 and 9 activity kits (Beyotime) according to the manufacture’s protocol. The lysate was mixed with corresponding substrate, incubated for 1 h at 37 °C, and measured OD405. The activities were normalized by the protein amount of the corresponding cell lysate, and expressed in units per microgram of protein.
Animals
BaF3/BCR-ABL cell was used as a model to generate leukemia in mouse studies as previously described [53]. Six- to 8-week-old female BalB/C mice were lethally irradiated (650 cGy), and the sorted test or control cells (5 × 104/mouse for miR-181a overexpression, 1 × 105/mouse for Serpine1 silencing) combined with non-irradiated mouse bone marrow cells (5 × 105/mouse) as donor cells were injected intravenously into the recipient mice (no less than 6 mice in each group). These mice were observed closely for signs of weight loss or lethargy. The diseased mice were dissected, and the spleens and livers were weighed. Cells from the spleen, liver, bone marrow, and peripheral blood were analyzed by flow cytometry to evaluate the infiltration of leukemic cells. All animals were maintained under pathogen-free conditions and in compliance with national and institutional guidelines. All protocols were approved by the Ethics Committee of Soochow University (SUDA20211224A01, Suzhou, China).
Statistical analysis
All values are represented as the mean ± SEM from more than three biological replicates, and statistical analysis was performed with Student’s t test, in which p < 0.05 was considered significant. The Kaplan–Meier method was used to study the survival tendency, and the P value was estimated using the log-rank test.
Results
The expression of miR-181a is suppressed by BCR-ABL activity in CML stem/progenitor cells
To investigate the role of miR-181a in CML stem/progenitor cells, the expression of miR-181a in CML stem/progenitor cells compared with normal control cells was assessed by RT-qPCR in a cohort of patients recruited from the First Affiliated Hospital of Soochow University during 2018–2022. The results showed that miR-181a expression was significantly lower in CML CD34+ cells (n = 11) than in normal bone marrow (NBM) CD34+ cells (n = 10) (Fig. 1A). Moreover, miR-181a expression was lower in CML CD34+38− cells than in their normal counterparts (Fig. 1B). In line with these data, miR-181a expression was decreased in BCR-ABL-expressing Lin−Sca-1+c-Kit+ (LSK) cells compared with control cells in the public database (GSE122432) (Fig. 1C); another set of data (GSE148567) showed that miR-181a expression was higher in CML patients underwent allogenetic transplantation than in newly diagnosed patients (Fig. 1D). To explore the possible relationship between miR-181a and BCR-ABL, we browsed the database to show that miR-181a expression was increased upon imatinib or dasatinib treatment in K562 cells compared with untreated cells (GSE28825) (Fig. 1E). Hence nilotinib (NL) was used to treat K562 cells, and the results showed that miR-181a expression was significantly enhanced (Fig. 1F). Similarly, imatinib treatment significantly increased miR-181a expression in CML CD34+ cells (n = 10) (Fig. 1G), though the extent was not as strong as that in K562 cells treated with NL.
miR-181a acts as a tumor suppressor in CML stem/progenitor cells
To study the functional role of miR-181a in leukemic stem/progenitor cells, miR-181a and scramble sequences were delivered into CML CD34+ cells by lentiviral vectors, and then the CFC production of the transduced CD34+ cells was assayed. The data showed that the CFC production of miR-181a-transduced cells was significantly lower than that of the control cells (Fig. 2A). Quiescent CML CD34+ cells were obtained and transfected with miR-181a and scramble oligonucleotides, and the results showed that miR-181a significantly suppressed the CFC production of quiescent CD34+ cells compared with the control (scramble) (Fig. 2B). miR-181a-transduced and control CML CD34+ cells were subjected to IM treatment, and the data showed that miR-181a enhanced the IM response of CML CD34+ cells (Fig. 2C). Similarly, miR-181a rendered quiescent CML CD34+ cells more sensitive to IM treatment (Fig. 2D).
Next, the function of miR-181a in NBM CD34+ cells was studied. miR-181a overexpression in NBM CD34+ significantly suppressed CFC production of these cells compared with the control (Fig. 2E). A sponge sequence, which entraps miR-181a was introduced to successfully inhibit miR-181a expression in NBM CD34+ cells (Fig. 2F). The sponge and BCR-ABL significantly enhanced the CFC production of NBM CD34+ cells compared with the control; moreover, the sponge collaborated with BCR-ABL to yield significantly more CFC production than the sponge or BCR-ABL alone (Fig. 2G).
Although the tumor-suppressor role of miR-181a in CML cells has been reported [17, 28,29,30,31,32], the effect of miR-181a on BCR-ABL-induced leukemogenesis has not been studied. Herein, we showed that miR-181a overexpression significantly suppressed the growth and CFC production of BaF3/BCR-ABL cells (Fig. 2H, I). In animal studies, Kaplan–Meier analysis indicated that miR-181a significantly delayed BCR-ABL-induced leukemia (Fig. 2J). The coefficients of both the liver and the spleen were significantly lower in the miR-181a-overexpressing group than in the control group (Fig. 2K, L). The infiltration of leukemic cells in the bone marrow, peripheral blood, liver, and spleen of the diseased mice in the miR-181a-overexpressing group was significantly lower than in the control group (Fig. 2M–P). Overall, these data demonstrated that miR-181a played a tumor-suppressor role in CML stem/progenitor cells.
SERPINE1 is a bona fide target of miR-181a in CML CD34+ cells
Identifying the direct target of miR-181a is the key to understanding how miR-181a regulates the growth and TKI response of CML stem/progenitor cells. Microarray data were generated to compare miR-181a-transduced K562 cells with control cells. A total of 181 transcripts were downregulated upon miR-181a. In silicon analysis showed that over 7 thousand transcripts are potential miR-181a direct targets in the TargetScan database. The intersection of downregulated transcripts upon miR-181a overexpression and the potential direct targets included 57 transcripts. When more stringent criteria were used, the candidate list was shortened to 15 transcripts (Fig. 3A), whose expression in miR-181a-transduced and control K562 cells was displayed in a heatmap (Fig. 3B). Among these candidates, GATA6 is a reported miR-181a target [54]. RT-qPCR analysis showed that GATA6 expression was significantly decreased in miR-181a-transduced cells than in control cells (Fig. S1), which supported the validity of our microarray data. SERPINE1 was chosen as it promotes cancer cell growth and its inhibitors are available. Hence, we performed studies to validate that both the transcript and protein expression of SERPINE1 was decreased in miR-181a-transduced cells compared with control cells (Fig. 3C). Conversely, the sponge against miR-181a significantly enhanced SERPINE1 expression in NBM CD34+ cells (Fig. 3D). Interestingly, SERPINE1 expression was significantly higher in CML CD34+ cells than in NBM CD34+ cells (28-fold, p < 0.05) (Fig. 3E), and SERPINE1 expression was significantly correlated with miR-181a expression in CML CD34+ cells (Fig. 3F).
A reporter vector containing the full-length 3′-UTR of SERPINE1 was delivered into miR-181a-overexpressing and control 293 T or K562 cells, and the results showed that miR-181a significantly decreased reporter activity compared with the control (Fig. 3G). Two potential miR-181a recognition motifs were predicted (Fig. 3H). Two constructs (~ 200 bp) containing the predicted motifs (wild-type, WT) and their mutants (MT), were subcloned into the reporter vector. The reporter assay in K562 cells showed that the reporter activity of construct-2 [containing motif-2 (2899–2921)] was significantly suppressed by miR-181a but not construct-1 [containing motif-1 (1904–1926)], and the construct-2 mutant was not regulated by miR-181a (Fig. 3I). Therefore, miR-181a directly regulates the expression of SERPINE1 in CML stem/progenitor cells through a specific recognition motif.
SERPINE1 silencing inhibits the growth of CML stem/progenitor cells and sensitizes them to IM treatment
To study the function of SERPINE1 in CML cells, two independent shRNA and control (scramble) sequences were delivered into K562 cells, and RT-qPCR and Western blotting showed that these two shRNAs effectively inhibited SERPINE1 expression (Fig. S2A, B). SERPINE1 silencing inhibited the growth and CFC production of K562 cells (Fig. S2C, D). As these two shRNA sequences displayed similar inhibitory effect, we conduced following studies with only one of them (shSERPINE1#1). The result showed that SERPINE1 silencing rendered K562 cells more sensitive to IM treatment (Fig. S2E). RT-qPCR showed that SERPINE1 expression in CML CD34+ cells was significantly suppressed by shSERPINE1#1 (Fig. 4A), which led to significant reduction of CFC production of CML CD34+ cells and hypersensitivity of these cell upon IM treatment (Fig. 4B, C). It was noted that SERPINE1 silencing also significantly suppressed the CFC production of normal CD34+ cells from healthy donors (Fig. S2E).
To examine the effect of Serpine1 silencing on BCR-ABL-induced leukemogenesis, two independent shRNA sequences against Serpine1 and the control (scramble) were delivered into BaF3/BCR-ABL cells, and both effectively inhibited Serpine1 expression (Fig. S3G). Serpine1 silencing significantly suppressed the growth and CFC production of BaF3/BCR-ABL cells (Fig. S2H, I). As these two shRNAs had similar inhibitory effects, we chose one (shSerpine1#1) to conduct animal studies. Kaplan–Meier analysis showed that Serpine1 silencing significantly inhibited BCR-ABL-induced leukemia (Fig. 4D). The coefficient of the spleen but not the liver was significantly decreased by Serpine1 silencing (Fig. 4E, F). Moreover, Serpine1 silencing led to significantly fewer leukemic cells in both the liver and spleen, as analyzed by flow cytometry (Fig. 4G–J).
Finally, a rescue experiment was performed to elucidate the role of SERPINE1 in miR-181a-mediated regulation of the growth and IM response of CML cells. The results showed that SERPINE1 overexpression did not enhance the growth of K562 cells or confer IM resistance to these cells; however, this action partially rescued the growth inhibition and IM hypersensitivity induced by miR-181a (Fig. 4K–M). Overall, this study identified a novel miR-181a/SERPINE1 axis that modulates the growth and TKI response of CML cells.
Tiplaxtinin and IM collaboratively inhibit the growth of CML stem/progenitor cells
SERPINE1 inhibitors exhibit anti-leukemia activity [47]; however, their effect on CML stem/progenitor cells has not yet been reported. Herein, two types of SERPINE1 inhibitors (tiplaxtinin and TM5441) were studied (Fig. S3A). The effects of these two inhibitors on the viability of K562 cells, KU812 cells and BaF3/BCR-ABL cells were analyzed (Fig. 5A, B, Fig. S3B, S3C, Fig. S4A). The results showed that both had inhibitory effects on the viability of CML cells and that CML cells were more sensitive to tiplaxtinin treatment than TM5441 treatment. The effect of tiplaxtinin on the growth and CFC production of K562 cells was also analyzed (Fig. S3D, E). Next, the effect of the combination of the SERPINE1 inhibitor and IM was studied, and both tiplaxtinin and TM5441 collaborated with IM to inhibit the viability of K562 cells and KU812 cells (Fig. 5C, Fig. S3F). The combination of tiplaxtinin and IM showed a stronger inhibitory effect than the combination of TM5441 and IM. Additionally, the combination of tiplaxtinin and IM showed a stronger inhibitory effect on BaF3/BCR-ABL cells than a single agent (Fig. S4B). Then, tiplaxtinin was chosen for the following studies. The results showed that tiplaxtinin inhibited the CFC production of CML CD34+ cells in a dose-dependent manner and exhibited a stronger inhibitory effect on leukemic cells than on normal control cells (Fig. 5D). The effect of the combination of tiplaxtinin and IM on the CFC production of NBM CD34+ cells, IM-sensitive CML CD34+ cells, and IM-resistant CML CD34+ cells was studied. In this assay, CML CD34+ cells were divided into two categories based on CFC survival measurement in the presence of 5 μM IM following the criteria of Jiang’s study [55]. The individual CML sample with a survival rate less than 60% was assigned as IM-sensitive; otherwise, the sample was IM-resistant. The combination treatment had a stronger inhibitory effect on both IM-sensitive and IM-resistant CML CD34+ cells than on NBM CD34+ cells (Fig. 5E), and effectively induced apoptosis in CML CD34+ cells (Fig. S5). Finally, the combination treatment manifested a much stronger inhibitory effect on leukemic quiescent CD34+ cells than on normal quiescent CD34+ cells (Fig. 5F).
Tiplaxtinin, a SERPINE1 inhibitor, activates caspase-9 to induce apoptosis of CML cells
A previous report showed that SERPINE1 protects cells from FasL/Fas-mediated apoptosis [36]. Therefore, the effect of SERPINE1 blockade on the apoptosis of CML cells was studied. The results showed that SERPINE1 silencing strongly induced apoptosis in K562 cells (Fig. 6A, Fig. S6A). Western blotting indicated that caspase-9 and PARP were activated, while the phosphorylation of STAT5 was not affected (Fig. 6B), which suggested a possible collaborative effect of the SERPINE1 inhibitor and IM on the growth of CML cells. Tiplaxtinin treatment strongly induced apoptosis in CML CD34+ cells but not in NBM CD34+ cells (Fig. 6C, Fig. S6B). Western blotting indicated that caspase-3, caspase-8, caspase-9, and PARP were all activated upon tiplaxtinin treatment (Fig. 6D). Next, the results showed that SERPINE1 silencing slightly increased the activity of caspase-8 while strongly enhanced caspase-9 activity (Fig. 6E). Similarly, caspase-9 activity was induced by tiplaxtinin in a time-dependent manner, while caspase-8 activity was induced to a much lesser extent than caspase-9 activity (Fig. 6F). As caspase-9 activation is tightly linked with internal apoptosis and mitochondrial dysfunction, the effects of tiaplaxtinin on mitochondrial activities were analyzed. The results showed that reactive oxygen species (ROS) generation was significantly enhanced by tiplaxtinin in a dose-dependent manner (Fig. 6G). The activity of mitochondria was decreased by tiplaxtinin treatment, as indicated by MitoTracker (Red CMXRos) staining (Fig. 6H, Fig. S6C). The membrane potential of mitochondria was impaired upon tiplaxtinin treatment, as shown by JC-1 staining (Fig. 6I, Fig. S6D). Western blotting showed that Bax expression was strongly increased, while Bcl-2 expression was not affected upon tiplaxtinin treatment, which strongly suggested that tiplaxtinin induced internal apoptosis. Moreover, γ-H2A.X expression was increased, suggesting the accumulation of DNA damage, possibly due to the enhancement of ROS (Fig. 6J). The analysis of protein extracts from the cytoplasm and mitochondria indicated the translocation of Bax from the mitochondria to the cytoplasm and the release of cytochrome C from the mitochondria to the cytoplasm (Fig. 6K), supporting the involvement of caspase-9-mediated internal apoptosis upon tiplaxtinin treatment. Finally, a pan-caspase inhibitor Z-VAD-FMK was used to perform rescue experiments. The co-treatment of tiplaxtinin and Z-VAD-FMK significantly decreased apoptosis and increased cell viability (Fig. 6L, M, Fig. S6E). Interestingly, Z-VAD-FMK significantly decreased the activation of caspase-9 but not caspase-8 upon tiplaxtinin (Fig. 6N), supporting the notion that caspase-9 activation plays a critical role in tiplaxtinin-induced apoptosis in CML cells.
Overall, we identified SERPINE1 as a bona fide target of miR-181a in CML stem/progenitor cells, which demonstrated the importance of SERPINE1 in BCR-ABL-mediated leukemogenesis. Interestingly, SERPINE1 inhibitors (e.g., tiplaxtinin) combined with TKI exhibited stronger anti-leukemia effect than a single agent, while sparing normal stem/progenitor cells (Fig. 7), which supported that the combination treatment may benefit CML management.
Discussion
Several reports have described the tumor-suppressor role of miR-181a in CML cells [17, 28,29,30,31,32]; however, the effect of miR-181a on BCR-ABL-induced leukemogenesis has not yet been elucidated. In the present study, we showed that miR-181a overexpression delayed the generation of BCR-ABL-induced leukemia in mice and significantly inhibited the infiltration of leukemic cells. Additionally, the role of miR-181a in CML stem/progenitor cells has not been well studied. Herein, we showed that miR-181a inhibition enhanced the growth of NBM CD34+ cells and that this effect was enhanced by BCR-ABL, which strongly suggested the tumor-suppressor role of miR-181a in BCR-ABL+ leukemic cells. Our data also showed that miR-181a inhibited quiescent CML CD34+ cells and sensitized them to IM treatment. Therefore, we provided important pieces of evidence to highlight the importance of miR-181a in CML stem/progenitor cells.
The key to understanding the molecular mechanism of miR-181a in CML stem/progenitor cells is to identify the crucial target of miR-181a. Previously, some miR-181a targets were identified, including Bcl-2, RalA, and Parp1 [28, 29, 32]. In this study, we identified SERPINE1 as a bona fide target of miR-181a as our data demonstrated that miR-181a regulated the expression of SERPINE1 through a specific recognition motif. Importantly, “rescue” experiments showed that SERPINE1 played a critical role in miR-181a-mediated CML cell growth and IM response.
Interestingly, the role of SERPINE1 in leukemic stem/progenitor cells was recently reported in a mice model [46]. Similarly, we showed that Serpine1 silencing attenuated BCR-ABL-induced leukemia in a model of BaF3 cells, which agreed with the previous study. Importantly, our results showed that SERPINE1 silencing suppressed the CFC production of CD34+ cells from CML patients, including the quiescent subset, which expanded the previous findings.
Studies have shown that SERPINE1 inhibitors exhibit anti-leukemia effects in mice model of CML, and the combination of SERPINE1 inhibitor and TKI achieves a higher molecular response rate in CML patients than monotherapy with TKIs [47, 48]. However, the direct effect of combination therapy on CML stem/progenitor cells is still elusive. In this study, we showed that the combination of SERPINE1 inhibitor and TKI effectively suppressed the growth of IM-resistant CML CD34+ cells and quiescent CML CD34+ cells, while it had a mild effect on normal control cells. Therefore, previous studies and ours strongly promoted the combination therapy in the future.
Overall, this study has revealed a novel regulatory connection between miR-181a and SERPINE1 in CML stem/progenitor cells and provided new pieces of evidence to promote the combination therapy of SERPINE1 inhibitors and TKIs in CML treatment.
Availability of data and materials
Data supporting the findings are included in this article and the Supplemental information. The raw data and experimental materials are available from the corresponding author upon reasonable request.
References
Druker BJ (2008) Translation of the Philadelphia chromosome into therapy for CML. Blood 112:4808–4817
Mughal TI, Radich JP, Deininger MW et al (2016) Chronic myeloid leukemia: reminiscences and dreams. Haematologica 101:541–558
Braun TP, Eide CA, Druker BJ (2020) Response and resistance to BCR-ABL1-targeted therapies. Cancer Cell 37:530–542
Graham SM, Jørgensen HG, Allan E et al (2002) Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood 99:319–325
Jiang X, Saw KM, Eaves A et al (2007) Instability of BCR-ABL gene in primary and cultured chronic myeloid leukemia stem cells. J Natl Cancer Inst 99:680–693
Jiang X, Zhao Y, Smith C et al (2007) Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies. Leukemia 21:926–935
Chu S, McDonald T, Lin A et al (2011) Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment. Blood 118:5565–5572
Corbin AS, Agarwal A, Loriaux M et al (2011) Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Invest 121:396–409
Hamilton A, Helgason GV, Schemionek M et al (2012) Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival. Blood 119:1501–1510
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Iwakawa HO, Tomari Y (2015) The functions of MicroRNAs: mRNA decay and translational repression. Trends Cell Biol 25:651–665
Bueno MJ, Pérez de Castro I, Gómez de Cedrón M et al (2008) Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression. Cancer Cell 13:496–506
Litwińska Z, Machaliński B (2017) miRNAs in chronic myeloid leukemia: small molecules, essential function. Leuk Lymphoma 58:1297–1305
Navabi A, Akbari B, Abdalsamadi M et al (2022) The role of microRNAs in the development, progression and drug resistance of chronic myeloid leukemia and their potential clinical significance. Life Sci 296:120437
Agirre X, Jiménez-Velasco A, San José-Enériz E et al (2008) Down-regulation of hsa-miR-10a in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth. Mol Cancer Res 6:1830–1840
Wang WZ, Pu QH, Lin XH et al (2015) Silencing of miR-21 sensitizes CML CD34+ stem/progenitor cells to imatinib-induced apoptosis by blocking PI3K/AKT pathway. Leuk Res 39:1117–1124
Ma W, Liu J, Xie J et al (2015) Modulating the growth and imatinib sensitivity of chronic myeloid leukemia stem/progenitor cells with pullulan/microRNA nanoparticles in vitro. J Biomed Nanotechnol 11:1961–1974
Wang LS, Li L, Li L et al (2015) MicroRNA-486 regulates normal erythropoiesis and enhances growth and modulates drug response in CML progenitors. Blood 125:1302–1313
Choi J, Kim YK, Park K et al (2016) MicroRNA-139-5p regulates proliferation of hematopoietic progenitors and is repressed during BCR-ABL-mediated leukemogenesis. Blood 128:2117–2129
Pellicano F, Park L, Hopcroft LEM et al (2018) hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival. Blood 131:1532–1544
Taverna S, Amodeo V, Saieva L et al (2014) Exosomal shuttling of miR-126 in endothelial cells modulates adhesive and migratory abilities of chronic myelogenous leukemia cells. Mol Cancer 13:169
Zhang B, Nguyen LXT, Li L et al (2018) Bone marrow niche trafficking of miR-126 controls the self-renewal of leukemia stem cells in chronic myelogenous leukemia. Nat Med 24:450–462
Lin H, Rothe K, Chen M et al (2020) The miR-185/PAK6 axis predicts therapy response and regulates survival of drug-resistant leukemic stem cells in CML. Blood 136:596–609
Silvestri G, Trotta R, Stramucci L et al (2020) Persistence of drug-resistant leukemic stem cells and impaired NK cell immunity in CML patients depend on MIR300 antiproliferative and PP2A-activating functions. Blood Cancer Discov 1:48–67
Alves R, Gonçalves AC, Jorge J et al (2019) MicroRNA signature refine response prediction in CML. Sci Rep 9:9666
Klümper T, Bruckmueller H, Diewock T et al (2020) Expression differences of miR-142-5p between treatment-naïve chronic myeloid leukemia patients responding and non-responding to imatinib therapy suggest a link to oncogenic ABL2, SRI, cKIT and MCL1 signaling pathways critical for development of therapy resistance. Exp Hematol Oncol 9:26
Yen R, Grasedieck S, Wu A et al (2022) Identification of key microRNAs as predictive biomarkers of Nilotinib response in chronic myeloid leukemia: a sub-analysis of the ENESTxtnd clinical trial. Leukemia 36:2443–2452
Li H, Hui L, Xu W (2012) miR-181a sensitizes a multidrug-resistant leukemia cell line K562/A02 to daunorubicin by targeting BCL-2. Acta Biochim Biophys Sin (Shanghai) 44:269–277
Fei J, Li Y, Zhu X et al (2012) miR-181a post-transcriptionally downregulates oncogenic RalA and contributes to growth inhibition and apoptosis in chronic myelogenous leukemia (CML). PLoS ONE 7:e32834
Wang G, Zhao R, Zhao X et al (2015) MicroRNA-181a enhances the chemotherapeutic sensitivity of chronic myeloid leukemia to imatinib. Oncol Lett 10:2835–2841
Gu C, Feng M, Yin Z et al (2016) RalA, a GTPase targeted by miR-181a, promotes transformation and progression by activating the Ras-related signaling pathway in chronic myelogenous leukemia. Oncotarget 7:20561–20573
Gu C, Liu Y, Yin Z et al (2019) Discovery of the oncogenic parp1, a target of bcr-abl and a potential therapeutic, in mir-181a/PPFIA1 signaling pathway. Mol Ther Nucl Acids 16:1–14
Su R, Li C, Wang X et al (2023) PPFIA1-targeting miR-181a mimic and saRNA overcome imatinib resistance in BCR-ABL1-independent chronic myeloid leukemia by suppressing leukemia stem cell regeneration. Mol Ther Nucl Acids 32:729–742
Van De Craen B, Declerck PJ, Gils A (2012) The biochemistry, physiology and pathological roles of PAI-1 and the requirements for PAI-1 inhibition in vivo. Thromb Res 130:576–585
Vaughan DE, Rai R, Khan SS et al (2017) Plasminogen activator inhibitor-1 is a marker and a mediator of senescence. Arterioscler Thromb Vasc Biol 37:1446–1452
Bajou K, Peng H, Laug WE et al (2008) Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis. Cancer Cell 14:324–334
Placencio VR, DeClerck YA (2015) Plasminogen activator inhibitor-1 in cancer: rationale and insight for future therapeutic testing. Cancer Res 75:2969–2974
Madunić J (2018) The urokinase plasminogen activator system in human cancers: an overview of its prognostic and predictive role. Thromb Haemost 118:2020–2036
Kubala MH, Punj V, Placencio-Hickok VR et al (2018) Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer. Cell Rep 25:2177-2191.e7
Mutoh M, Niho N, Komiya M et al (2008) Plasminogen activator inhibitor-1 (Pai-1) blockers suppress intestinal polyp formation in Min mice. Carcinogenesis 29:824–829
Giacoia EG, Miyake M, Lawton A et al (2014) PAI-1 leads to G1-phase cell-cycle progression through cyclin D3/cdk4/6 upregulation. Mol Cancer Res 12:322–334
Placencio VR, Ichimura A, Miyata T et al (2015) Small molecule inhibitors of plasminogen activator inhibitor-1 elicit anti-tumorigenic and anti-angiogenic activity. PLoS ONE 10:e0133786
Tseng YJ, Lee CH, Chen WY et al (2021) Inhibition of PAI-1 blocks PD-L1 endocytosis and improves the response of melanoma cells to immune checkpoint blockade. J Invest Dermatol 141:2690-2698.e6
Kubala MH, DeClerck YA (2019) The plasminogen activator inhibitor-1 paradox in cancer: a mechanistic understanding. Cancer Metastasis Rev 38:483–492
Yahata T, Ibrahim AA, Muguruma Y et al (2017) TGF-β-induced intracellular PAI-1 is responsible for retaining hematopoietic stem cells in the niche. Blood 130:2283–2294
Yahata T, Ibrahim AA, Hirano KI et al (2021) Targeting of plasminogen activator inhibitor-1 activity promotes elimination of chronic myeloid leukemia stem cells. Haematologica 106:483–494
Sasaki K, Fujiwara T, Ochi T et al (2022) TM5614, an inhibitor of plasminogen activator inhibitor-1, exerts an antitumor effect on chronic myeloid leukemia. Tohoku J Exp Med 257:211–224
Takahashi N, Kameoka Y, Onizuka M et al (2023) Deep molecular response in patients with chronic phase chronic myeloid leukemia treated with the plasminogen activator inhibitor-1 inhibitor TM5614 combined with a tyrosine kinase inhibitor. Cancer Med 12:4250–4258
Holyoake T, Jiang X, Eaves C et al (1999) Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 94:2056–2064
Zhang X, Ma W, Cui J et al (2015) Regulation of p21 by TWIST2 contributes to its tumor-suppressor function in human acute myeloid leukemia. Oncogene 34:3000–3010
Chorzalska A, Kim JF, Roder K et al (2017) Long-term exposure to imatinib mesylate downregulates hippo pathway and activates YAP in a model of chronic myelogenous leukemia. Stem Cells Dev 26:656–677
Zhou H, Ge Y, Sun L et al (2014) Growth arrest specific 2 is up-regulated in chronic myeloid leukemia cells and required for their growth. PLoS ONE 9:e86195
Ji D, Zhang P, Ma W et al (2020) Oncogenic heterogeneous nuclear ribonucleoprotein D-like modulates the growth and imatinib response of human chronic myeloid leukemia CD34+ cells via pre-B-cell leukemia homeobox 1. Oncogene 39:443–453
Bhushan R, Grünhagen J, Becker J et al (2013) miR-181a promotes osteoblastic differentiation through repression of TGF-β signaling molecules. Int J Biochem Cell Biol 45:696–705
Jiang X, Forrest D, Nicolini F et al (2010) Properties of CD34+ CML stem/progenitor cells that correlate with different clinical responses to imatinib mesylate. Blood 116:2112–2121
Acknowledgements
The authors are grateful to Drs. Jian Liu and Haiyan Xu from Peking Union Medical College for providing critical reagents.
Funding
This work was supported by the National Key R&D Program of China (2022YFC2502700), National Natural Science Foundation of China (Nos. 81800151, 32070793 and 82270191), Translational Research Grant of National Clinical Research Center for Hematologic Diseases (NCRCH, Nos. 2020WSB09, 2021ZKMB02, 2020WSC06 and 2020WSB02), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Innovation Capability Development Project of Jiangsu Province (No. BM2015004).
Author information
Authors and Affiliations
Contributions
XZ, WM, XW, YW, and PC performed the experimental studies and analyzed the data, QL and YL performed the bioinformatics analysis, XH and HZ supervised the quality of clinical samples, XZ, XH, YZ, and HZ analyzed the data, prepared the manuscript and supervised the experimental study. All authors read and approved the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Consent for publication
All authors agree for publication.
Ethics approval and consent to participate
Bone marrow cells from human CML patients and healthy donors were obtained in accordance with the Declaration of Helsinki, and with written informed consent approved by the Ethical Committee of Soochow University (SUDA20211224H03, Suzhou, China).
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Zhang, X., Ma, W., Xue, W. et al. miR-181a plays the tumor-suppressor role in chronic myeloid leukemia CD34 + cells partially via SERPINE1. Cell. Mol. Life Sci. 81, 10 (2024). https://doi.org/10.1007/s00018-023-05036-8
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00018-023-05036-8