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AKT pathway
Biochemical and Biophysical Research Communications 519 (2019) 234e239
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TAL1 mediates imatinib-induced CML cell apoptosis via the PTEN/ PI3K/AKT pathway Yifan Wu a, 1, Yanyun Hu a, 1, Xibao Yu a, b, 1, Yikai Zhang a, Xin Huang c, Shaohua Chen a, Yangqiu Li a, **, Chengwu Zeng a, * a b c
Key Laboratory for Regenerative Medicine of Ministry of Education, Institute of Hematology, Jinan University, Guangzhou, 510632, China Department of Experimental Research, Sun Yat-sen University Cancer Center, State Key Laboratory Oncology in South China, Guangzhou, 510060, China Department of Hematology, Guangdong General Hospital (Guangdong Academy of Medical Sciences), Guangzhou, 510080, PR China
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Article history: Received 20 August 2019 Accepted 31 August 2019 Available online 4 September 2019
Chronic myeloid leukemia (CML) is associated with chromosomal translocation t(9; 22), which results in formation of the BCR-ABL oncogene. CML is treated with tyrosine kinase inhibitors (TKIs), which target BCR-ABL, to eradicate BCR-ABL þ cells. However, the TKI imatinib (IM) fails to eliminate quiescent leukemia stem cells (LSCs) in CML. In this study, we demonstrate that transcription factor TAL1 is downregulated in CML LSCs by BCR-ABL, and IM triggers TAL1 mRNA expression. In addition, loss of TAL1 abrogates IM-induced CML cell apoptosis. RNA-seq analysis suggests that TAL1 expression may affect PI3K/AKT pathway. Moreover, depletion of TAL1 inhibits the expression of PTEN, which is a negative regulator of the PI3K/AKT pathway. Our results reveal an unexpected involvement of TAL1 in CML etiology and demonstrate that TAL1 may regulate PTEN expression and lead to inhibition of the PI3K/AKT pathway in the response of CML cells to TKI. These results implicate regulation of PTEN expression as a novel mechanism for the transcriptional regulatory networks of TAL1 in CML. © 2019 Elsevier Inc. All rights reserved.
Keywords: Chronic myeloid leukemia TAL1 Leukemia stem cell Imatinib PI3K/AKT
1. Introduction Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph), which results in formation of the BCRABL oncogene. BCR-ABL induces uncontrolled cell proliferation and suppresses apoptosis, which is the pathogenesis of CML [1,2]. CML is treated with imatinib (IM), a tyrosine kinase inhibitor (TKI) that competively binds to the ATP-binding site of BCR-ABL, inhibiting tyrosine kinase activity and blocking signaling pathways mediated by tyrosine phosphorylation [3]. However, quiescent CML leukemia stem cells (LSCs) are resistant to apoptosis following TKI treatment despite effective inhibition of BCR-ABL kinase activity [4e6]. Therefore, seeking alternative targets and additional therapeutic strategies for CML is of interest. Recent studies have shown that PTEN suppresses the survival
* Corresponding author. Institute of Hematology, School of Medicine, Jinan University, 601 Huangpu Avenue West, 510632, Guangzhou, PR China. ** Corresponding author. Institute of Hematology, School of Medicine, Jinan University, 601 Huangpu Avenue West, 510632, Guangzhou, PR China. E-mail addresses: [email protected] (Y. Li), [email protected] (C. Zeng). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.08.164 0006-291X/© 2019 Elsevier Inc. All rights reserved.
and self-renewal of CML LSCs [7e9]. PTEN has been shown to be down-regulated by BCR-ABL in CML [7,10,11]. However, the mechanisms involved in down-regulating PTEN by BCR-ABL remain unclear. TAL1 is essential for generating embryonic hematopoietic stem cells (HSCs) [12,13]. TAL1 also appears to control hematopoietic specification in embryogenesis [14]. TAL1 is normally expressed in hematopoietic stem cells (HSCs), progenitor cells, and erythromegakaryocytic lineages, but it is silenced during T-cell development [15]. Aberrantly expressed TAL1 exhibits oncogenic properties in lymphoid tissue. TAL1 is considered a cause of T-cell acute lymphoblastic leukemia (T-ALL), and wild-type TAL1 is aberrantly expressed in over 60% of T-ALL patients where it promotes the proliferation of T-ALL cells via perturbation of the transcriptional regulatory network [16e18]. However, whether TAL1 is involved in CML generation is unknown, and the mechanism by which TAL1 functions in CML tumorigenesis is also unknown. To understand the role of TAL1 in response to IM and explain its potential role in CML LSC permanence after treatment, we investigated the expression of TAL1 in CML LSCs and explored the role of TAL1 in mediating the effects of PTEN on CML cells.
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2. Materials and methods 2.1. Patients and samples Bone marrow (BM) samples were obtained from two patients with CML in chronic phase (CML-CP) were used in this study. This study was approved by the Ethic committee of First Affiliated Hospital, Medical School of Jinan university. Studies were conducted in accordance with the Declaration of Helsinki. 2.2. Lin-/CD34þ/CD38-/CD26 þ LSC isolation by FACS Mononuclear cells (MNCs) were isolated from BM samples using Ficoll. Primary human Lin-/CD34þ/CD38-/CD26 þ and Lin-/CD34þ/ CD38-/CD26-cells were obtained by multicolor flow cytometry as previously described [19]. Antibodies for lineage cocktail FITC and CD26 (2A6)-PE were from ThermoFisher. Antibodies for CD34-APC and CD38 PE-Cy7 were from BD Biosciences. 2.3. Cell lines and cultures KBM5 cells were provided by Jingxuan Pan (State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China) and cultured in Iscove's modified Dulbecco's medium supplemented with 10% FBS. K562, KU812, 32D-Bcr-Abl and 32D-T315I (provided by Prof. Lin Qiu, Harbin Institute of Hematolgy & Oncology, Harbin, China) were cultured in RPMI 1640 containing 10% fetal boving serum at 37 C in a 5% CO2 incubator. IM was purchased from Sigma-Aldrich and used at a final concentration of 1 mM. 2.4. RNA extraction and Quantitative real-time RT-PCR Total RNA was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA from Lin-/ CD34þ/CD38-/CD26 þ and Lin-/CD34þ/CD38-/CD26-cells was purified using the RNeasy Mini Kit (Qiagen). RNA was reverse transcribed into cDNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems) [5]. Quantitative real-time RT-PCR (qRT-PCR) was performed with SYBR Green (TIANGEN, China), the list of primer sequences are shown in Supplementary Table S1. 2.5. RNA interference siRNA and negative control siRNA were purchased from RiboBio (Guangzhou, China). The siRNA oligonucleotides were transfected into cells using the Neon® Transfection System (Invitrogen) following the manufacturer's protocol [20e22]. The siRNA sequences used to target TAL1 were as follows: siTAL1-1, GAAGCTCAGCAAGAATGAG and siTAL1-2, CCAAAGTTGTGCGGCGTAT; The siRNA sequences used to target PTEN were as follows: siPTEN-1, CACCACAGCTAGAACTTAT and siPTEN-2, CCAGTCAGAGGCGCTA TGT. The following siRNA sequences were used for AKT1 (ACTCCATGACAAAGCAGAG), AKT2 (TGCCCTTCTACAACCAGGA). 2.6. Flow cytometry analysis Cells were incubated and stained by the PI Annexin-V Apoptosis Detection Kit (BD Biosciences). Analysis was performed by flow cytometry using the manufacturer's protocol. 2.7. Western blot analysis Total cellular protein was extracted by incubating cells in RIPA
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lysis buffer (Cell Signaling Technology). Protein extracts were separated in a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane and probed with primary antibodies. Antibodies for TAL1, PTEN, and GAPDH were obtained from Cell Signaling Technology. 2.8. Statistical analysis When comparing two groups, the Student's t-test was used. All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc, San Diego, CA), and the results were considered significant when the p-value was <0.05. 3. Results 3.1. TAL1 is decreased in CML CD34þ/CD38-/CD26 þ cells CML is a stem cell (SC) neoplasm characterized by the BCR-ABL oncogene. In patients with CML-CP, LSCs reside in a CD34þ/CD38compartment of malignant clones. Recent studies have reported CD34þ/CD38-/CD26 þ CML LSCs with significantly high purity and leukemia-initiating potential [19,23]. Consistent with these results, we found BCR-ABL only in Lin-/CD34þ/CD38-/CD26 þ cells but not in CD34þ/CD38-/CD26-cells (Fig. 1A, upper panel). Based on these observations, we compared TAL1 expression in Lin-/CD34þ/CD38-/ CD26 þ CML LSCs and Lin-/CD34þ/CD38-/CD26- SCs from CML patients. As assessed by qRT-PCR, the TAL1 mRNA level was significantly lower in Lin-/CD34þ/CD38-/CD26 þ CML LSCs (Fig. 1A, lower panel). In addition, TAL1 was increased in K562 and KBM5 cells treated with IM (Fig. 1B and C). This effect was also observed in murine 32D myeloid cells stably expressing wild-type BCR-ABL (32D-BCR-ABL) (Fig. 1D). Next, we knocked down BCR-ABL expression by siRNA in K562 cells, and the loss of BCR-ABL increased TAL1 expression (Fig. 1E). These findings demonstrate that TAL1 may be important in CML pathogenesis. 3.2. Knockdown of TAL1 impairs IM-mediated apoptosis in CML cells We next explored the functional role of TAL1 in IM-induced apoptosis in CML cells. TAL1 was shown to be effectively knocked down by siRNA (Fig. 2A). Transfected cells were treated with IM, and after 48 h, apoptotic cells were detected by flow cytometry. The results demonstrated that TAL1 loss combined with IM treatment inhibits the apoptosis of K562 cells (Fig. 2B). 3.3. Gene expression analysis upon TAL1 knockdown To determine whether TAL1 is functionally involved in the regulation of genes that mediate IM-induced apoptosis, we analyzed differential gene expression using RNA-seq data from TAL1-knockdown cells treated with or without IM. Notably, pathway analysis using DAVID Bioinformatics revealed that the PI3K/AKT pathway is regulated after TAL1 knockdown (Fig. 3A and B). TAL1 knockdown significantly altered the expression of PI3K/ AKT pathway genes in cells with or without IM treatment (Fig. 3C, D and Supplementary Table S2). Specifically, we found that ablation of TAL1 represses the expression of PTEN and the long non-coding RNA (lncRNA) BGLT3 (a positive regulator of PTEN [2,24]) in K562 (Fig. 3E and F) and KU812 cells (Fig. 3G). As expected, there was significantly increased expression of PTEN and BGLT3 in TAL1overexpressing K562 cells (Fig. 3H). These results suggest that upregulation of PTEN and BGLT3 by TAL1 inhibits PI3K/AKT pathway.
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Fig. 1. TAL1 is significantly downregulated in CD34þ/CD38-/CD26 þ LSCs. (A) CD34þ/CD38-/CD26 þ LSCs from two patients with CML (patients 1, 2) were examined for the expression of TAL1 by qRT-PCR. The upper panel shows the expression of BCR-ABL in purified CD34þ/CD38-/CD26 þ CML LSCs (left) and CD34þ/CD38-/CD26- SCs (right) from CML patients. Lower panel: The TAL1 mRNA level was lower in BCR-ABL þ CML CD34þ/CD38-/CD26 þ cells than in CD34þ/CD38-/CD26-cells. The qRT-PCR analysis of TAL1 in K562 (B) and KBM5 (C) cells treated with 1 mM IM for the indicated times. (D) 32D myeloid cells stably expressing either 210 kDa wild-type BCR-ABL (32D-BCR-ABL) or T315I BCR-ABL (32DT315I) were treated with IM for 24 h. TAL1 was measured by qRT-PCR. (E) The TAL1 expression level was detected in K562 cells transfected with siRNA specifically targeting BCR-ABL (si-BCR-ABL) or negative control siRNA (si-NC). Analysis of qRT-PCR data showed that the TAL1 expression level in cells transfected with si-BCR-ABL was significantly higher than that in cells transfected with si-NC.
Fig. 2. Knockdown of TAL1 inhibits IM-induced apoptosis. (A) 48 h after transfection, knockdown efficiency was confirmed by qRT-PCR and Western blot. (B) K562 cells were transfected with control siRNA or TAL1 siRNA followed by a 48 h IM treatment. Apoptosis was assessed by flow cytometry. Data are mean ± SEM of 3 independent experiments.
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Fig. 3. LncRNA-BGLT3 and PTEN are TAL1 target genes. (A) Pathway analysis of the genes regulated by siTAL1. (B) Pathway analysis of the genes regulated by siTAL1 upon IM treatment. (C, D) TAL1 mediated a large number of PI3K/AKT pathway genes (see Supplementary Table S2). (E) qPCR analysis of the expression of PTEN and the lncRNA-BGLT3 in K562 cells following transfection with control siRNA or TAL1 siRNA, demonstrating marked dependence on TAL1. (F) Western blot analysis of PTEN in TAL1 knockdown K562 cells. (G) The expression levels of PTEN and the lncRNA-BGLT3 were detected in KU812 cells transfected with siTAL1 or siNC. (H) Changes in PTEN and BGLT3 expression in K562 cells upon TAL1 overexpression (TAL1-OE).
3.4. TAL1 depletion inhibits IM-induced apoptosis via the PTEN/ PI3K/AKT pathway To determine whether reduced TAL1 results in decreased PTEN, TAL1 knockdown cells were treated with or without IM and analyzed for PTEN expression by Western blot. As expected, PTEN levels were low in TAL1 knockdown cells with treated with IM (Fig. 4A). Given the importance of the PI3K/AKT pathway in the regulation of apoptosis and our observation that TAL1 knockdown attenuates IM-induced apoptosis and regulates PTEN expression, we hypothesized that TAL1 modulates IM-induced apoptosis via the PI3K/AKT pathway. To confirm this hypothesis, we silenced PTEN in K562 cells, and after transfection for 24 h, the cells were treated with IM for 48 h. We found that siRNA-mediated depletion of PTEN prevented the apoptotic effects induced by IM in K562 cells (Fig. 4B). Furthermore, loss of AKT significantly increased the sensitivity of the K562 cells to IM-induced apoptosis (Fig. 4C). Similar to the results from AKT knocked down cells, number of the apoptotic cells were obviously increased upon combined treatment with IM and AKT inhibitors (Fig. 4D). In addition, combined IM/ LY294002 treatment was more efficient than IM alone (Fig. 4E). Importantly, TAL1 knockdown significantly inhibited the reduction
of AKT phosphorylation by IM (Fig. 4F). Thus, TAL1-mediated PTEN is essential for IM-induced apoptosis. 4. Discussion The transcription factor TAL1 is a critical regulator of hematopoietic gene expression and can act as an oncogene in T-ALL if it is aberrantly expressed [17]. However, the role of TAL1 in other malignant hematopoietic diseases is unknown. In this study, we found that TAL1 is significantly downregulated in CML LSCs. We showed that TAL1 is an essential mediator for regulating IM-induced apoptosis in CML. Moreover, we demonstrated that TAL1 inhibits the PI3K/AKT pathway, which is mediated by PTEN, to promote CML apoptosis. Using RNA interference to knock down TAL1 expression in human CML cell lines, we demonstrated that TAL1 inhibits IMinduced apoptosis. To study the TAL1 network that regulates the apoptosis in CML cells, we performed gene expression profiling of K562 cell lines expressing TAL1. Functional annotation of these genes led to the identification of biological categories that are consistent with TAL1 function, providing confidence that TAL1 regulates apoptosis via the PI3K/AKT pathway. Indeed, TAL1 loss
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Fig. 4. Blockade of PI3K/AKT sensitizes CML cells to IM-induced apoptosis. (A) TAL1 knockdown in K562 cells repressed the expression of PTEN in response to IM stimuli. (B) K562 cells were transfected with control siRNA or PTEN siRNA followed by 48 h IM treatment. (C) Each of the K562 cells transfected with si-AKT1, si-AKT2 or si-NC were divided to two groups, one treated with IM and the other one untreated. Apoptosis was assessed by flow cytometry. (D) AKT inhibitors enhanced IM-induced apoptosis in K562 cells. K562 cells were treated with IM, Perifosine (1 mM), Triciribine (1 mM), the combination of IM and Perifosine, or the combination of IM and Triciribine for 48 h. (E) Apoptosis was significantly increased in K562 cells after co-treatment with IM and LY294002. K562 cells were treated with 1 mM IM alone or in combination with LY294002 (10 mM) for 48 h. (F) Western blot analysis of TAL1 knockdown in the presence or absence of IM. (G) Proposed model depicting the regulation and role of TAL1 in CML cells.
repressed lncRNA-BGLT3/PTEN expression and led to high expression of pAKT. Similarly, PI3K/AKT pathway inhibitors promoted IMinduced apoptosis. This observation strengthens the notion that TAL1 is an essential mediator of the PI3K/AKT pathway for regulating IM-induced apoptosis in CML. Our study demonstrates that the TAL1 mRNA level is decreased in CML LSCs, indicating that the CML oncogene BCR-ABL may regulates TAL1 at the transcriptional level. Specifically, we observed that TAL1 knockdown could strongly decrease PTEN, which is an endogenous inhibitor of the PI3K/AKT signaling pathway [25e27]. In addition, we also observed that ablation of TAL1 represses the expression of lncRNA-BGLT3 which acts as a ceRNA to regulate PTEN levels. Interestingly, PTEN is also down-regulated in CML LSCs, suggesting that the PTEN down-regulation regulated by BCRABL may be mediated by TAL1 in CML. A number of studies have suggested a critical role for low PTEN in the survival of CML LSCs [7,25,28]. It is remarkable that immature quiescent human HSCs exhibit weak TAL1 expression [29], and loss of TAL1 expression retains hematopoietic stem/progenitor cells (HSPCs) in a quiescent state [30]. Thus, it is possible that TAL1-mediated PTEN downregulation contributes to CML LSC survival and resistance to TKI treatment (Fig. 4G). Although PTEN has been identified as a direct target of TAL1 in TALL cells [31,32], the relative contributions of PTEN and TAL1 to CML leukemogenesis remain less clear. We observed that TAL1 acts as an activator of PTEN expression, which is distinct from its role reported in T-ALL cells [31,33]. Several studies have suggested that TAL1 could act either as a transcriptional activator or as a repressor in a context-dependent fashion. Specifically, TAL1 can be phosphorylated. This phosphorylation regulates the transcriptional activities of TAL1, suggesting that phosphorylation may dictate the
ability of TAL1 to be associated with transcriptional corepressors or coactivators at the PTEN promoter. Our study together with the study of TAL1 in T-ALL highlights the complexity and functional importance of TAL1 regulation and signalling pathways in hematopoietic malignancies and may provide novel insights into the genetic/epigenetic differences between T-ALL and CML. In summary, to our best knowledge, we for the first time report that TAL1 is significantly downregulated in CML LSCs and demonstrate that TAL1 regulates PTEN expression and leads to inhibition of the PI3K pathway in IM-induced CML apoptosis. This study reveals an unexpected contribution of TAL1 in CML and implicates regulation of PTEN expression as a novel mechanism for the transcriptional regulatory networks of TAL1. Funding This work was supported by the National Natural Science Foundation of China (Nos. 81400102, 81770158 and U1301226); the Guangdong Science & Technology Project (No. 2015A050502029); the Pearl River S&T Nova Program of Guangzhou, China (No. 201906010002) and the Fundamental Research Funds for the Central Universities (No. 17817015). Conflicts of interest The authors declare no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.164.
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TAL1 mediates imatinib-induced CML cell apoptosis via the PTEN/ PI3K/AKT pathway Yifan Wu a, 1, Yanyun Hu a, 1, Xibao Yu a, b, 1, Yikai Zhang a, Xin Huang c, Shaohua Chen a, Yangqiu Li a, **, Chengwu Zeng a, * a b c
Key Laboratory for Regenerative Medicine of Ministry of Education, Institute of Hematology, Jinan University, Guangzhou, 510632, China Department of Experimental Research, Sun Yat-sen University Cancer Center, State Key Laboratory Oncology in South China, Guangzhou, 510060, China Department of Hematology, Guangdong General Hospital (Guangdong Academy of Medical Sciences), Guangzhou, 510080, PR China
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a b s t r a c t
Article history: Received 20 August 2019 Accepted 31 August 2019 Available online 4 September 2019
Chronic myeloid leukemia (CML) is associated with chromosomal translocation t(9; 22), which results in formation of the BCR-ABL oncogene. CML is treated with tyrosine kinase inhibitors (TKIs), which target BCR-ABL, to eradicate BCR-ABL þ cells. However, the TKI imatinib (IM) fails to eliminate quiescent leukemia stem cells (LSCs) in CML. In this study, we demonstrate that transcription factor TAL1 is downregulated in CML LSCs by BCR-ABL, and IM triggers TAL1 mRNA expression. In addition, loss of TAL1 abrogates IM-induced CML cell apoptosis. RNA-seq analysis suggests that TAL1 expression may affect PI3K/AKT pathway. Moreover, depletion of TAL1 inhibits the expression of PTEN, which is a negative regulator of the PI3K/AKT pathway. Our results reveal an unexpected involvement of TAL1 in CML etiology and demonstrate that TAL1 may regulate PTEN expression and lead to inhibition of the PI3K/AKT pathway in the response of CML cells to TKI. These results implicate regulation of PTEN expression as a novel mechanism for the transcriptional regulatory networks of TAL1 in CML. © 2019 Elsevier Inc. All rights reserved.
Keywords: Chronic myeloid leukemia TAL1 Leukemia stem cell Imatinib PI3K/AKT
1. Introduction Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph), which results in formation of the BCRABL oncogene. BCR-ABL induces uncontrolled cell proliferation and suppresses apoptosis, which is the pathogenesis of CML [1,2]. CML is treated with imatinib (IM), a tyrosine kinase inhibitor (TKI) that competively binds to the ATP-binding site of BCR-ABL, inhibiting tyrosine kinase activity and blocking signaling pathways mediated by tyrosine phosphorylation [3]. However, quiescent CML leukemia stem cells (LSCs) are resistant to apoptosis following TKI treatment despite effective inhibition of BCR-ABL kinase activity [4e6]. Therefore, seeking alternative targets and additional therapeutic strategies for CML is of interest. Recent studies have shown that PTEN suppresses the survival
* Corresponding author. Institute of Hematology, School of Medicine, Jinan University, 601 Huangpu Avenue West, 510632, Guangzhou, PR China. ** Corresponding author. Institute of Hematology, School of Medicine, Jinan University, 601 Huangpu Avenue West, 510632, Guangzhou, PR China. E-mail addresses: [email protected] (Y. Li), [email protected] (C. Zeng). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrc.2019.08.164 0006-291X/© 2019 Elsevier Inc. All rights reserved.
and self-renewal of CML LSCs [7e9]. PTEN has been shown to be down-regulated by BCR-ABL in CML [7,10,11]. However, the mechanisms involved in down-regulating PTEN by BCR-ABL remain unclear. TAL1 is essential for generating embryonic hematopoietic stem cells (HSCs) [12,13]. TAL1 also appears to control hematopoietic specification in embryogenesis [14]. TAL1 is normally expressed in hematopoietic stem cells (HSCs), progenitor cells, and erythromegakaryocytic lineages, but it is silenced during T-cell development [15]. Aberrantly expressed TAL1 exhibits oncogenic properties in lymphoid tissue. TAL1 is considered a cause of T-cell acute lymphoblastic leukemia (T-ALL), and wild-type TAL1 is aberrantly expressed in over 60% of T-ALL patients where it promotes the proliferation of T-ALL cells via perturbation of the transcriptional regulatory network [16e18]. However, whether TAL1 is involved in CML generation is unknown, and the mechanism by which TAL1 functions in CML tumorigenesis is also unknown. To understand the role of TAL1 in response to IM and explain its potential role in CML LSC permanence after treatment, we investigated the expression of TAL1 in CML LSCs and explored the role of TAL1 in mediating the effects of PTEN on CML cells.
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2. Materials and methods 2.1. Patients and samples Bone marrow (BM) samples were obtained from two patients with CML in chronic phase (CML-CP) were used in this study. This study was approved by the Ethic committee of First Affiliated Hospital, Medical School of Jinan university. Studies were conducted in accordance with the Declaration of Helsinki. 2.2. Lin-/CD34þ/CD38-/CD26 þ LSC isolation by FACS Mononuclear cells (MNCs) were isolated from BM samples using Ficoll. Primary human Lin-/CD34þ/CD38-/CD26 þ and Lin-/CD34þ/ CD38-/CD26-cells were obtained by multicolor flow cytometry as previously described [19]. Antibodies for lineage cocktail FITC and CD26 (2A6)-PE were from ThermoFisher. Antibodies for CD34-APC and CD38 PE-Cy7 were from BD Biosciences. 2.3. Cell lines and cultures KBM5 cells were provided by Jingxuan Pan (State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China) and cultured in Iscove's modified Dulbecco's medium supplemented with 10% FBS. K562, KU812, 32D-Bcr-Abl and 32D-T315I (provided by Prof. Lin Qiu, Harbin Institute of Hematolgy & Oncology, Harbin, China) were cultured in RPMI 1640 containing 10% fetal boving serum at 37 C in a 5% CO2 incubator. IM was purchased from Sigma-Aldrich and used at a final concentration of 1 mM. 2.4. RNA extraction and Quantitative real-time RT-PCR Total RNA was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Total RNA from Lin-/ CD34þ/CD38-/CD26 þ and Lin-/CD34þ/CD38-/CD26-cells was purified using the RNeasy Mini Kit (Qiagen). RNA was reverse transcribed into cDNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems) [5]. Quantitative real-time RT-PCR (qRT-PCR) was performed with SYBR Green (TIANGEN, China), the list of primer sequences are shown in Supplementary Table S1. 2.5. RNA interference siRNA and negative control siRNA were purchased from RiboBio (Guangzhou, China). The siRNA oligonucleotides were transfected into cells using the Neon® Transfection System (Invitrogen) following the manufacturer's protocol [20e22]. The siRNA sequences used to target TAL1 were as follows: siTAL1-1, GAAGCTCAGCAAGAATGAG and siTAL1-2, CCAAAGTTGTGCGGCGTAT; The siRNA sequences used to target PTEN were as follows: siPTEN-1, CACCACAGCTAGAACTTAT and siPTEN-2, CCAGTCAGAGGCGCTA TGT. The following siRNA sequences were used for AKT1 (ACTCCATGACAAAGCAGAG), AKT2 (TGCCCTTCTACAACCAGGA). 2.6. Flow cytometry analysis Cells were incubated and stained by the PI Annexin-V Apoptosis Detection Kit (BD Biosciences). Analysis was performed by flow cytometry using the manufacturer's protocol. 2.7. Western blot analysis Total cellular protein was extracted by incubating cells in RIPA
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lysis buffer (Cell Signaling Technology). Protein extracts were separated in a sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane and probed with primary antibodies. Antibodies for TAL1, PTEN, and GAPDH were obtained from Cell Signaling Technology. 2.8. Statistical analysis When comparing two groups, the Student's t-test was used. All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc, San Diego, CA), and the results were considered significant when the p-value was <0.05. 3. Results 3.1. TAL1 is decreased in CML CD34þ/CD38-/CD26 þ cells CML is a stem cell (SC) neoplasm characterized by the BCR-ABL oncogene. In patients with CML-CP, LSCs reside in a CD34þ/CD38compartment of malignant clones. Recent studies have reported CD34þ/CD38-/CD26 þ CML LSCs with significantly high purity and leukemia-initiating potential [19,23]. Consistent with these results, we found BCR-ABL only in Lin-/CD34þ/CD38-/CD26 þ cells but not in CD34þ/CD38-/CD26-cells (Fig. 1A, upper panel). Based on these observations, we compared TAL1 expression in Lin-/CD34þ/CD38-/ CD26 þ CML LSCs and Lin-/CD34þ/CD38-/CD26- SCs from CML patients. As assessed by qRT-PCR, the TAL1 mRNA level was significantly lower in Lin-/CD34þ/CD38-/CD26 þ CML LSCs (Fig. 1A, lower panel). In addition, TAL1 was increased in K562 and KBM5 cells treated with IM (Fig. 1B and C). This effect was also observed in murine 32D myeloid cells stably expressing wild-type BCR-ABL (32D-BCR-ABL) (Fig. 1D). Next, we knocked down BCR-ABL expression by siRNA in K562 cells, and the loss of BCR-ABL increased TAL1 expression (Fig. 1E). These findings demonstrate that TAL1 may be important in CML pathogenesis. 3.2. Knockdown of TAL1 impairs IM-mediated apoptosis in CML cells We next explored the functional role of TAL1 in IM-induced apoptosis in CML cells. TAL1 was shown to be effectively knocked down by siRNA (Fig. 2A). Transfected cells were treated with IM, and after 48 h, apoptotic cells were detected by flow cytometry. The results demonstrated that TAL1 loss combined with IM treatment inhibits the apoptosis of K562 cells (Fig. 2B). 3.3. Gene expression analysis upon TAL1 knockdown To determine whether TAL1 is functionally involved in the regulation of genes that mediate IM-induced apoptosis, we analyzed differential gene expression using RNA-seq data from TAL1-knockdown cells treated with or without IM. Notably, pathway analysis using DAVID Bioinformatics revealed that the PI3K/AKT pathway is regulated after TAL1 knockdown (Fig. 3A and B). TAL1 knockdown significantly altered the expression of PI3K/ AKT pathway genes in cells with or without IM treatment (Fig. 3C, D and Supplementary Table S2). Specifically, we found that ablation of TAL1 represses the expression of PTEN and the long non-coding RNA (lncRNA) BGLT3 (a positive regulator of PTEN [2,24]) in K562 (Fig. 3E and F) and KU812 cells (Fig. 3G). As expected, there was significantly increased expression of PTEN and BGLT3 in TAL1overexpressing K562 cells (Fig. 3H). These results suggest that upregulation of PTEN and BGLT3 by TAL1 inhibits PI3K/AKT pathway.
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Fig. 1. TAL1 is significantly downregulated in CD34þ/CD38-/CD26 þ LSCs. (A) CD34þ/CD38-/CD26 þ LSCs from two patients with CML (patients 1, 2) were examined for the expression of TAL1 by qRT-PCR. The upper panel shows the expression of BCR-ABL in purified CD34þ/CD38-/CD26 þ CML LSCs (left) and CD34þ/CD38-/CD26- SCs (right) from CML patients. Lower panel: The TAL1 mRNA level was lower in BCR-ABL þ CML CD34þ/CD38-/CD26 þ cells than in CD34þ/CD38-/CD26-cells. The qRT-PCR analysis of TAL1 in K562 (B) and KBM5 (C) cells treated with 1 mM IM for the indicated times. (D) 32D myeloid cells stably expressing either 210 kDa wild-type BCR-ABL (32D-BCR-ABL) or T315I BCR-ABL (32DT315I) were treated with IM for 24 h. TAL1 was measured by qRT-PCR. (E) The TAL1 expression level was detected in K562 cells transfected with siRNA specifically targeting BCR-ABL (si-BCR-ABL) or negative control siRNA (si-NC). Analysis of qRT-PCR data showed that the TAL1 expression level in cells transfected with si-BCR-ABL was significantly higher than that in cells transfected with si-NC.
Fig. 2. Knockdown of TAL1 inhibits IM-induced apoptosis. (A) 48 h after transfection, knockdown efficiency was confirmed by qRT-PCR and Western blot. (B) K562 cells were transfected with control siRNA or TAL1 siRNA followed by a 48 h IM treatment. Apoptosis was assessed by flow cytometry. Data are mean ± SEM of 3 independent experiments.
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Fig. 3. LncRNA-BGLT3 and PTEN are TAL1 target genes. (A) Pathway analysis of the genes regulated by siTAL1. (B) Pathway analysis of the genes regulated by siTAL1 upon IM treatment. (C, D) TAL1 mediated a large number of PI3K/AKT pathway genes (see Supplementary Table S2). (E) qPCR analysis of the expression of PTEN and the lncRNA-BGLT3 in K562 cells following transfection with control siRNA or TAL1 siRNA, demonstrating marked dependence on TAL1. (F) Western blot analysis of PTEN in TAL1 knockdown K562 cells. (G) The expression levels of PTEN and the lncRNA-BGLT3 were detected in KU812 cells transfected with siTAL1 or siNC. (H) Changes in PTEN and BGLT3 expression in K562 cells upon TAL1 overexpression (TAL1-OE).
3.4. TAL1 depletion inhibits IM-induced apoptosis via the PTEN/ PI3K/AKT pathway To determine whether reduced TAL1 results in decreased PTEN, TAL1 knockdown cells were treated with or without IM and analyzed for PTEN expression by Western blot. As expected, PTEN levels were low in TAL1 knockdown cells with treated with IM (Fig. 4A). Given the importance of the PI3K/AKT pathway in the regulation of apoptosis and our observation that TAL1 knockdown attenuates IM-induced apoptosis and regulates PTEN expression, we hypothesized that TAL1 modulates IM-induced apoptosis via the PI3K/AKT pathway. To confirm this hypothesis, we silenced PTEN in K562 cells, and after transfection for 24 h, the cells were treated with IM for 48 h. We found that siRNA-mediated depletion of PTEN prevented the apoptotic effects induced by IM in K562 cells (Fig. 4B). Furthermore, loss of AKT significantly increased the sensitivity of the K562 cells to IM-induced apoptosis (Fig. 4C). Similar to the results from AKT knocked down cells, number of the apoptotic cells were obviously increased upon combined treatment with IM and AKT inhibitors (Fig. 4D). In addition, combined IM/ LY294002 treatment was more efficient than IM alone (Fig. 4E). Importantly, TAL1 knockdown significantly inhibited the reduction
of AKT phosphorylation by IM (Fig. 4F). Thus, TAL1-mediated PTEN is essential for IM-induced apoptosis. 4. Discussion The transcription factor TAL1 is a critical regulator of hematopoietic gene expression and can act as an oncogene in T-ALL if it is aberrantly expressed [17]. However, the role of TAL1 in other malignant hematopoietic diseases is unknown. In this study, we found that TAL1 is significantly downregulated in CML LSCs. We showed that TAL1 is an essential mediator for regulating IM-induced apoptosis in CML. Moreover, we demonstrated that TAL1 inhibits the PI3K/AKT pathway, which is mediated by PTEN, to promote CML apoptosis. Using RNA interference to knock down TAL1 expression in human CML cell lines, we demonstrated that TAL1 inhibits IMinduced apoptosis. To study the TAL1 network that regulates the apoptosis in CML cells, we performed gene expression profiling of K562 cell lines expressing TAL1. Functional annotation of these genes led to the identification of biological categories that are consistent with TAL1 function, providing confidence that TAL1 regulates apoptosis via the PI3K/AKT pathway. Indeed, TAL1 loss
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Fig. 4. Blockade of PI3K/AKT sensitizes CML cells to IM-induced apoptosis. (A) TAL1 knockdown in K562 cells repressed the expression of PTEN in response to IM stimuli. (B) K562 cells were transfected with control siRNA or PTEN siRNA followed by 48 h IM treatment. (C) Each of the K562 cells transfected with si-AKT1, si-AKT2 or si-NC were divided to two groups, one treated with IM and the other one untreated. Apoptosis was assessed by flow cytometry. (D) AKT inhibitors enhanced IM-induced apoptosis in K562 cells. K562 cells were treated with IM, Perifosine (1 mM), Triciribine (1 mM), the combination of IM and Perifosine, or the combination of IM and Triciribine for 48 h. (E) Apoptosis was significantly increased in K562 cells after co-treatment with IM and LY294002. K562 cells were treated with 1 mM IM alone or in combination with LY294002 (10 mM) for 48 h. (F) Western blot analysis of TAL1 knockdown in the presence or absence of IM. (G) Proposed model depicting the regulation and role of TAL1 in CML cells.
repressed lncRNA-BGLT3/PTEN expression and led to high expression of pAKT. Similarly, PI3K/AKT pathway inhibitors promoted IMinduced apoptosis. This observation strengthens the notion that TAL1 is an essential mediator of the PI3K/AKT pathway for regulating IM-induced apoptosis in CML. Our study demonstrates that the TAL1 mRNA level is decreased in CML LSCs, indicating that the CML oncogene BCR-ABL may regulates TAL1 at the transcriptional level. Specifically, we observed that TAL1 knockdown could strongly decrease PTEN, which is an endogenous inhibitor of the PI3K/AKT signaling pathway [25e27]. In addition, we also observed that ablation of TAL1 represses the expression of lncRNA-BGLT3 which acts as a ceRNA to regulate PTEN levels. Interestingly, PTEN is also down-regulated in CML LSCs, suggesting that the PTEN down-regulation regulated by BCRABL may be mediated by TAL1 in CML. A number of studies have suggested a critical role for low PTEN in the survival of CML LSCs [7,25,28]. It is remarkable that immature quiescent human HSCs exhibit weak TAL1 expression [29], and loss of TAL1 expression retains hematopoietic stem/progenitor cells (HSPCs) in a quiescent state [30]. Thus, it is possible that TAL1-mediated PTEN downregulation contributes to CML LSC survival and resistance to TKI treatment (Fig. 4G). Although PTEN has been identified as a direct target of TAL1 in TALL cells [31,32], the relative contributions of PTEN and TAL1 to CML leukemogenesis remain less clear. We observed that TAL1 acts as an activator of PTEN expression, which is distinct from its role reported in T-ALL cells [31,33]. Several studies have suggested that TAL1 could act either as a transcriptional activator or as a repressor in a context-dependent fashion. Specifically, TAL1 can be phosphorylated. This phosphorylation regulates the transcriptional activities of TAL1, suggesting that phosphorylation may dictate the
ability of TAL1 to be associated with transcriptional corepressors or coactivators at the PTEN promoter. Our study together with the study of TAL1 in T-ALL highlights the complexity and functional importance of TAL1 regulation and signalling pathways in hematopoietic malignancies and may provide novel insights into the genetic/epigenetic differences between T-ALL and CML. In summary, to our best knowledge, we for the first time report that TAL1 is significantly downregulated in CML LSCs and demonstrate that TAL1 regulates PTEN expression and leads to inhibition of the PI3K pathway in IM-induced CML apoptosis. This study reveals an unexpected contribution of TAL1 in CML and implicates regulation of PTEN expression as a novel mechanism for the transcriptional regulatory networks of TAL1. Funding This work was supported by the National Natural Science Foundation of China (Nos. 81400102, 81770158 and U1301226); the Guangdong Science & Technology Project (No. 2015A050502029); the Pearl River S&T Nova Program of Guangzhou, China (No. 201906010002) and the Fundamental Research Funds for the Central Universities (No. 17817015). Conflicts of interest The authors declare no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.164.
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