Inhibition of TERT suppressed proliferation and induced apoptosis in chronic myeloid leukemia cells

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The precise role of telomerase and its catalytic subunit, human telomerase reverse transcriptase (hTERT), in chronic myeloid leukemia (CML) has not been thoroughly elucidated. However, telomerase activity is recognized as a mechanism underlying resistance to imatinib (IM), a tyrosine kinase inhibitor. The loss of telomerase activity in CML has also been associated with acquiring infinite proliferative potential, which is closely linked to maintenance through telomerase reactivation. Methods K562 cells, along with their IM-resistant derivatives (K562R), were subjected to si-RNA targeting hTERT or a combination treatment involving IM and the hTERT inhibitor BIBR1532. To evaluate the role of hTERT in drug resistance, cell viability following exposure to si-hTERT, IM, and the combination of IM and BIBR1532 was evaluated using the Cell Counting Kit-8 (CCK-8) assay and colony-formation assays. The percentage of apoptosis was quantified using the Annexin V assay. Additionally, the transcriptional and protein expression levels of p73, p21, FOXO3a, c-Myc, hTERT, and other apoptosis-related target genes were analyzed through quantitative polymerase chain reaction (qPCR) and Western blotting (WB). Results In K562R and K562 cell lines, telomerase activity was significantly reduced following the pharmacological inhibition of telomerase using the inhibitor BIBR1532, as well as through the knockdown of hTERT. Flow cytometry analysis revealed that the knockdown of hTERT induced apoptosis in chronic myeloid leukemia (CML) cells and results in cell-cycle arrest at the G2 phase. Furthermore, the inhibition of hTERT was found to suppress the transcriptional activity of c-Myc while concurrently reactivating the transcription factors p73, p21, and Foxo3a. The combination of BIBR1532 with imatinib exhibited a synergistic anticancer effect on CML cells. Conclusions Overall, the combination of BIBR1532 is a novel therapeutic strategy for CML that may soon be clinically accessible. CML telomerase hTERT BIBR1532 p73 p21 Foxo3a Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The aberrant chimeric BCR-ABL oncoprotein, resulting from the reciprocal translocation between chromosomes 9 and 22, exhibits constitutive high kinase activity. Activated BCR-ABL1 facilitates the proliferation of chronic myeloid leukemia (CML) cells and impedes their capacity to undergo apoptosis by activating several downstream signaling pathways [ 1 – 2 ]. Tyrosine kinase inhibitors (TKIs), such as imatinib (IM) and nilotinib, have proven effective in treating CML during the chronic phase; however, approximately 15–20% of patients, particularly those in the accelerated stages of the disease, develop resistance to IM and ultimately experience relapse or progression to blast crisis [ 3 – 8 ]. Roughly 50% of TKI resistance cases are BCR-ABL dependent, arising from point mutations in the Abl kinase domain or amplification of the BCR-ABL gene, which leads to the reactivation of BCR-ABL kinase activity [ 9 ]. The remaining cases of resistance involve various critical signaling pathways associated with cellular proliferation and/or survival in cancer. The progression of CML from the chronic phase to advanced stages is driven by both BCR-ABL-dependent and independent mechanisms, which also exhibit a lack of response to specific TKIs. Human telomerase reverse transcriptase (hTERT) and the RNA component (hTERC) assemble to form the ribonucleoprotein complex known as telomerase. Numerous clinical studies have indicated that increased telomerase activity in human cancers is associated with unfavorable therapeutic outcomes and disease recurrence [ 10 – 15 ]. When telomerase activity or hTERT expression is inhibited, cancer cells experience progressive telomere shortening, which ultimately results in cellular senescence or apoptosis, leading to a loss of tumorigenic potential. Previous research has established a correlation between elevated telomerase activity and aggressive disease progression, as well as relapse in patients with acute promyelocytic leukemia [ 16 ]. Additionally, the overexpression of hTERT may facilitate IM resistance in CML cells [ 17 – 20 ]. However, the precise mechanisms by which telomerase contributes to the development of resistance in CML remain unclear. The primary functions of telomerase reverse transcriptase (hTERT) include the maintenance of telomere length and the synthesis of telomeric DNA repeats. However, an increasing body of research suggests that hTERT may also engage in critical biological processes beyond its enzymatic role in telomere maintenance. Recent studies indicate that telomerase reverse transcriptase can act as a transcriptional modulator within the nucleus [ 21 – 22 ]. Significant efforts have been dedicated to investigating the potential therapeutic applications of anti-telomerase agents. The present study aimed to elucidate the function and mechanism of hTERT in the context of resistance to CML. Materials and Methods Cell Culture Standard cell-culture conditions were employed for the cultivation of chronic myeloid leukemia-sensitive K562 cells and their resistant counterparts, K562R. The culture medium utilized was RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . Stock solutions of BIBR1532 and IM were prepared at concentrations of 10 mM and 200 µM, respectively, and stored at -20°C. To achieve the desired concentrations for drug treatment, the culture medium was supplemented with an appropriate volume of the respective stock solution of the drugs. Small molecules, siRNA, and antibodies MeChemExpress (MCE, USA) supplied the telomerase-specific inhibitor BIBR1532. Novartis provided the IM (Basel, Switzerland). Maobai Technologies (Chongqing, China) was the source of the siRNA targeting TERT. A prior description of the TERT siRNA target sequences is available in reference [ 23 ]. The following antibodies were utilized in this study: anti-myc (Abcam, USA); anti-β-actin (Zhong Shan Jin Qiao, China); anti-FOXO3A (Wanlei, China); anti-phosphorylated Bcr-Abl (Y412); anti-phosphorylated Stat5, anti-Stat5, anti-P73, anti-P21, and anti-P27 (Cell Signaling Technology, USA). Small-Molecule Therapeutics and RNA Interference A total of 1 × 10^6 cells were plated on 24-well plates and transfected with 30 µmol of shRNA, following the manufacturer's guidelines. After 48h of transfection, cells were harvested for immunofluorescence analysis, clonal-formation assays, western blot analysis, and viability assessments. RPMI-1640 was utilized to dissolve the small-molecule inhibitors IM and BIBR1532. At the specified time point, the cells treated with these inhibitors were collected for further examination. CCK-8 Cell Count Assay The Cell Counting Kit-8 (CCK-8) assay was employed to evaluate the inhibitory effects of IM, BIBR1532, and small interfering RNA (siRNA) targeting human hTERT on the proliferation of K562 and K562R cell lines. A total of 5,000 cells per well were plated on 96-well plates, and the cells were treated with either siRNA or the specified concentrations of the drugs for durations of 0h, 24h, 48h, and 72h. Following treatment, 10 µL of CCK-8 solution was added to each well, and the plates were incubated at 37°C in a 5% CO 2 atmosphere. After a two-hour incubation period, the absorbance of the wells was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Indirect immunofluorescence K562 and K562R cells were cultured on 12-well plates for a duration of 12h. Subsequently, 20 µM BIBR1532 was introduced into the culture medium for drug treatment, and the cells were incubated for 24h at 37°C in a 5% CO 2 atmosphere. Following this incubation period, the cells on the 12-well plates were removed and subjected to three washes with phosphate-buffered saline (PBS). The cells were then fixed for 1h using 4% paraformaldehyde at room temperature. Permeabilization was achieved with 1% Triton X-100 for 15 minutes, after which the cells were blocked with 10% normal goat serum for 60 minutes at 4°C. The cells were subsequently incubated overnight at 4°C with an anti-Abl antibody and the cells were treated with a Cy3-conjugated goat anti-mouse IgG (H + L) secondary antibody at room temperature for 60 minutes. Coverslips were affixed on the glass slides using a gel-mounting medium after DAPI staining of the DNA. Epifluorescence microscopy was performed using Nikon microscopes. Cell Death Assays and Cell Cycle Analysis Flow cytometry (FCM) was employed to assess cell apoptosis and cell-cycle dynamics. Following a 48h treatment with BIBR1532 or IM, K562 and K562R cells were harvested. Then the cells were subsequently washed twice with cold phosphate-buffered saline (PBS), resuspended in 300 µL of cold PBS, and treated with 7-AAD (7-amino-actinomycin D) and Annexin PE. To facilitate cell-cycle analysis, K562 and K562R cells were fixed in 70% ethanol and incubated overnight at 4°C. The results from three independent experiments were analyzed using a fluorescence-activated cell (FAC) sorter. Colony Formation Assays Six-well plates were inoculated with 500 K562 and K562R cells. The cells were subjected to analysis and enumeration fourteen days following treatment with hTERT shRNAs. Western Blot K562 and K562R cells, which had been treated with BIBR1532 or small interfering RNA, were harvested 48h post-treatment. The extraction of the cells was performed in accordance with the manufacturer's instructions utilizing RIPA buffer. Protein concentration was determined by BCA assay. And then an equivalent amount of protein was loaded onto 10% SDS-PAGE gels. The proteins were subsequently transferred on to PVDF membranes, which were then probed with a primary antibody after incubation with a secondary antibody, detection was conducted using HRP substrate. Before probing, the membranes were blocked with 5% BSA in TBST. RNA purification, reverse transcription, and real-time polymerase chain reaction (PCR) amplification. Total RNA was extracted 24h post-treatment with BIBR1532 using Trizol (Invitrogen Life Technologies). Reverse transcription was conducted utilizing the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio). Quantitative reverse-transcription PCR was performed on the complementary DNA using SYBR Premix Ex Taq technology (Takara Bio) and a light-cycler device (Roche Diagnostics). The protocol included an initial activation step of 30s at 95 ℃, followed by 40 cycles comprising denaturation for 15s at 95℃ and a combined annealing/extension step for 60s at 60 ℃. The specificity of the products was confirmed through melting curve analysis, and the relative quantification values were calculated using the 2 –∆∆Ct method. The primers for the target gene were previously described [ 24 ]. Statistical Analysis The results are presented as the mean accompanied by the standard deviation (SD). Comparisons were conducted using the independent samples Student's t-test, as implemented in GraphPad Prism 5. A p-value of less than 0.05 was considered indicative of statistical significance. Results K562R cells with higher TERT expression exhibit Bcr-Abl kinase-independent TKI resistance For 24h and 48h, K562 cells and their IM-resistant derivative, K562R, were cultivated in the presence and absence of IM. Notably, in the absence of IM, K562R demonstrated superior proliferative capacity over K562 at both 24 and 48 hours. Intriguingly, even when subjected to 1.25 µM of IM, K562R exhibited a notably higher level of viability compared to K562 (Fig. 1 A). The induction of tyrosine kinase-independent resistance has been implicated in the upregulation of Stat5 and c-Myc, both of which also serve as regulators of TERT expression. Our western blotting analysis revealed that K562R cells exhibit elevated levels of phosphorylated Bcr-Abl, phosphorylated Stat5, and phosphorylated c-Myc compared to K562 cells, further corroborating the involvement of these signaling pathways in conferring resistance. Simultaneously, treatment with a low dose of IM (1µM) failed to completely diminish the phosphorylation level in K562R cells, as indicated in Fig. 1 B. Immunofluorescence analysis further underscored this finding, revealing that K562R cells display a higher abundance of Bcr-Abl protein compared to K562 cells (Fig. 1 C). To delve deeper, we examined whether the presence or absence of IM impacted TERT protein expression in both K562 and K562R cells. The results presented in Figs. 1 D and 1 E indicate that TERT expression is upregulated in K562R cells compared to K562 cells, suggesting a potential influence of IM on TERT expression. Collectively, these findings reveal that K562R cells exhibit greater resistance to IM and possess higher viability than K562 cells. Inhibition of TERT potently reduced proliferation activity in K562R and K562 cells through activation of p73, p21, and FOXO3a K562 and K562R cells were treated with si-TERT for 24 hours to examine the impact of TERT knock-down on the growth of CML cell lines. Following this, WB was used to measure TERT expression. Figure 2 A illustrates how si-TERT-3 dramatically reduced TERT expression in K562R and K562 cells. According to the CCK-8 assay, si-TERT decreases K562 and K562R cell viability (Fig. 2 B, 2 C). Furthermore, the colony formation assay indicates that interfering with TERT can dramatically reduce the in vitro proliferation of CML cells (2D, 2E). To further evaluate the proliferation of CML cells after inhibition of hTERT, we examined the molecules that control the cell cycle. Remarkably, a decrease in the mRNA expression of c-Myc and a significant increase in the mRNA expression levels of p73, p21, and FOXO3a were observed following a 24-hour disruption of the TERT-3 (Fig. 2 F-I). WB results also demonstrated that TERT inhibition raised the expression of P73, P21, and FOXO3a while decreasing the expression of Stat5, c-Myc, and other proteins (Fig. 2 J). Taken together, our data revealed that the proliferative effect of TERT in CML cells is probably mediated through the activation of p73 and p21. BIBR1532 enhanced the IM-induced effect and induced G2 arrest in K562R cells. BIBR1532 is the most potent non-peptidic, non-nucleoside small molecule inhibitor of telomerase catalytic subunit (hTERT) discovered thus far. Its anticancer value has recently been evaluated in considerable preclinical studies, indicating the potent ability of this inhibitor to repress tumor cell growth in several types of cancers. To determine to what extent the synergistic effect of BIBR1532 would impact the expression of TERT in CML cells, the mRNA and protein expression of TERT were measured by RT-PCR and WB. As shown in Fig. 3 A- 3 C, BIBR1532 showed significantly restricted TERT expression. To address whether TERT inhibition could improve the cytotoxic effect of the IM used in CML treatment, we analyzed the effects of both individual and combination treatments of BIBR1532 and IM on the viability and cell count of CML cells. Our results (Fig. 3 D, 3 E) revealed that single agents of BIBR1532 exerted dose-dependent growth suppressive and cytotoxic effects in K562 and K562R. Our data also demonstrated that BIBR1532 markedly augmented the sensitivity of cells to IM at 10 µM, as revealed by the decreased viability and number of viable cells. Measuring the effects of BIBR1532 on cell cycle progression displayed that BIBR1532 increased the G2 cell population in K562 and K562R cells than untreated groups (Fig. 3 G, 3 H). Moreover, analysis of the cell cycle also exhibited that treatment of the cells with BIBR1532 significantly increased the percentage of cell populations in the G2 phase coupled with a decreased percentage of cells in the G1 and S phases, as well (Fig. 3 I). BIBR1532 enhanced IM-induced apoptosis in K562R cells To determine whether the cytotoxic effect induced by the drug combination is mediated through the induction of apoptosis, drug-treated cells were subjected to an annexin-V-staining assay. We discovered that simultaneous treatment of cells with BIBR1532 and IM resulted in a marked increase in the percentage of apoptosis compared to the IM-treated group (Fig. 4 A-E), which indicated that BIBR1532 enhanced IM-induced apoptosis in K562R cells. As illustrated in Figs. 4 F and 4 G, BIBR1532 enhanced the mRNA expression level of pro-apoptotic members, such as Bad and Bax. These results recommend that BIBR1532 induces apoptosis of K562 and K562R cells via activation of Bad and Bax. Discussion Imatinib, the most typical clinical treatment, promotes the response rate of most chronic myeloid leukemia patients. However, 15–20% of the patients, acquire resistance to imatinib, so it is urgent to seek novel drug targets or combined treatment therapy. Activation or upregulation of telomerase is considered as a critical role in the advancement of most human malignancies. While the implication of telomerase in the development of imatinib resistance has already been suggested [ 25 – 28 ], however, there is no direct obvious demonstration of the influence of telomerase inhibition has been given. In our study, we explore the consequence of the knockdown of hTERT or BIBR1532, a small molecule inhibitor of telomerase catalytic subunit (TERT), on proliferation and apoptosis in K562 and K562R cells. First of all, we investigated that K562R cells exhibit more potent viability and more resistance to IM than K562 cells. Meanwhile, western blot results indicate that resistance to IM may involve Stat5 and c-Myc. To investigate the effect of TERT knock-down in the proliferation of CML cell lines, K562 and K562R cells were treated with si-TERT for 24h, and then, the expression of TERT was determined by using WB. Interestingly, 24 h of interference with the TERT-3 led to a significant increase in the mRNA expression level of p73, p21, and FOXO3a and a decrease in the mRNA expression of c-Myc. Based on synergistic experiments, we encountered an enhanced reduction in the viability of K562R cells when IM was used in combination with BIBR1532. Our results also delineated that BIBR1532 significantly induces chronic myeloid leukemia cell apoptosis and cell cycle arrest, as evidenced by elevated G2 cell population, and decreased number of inhibitor-treated viable cells. The transcription factor p73, which is a potent surrogate for p53, elicits anticancer effects through either activation of programmed cell death or induction of cell cycle arrest by regulating the expression level of a large cohort of target genes. It has been reported that p73 could provide a signal that up-regulates FOXO3a, which in turn induces G1 cell cycle arrest mainly through activation of p21, as a key regulator of cell cycle progression at the G2 phase[ 29 – 30 ]. Consistent with this, our data showed that knockdown of hTERT not only elevated the transcriptional activity of both FOXO3a and p21 but also induced a robust increase in the cell population of the G2 phase. Moreover, Kartasheva revealed that the mRNA level of c-Myc[ 31 ], as a strong regulator of TERT transcription, was reduced by p73. In agreement with the inductive effect on p73, our results showed that si-hTERT significantly suppressed the c-Myc mRNA level. It has been reported that high expression of Stat5 and c-Myc are critical parameters that determine the sensitivity of Bcr-Abl (+) cells against IM. Juin Hsien Chai has reported that the TERT promoter was significantly activated by STAT5a and c-Myc[ 32 ]. We then detected the protein expression of Stat5 and c-Myc after being treated with si-hTERT. Our results showed that inhibition of telomerase by BIBR1532 significantly reduced the protein level of c-Myc, Stat5, and phosphorylation of Stat5. To our surprise, the level of Erk and p-Erk also decreased. These results indicate the complicated mechanism involved in the telomerase-related CML resistance, but the underlying mechanism is still ambiguous. Our study explores the effect of BIBR1532 in CML cells on proliferation and apoptosis. BIBR1532 enhances imatinib sensibility in K562 and K562R cells by inhibiting telomerase activity. Taken together, BIBR1532 may produce a synergistic anticancer effect in CML cells and telomerase activity may be a novel therapy target for the treatment of CML. Declarations Ethics approval and consent to participate Not applicable Patient consent for publication Not applicable Conflict of interest statement The authors declare that they have no competing interests. Acknowledgments This work was funded by the Natural Science Foundation of Chongqing, China (No.cstc2021jcyj-msxmX0289). We would like to thank Mrs. Deng for their valuable contributions to this research. We gratefully acknowledge the Laboratory of the Second Affiliated Hospital of Chongqing Medical University for providing the necessary equipment for this study. Funding This work was funded by the Natural Science Foundation of Chongqing, China (No.cstc2021jcyj-msxmX0289). Data availability statement The data associated with our study are all included in the article/supp material/referenced in the article. No datasets were generated or analyzed during the current study. Authors' contributions Teng Wang conceived and designed the research project. Wen Liu and Deng Fang completed the experiments and wrote the manuscript. Teng Wang analyzed and interpreted the data and supervised the entire study process and revised the manuscript. All authors read and approved the final manuscript. References Braun TP, Eide CA, Druker BJ. Response and Resistance to BCR-ABL1-Targeted Therapies. Cancer Cell. 2020 Apr 13;37(4):530-542. Hazlehurst LA, Bewry NN, Nair RR, et al. Signaling networks associated with BCR-ABL-dependent transformation. Cancer Control. 2009;16(2):100-7. Osman AEG, Deininger MW. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5754607","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":397349535,"identity":"b1b10024-8d30-4432-b53b-c887d4a20c69","order_by":0,"name":"Wen Liu","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Liu","suffix":""},{"id":397349537,"identity":"015db03c-afa8-4c3e-a8a5-8d3ba188faff","order_by":1,"name":"Fang Deng","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Deng","suffix":""},{"id":397349538,"identity":"8123452b-9d36-4243-8259-52ef9c9797ba","order_by":2,"name":"Teng Wang","email":"data:image/png;base64,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","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Teng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-01-03 02:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5754607/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5754607/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73055228,"identity":"ec70ef18-e641-4d65-b626-f58cf7b725ec","added_by":"auto","created_at":"2025-01-06 10:02:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3880832,"visible":true,"origin":"","legend":"\u003cp\u003eK562R cells show more resistance to immunization than K562 cells do. CCK-8 was used to measure the proliferation of A, K562, and K562R cells in the absence of IM therapy. B, the K562 and K562R cells' protein expression of p-Bcr-Abl, c-Myc, and p-Stat5. C, Indirect Immunofluorescence-Based Bcr-Abl Subcellular Location and Protein Level. D and E represent TERT protein expression in K562 and K562R, either with or without IM.\u003c/p\u003e","description":"","filename":"Figure1TERT.png","url":"https://assets-eu.researchsquare.com/files/rs-5754607/v1/0b86dc57ea9cf88ec6da5386.png"},{"id":73055229,"identity":"28730758-a8ab-456e-ada8-f9a95ee030f0","added_by":"auto","created_at":"2025-01-06 10:02:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1724251,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of TERT potently reduced proliferation activity in K562R and K562 cells through activation of p73, p21, and FOXO3a. A Western Blotting to detect the expression of TERT in K562 and K562R cells with or without TERT knock-down. B and C, si-TERT-3 significantly reduce cell numbers of K562 and K562R cells. D-E, inhibition of TERT reduces the proliferative ability of CML colony formation. F-I, RT-PCR to detect the mRNA expression of the cell cycle regulator. J, Western Blotting to detect the protein expression of cell cycle regulator. Values are given as mean ± SD of three independent experiments. *p ≤ 0.05; **p≤ 0.005; ***p ≤ 0.001 represent significant changes from untreated control.\u003c/p\u003e","description":"","filename":"Figure2TERT.png","url":"https://assets-eu.researchsquare.com/files/rs-5754607/v1/afae3f51d9ffc096448f9c51.png"},{"id":73054505,"identity":"1e8d2a56-5e28-4cf6-9d7d-44c7fb5c07d8","added_by":"auto","created_at":"2025-01-06 09:54:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":904460,"visible":true,"origin":"","legend":"\u003cp\u003eBIBR1532 enhanced IM-induced growth suppressive effect and induced G2 arrest in K562R cells. A-C, RT-PCR, and Western blotting were used to determine the mRNA and protein expression of TERT. D-F, CCK-8 assay was used to detect the BIBR1532 on proliferation activity of CML cells. G-I, Flow cytometry was used to evaluate the cell cycle of CML cells after treatment of BIBR1532 (10μM). Statistical results were analyzed by GraphPad Prism 5. Values are given as mean ± SD of three independent experiments. *p ≤ 0.05, ***p ≤ 0.001 represent significant changes from untreated control.\u003c/p\u003e","description":"","filename":"Figure3TERT.png","url":"https://assets-eu.researchsquare.com/files/rs-5754607/v1/e42f285b69981c59041119c9.png"},{"id":73054503,"identity":"c1c1cee4-f297-4ffb-af53-94317e139b16","added_by":"auto","created_at":"2025-01-06 09:54:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1319055,"visible":true,"origin":"","legend":"\u003cp\u003eBIBR1532 enhanced IM-induced apoptosis in K562R cells. A-E, apoptosis assays of K562 and K562R treated with BIBR1532 combined IM. F-G, the mRNA level of pro-apoptotic members of Bcl-2 family Bad and Bax. Values are given as mean ± SD of three independent experiments. ***p ≤ 0.001 represents significant changes from untreated control.\u003c/p\u003e","description":"","filename":"Figure4TERT.png","url":"https://assets-eu.researchsquare.com/files/rs-5754607/v1/78e5467e4e505cead371bb18.png"},{"id":74336221,"identity":"8dec50aa-f1b3-4295-b309-c71813f7ae3e","added_by":"auto","created_at":"2025-01-21 07:40:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7692656,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5754607/v1/0944696a-2252-43c5-8c67-3dc80619fc48.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of TERT suppressed proliferation and induced apoptosis in chronic myeloid leukemia cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe aberrant chimeric BCR-ABL oncoprotein, resulting from the reciprocal translocation between chromosomes 9 and 22, exhibits constitutive high kinase activity. Activated BCR-ABL1 facilitates the proliferation of chronic myeloid leukemia (CML) cells and impedes their capacity to undergo apoptosis by activating several downstream signaling pathways [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Tyrosine kinase inhibitors (TKIs), such as imatinib (IM) and nilotinib, have proven effective in treating CML during the chronic phase; however, approximately 15\u0026ndash;20% of patients, particularly those in the accelerated stages of the disease, develop resistance to IM and ultimately experience relapse or progression to blast crisis [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Roughly 50% of TKI resistance cases are BCR-ABL dependent, arising from point mutations in the Abl kinase domain or amplification of the BCR-ABL gene, which leads to the reactivation of BCR-ABL kinase activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The remaining cases of resistance involve various critical signaling pathways associated with cellular proliferation and/or survival in cancer. The progression of CML from the chronic phase to advanced stages is driven by both BCR-ABL-dependent and independent mechanisms, which also exhibit a lack of response to specific TKIs.\u003c/p\u003e \u003cp\u003eHuman telomerase reverse transcriptase (hTERT) and the RNA component (hTERC) assemble to form the ribonucleoprotein complex known as telomerase. Numerous clinical studies have indicated that increased telomerase activity in human cancers is associated with unfavorable therapeutic outcomes and disease recurrence [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. When telomerase activity or hTERT expression is inhibited, cancer cells experience progressive telomere shortening, which ultimately results in cellular senescence or apoptosis, leading to a loss of tumorigenic potential. Previous research has established a correlation between elevated telomerase activity and aggressive disease progression, as well as relapse in patients with acute promyelocytic leukemia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, the overexpression of hTERT may facilitate IM resistance in CML cells [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the precise mechanisms by which telomerase contributes to the development of resistance in CML remain unclear.\u003c/p\u003e \u003cp\u003eThe primary functions of telomerase reverse transcriptase (hTERT) include the maintenance of telomere length and the synthesis of telomeric DNA repeats. However, an increasing body of research suggests that hTERT may also engage in critical biological processes beyond its enzymatic role in telomere maintenance. Recent studies indicate that telomerase reverse transcriptase can act as a transcriptional modulator within the nucleus [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Significant efforts have been dedicated to investigating the potential therapeutic applications of anti-telomerase agents. The present study aimed to elucidate the function and mechanism of hTERT in the context of resistance to CML.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eStandard cell-culture conditions were employed for the cultivation of chronic myeloid leukemia-sensitive K562 cells and their resistant counterparts, K562R. The culture medium utilized was RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum. The cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Stock solutions of BIBR1532 and IM were prepared at concentrations of 10 mM and 200 \u0026micro;M, respectively, and stored at -20\u0026deg;C. To achieve the desired concentrations for drug treatment, the culture medium was supplemented with an appropriate volume of the respective stock solution of the drugs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSmall molecules, siRNA, and antibodies\u003c/h3\u003e\n\u003cp\u003eMeChemExpress (MCE, USA) supplied the telomerase-specific inhibitor BIBR1532. Novartis provided the IM (Basel, Switzerland). Maobai Technologies (Chongqing, China) was the source of the siRNA targeting TERT. A prior description of the TERT siRNA target sequences is available in reference [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The following antibodies were utilized in this study: anti-myc (Abcam, USA); anti-β-actin (Zhong Shan Jin Qiao, China); anti-FOXO3A (Wanlei, China); anti-phosphorylated Bcr-Abl (Y412); anti-phosphorylated Stat5, anti-Stat5, anti-P73, anti-P21, and anti-P27 (Cell Signaling Technology, USA).\u003c/p\u003e\n\u003ch3\u003eSmall-Molecule Therapeutics and RNA Interference\u003c/h3\u003e\n\u003cp\u003eA total of 1 \u0026times; 10^6 cells were plated on 24-well plates and transfected with 30 \u0026micro;mol of shRNA, following the manufacturer's guidelines. After 48h of transfection, cells were harvested for immunofluorescence analysis, clonal-formation assays, western blot analysis, and viability assessments. RPMI-1640 was utilized to dissolve the small-molecule inhibitors IM and BIBR1532. At the specified time point, the cells treated with these inhibitors were collected for further examination.\u003c/p\u003e\n\u003ch3\u003eCCK-8 Cell Count Assay\u003c/h3\u003e\n\u003cp\u003eThe Cell Counting Kit-8 (CCK-8) assay was employed to evaluate the inhibitory effects of IM, BIBR1532, and small interfering RNA (siRNA) targeting human hTERT on the proliferation of K562 and K562R cell lines. A total of 5,000 cells per well were plated on 96-well plates, and the cells were treated with either siRNA or the specified concentrations of the drugs for durations of 0h, 24h, 48h, and 72h. Following treatment, 10 \u0026micro;L of CCK-8 solution was added to each well, and the plates were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. After a two-hour incubation period, the absorbance of the wells was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.\u003c/p\u003e\n\u003ch3\u003eIndirect immunofluorescence\u003c/h3\u003e\n\u003cp\u003eK562 and K562R cells were cultured on 12-well plates for a duration of 12h. Subsequently, 20 \u0026micro;M BIBR1532 was introduced into the culture medium for drug treatment, and the cells were incubated for 24h at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Following this incubation period, the cells on the 12-well plates were removed and subjected to three washes with phosphate-buffered saline (PBS). The cells were then fixed for 1h using 4% paraformaldehyde at room temperature. Permeabilization was achieved with 1% Triton X-100 for 15 minutes, after which the cells were blocked with 10% normal goat serum for 60 minutes at 4\u0026deg;C. The cells were subsequently incubated overnight at 4\u0026deg;C with an anti-Abl antibody and the cells were treated with a Cy3-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody at room temperature for 60 minutes. Coverslips were affixed on the glass slides using a gel-mounting medium after DAPI staining of the DNA. Epifluorescence microscopy was performed using Nikon microscopes.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Death Assays and Cell Cycle Analysis\u003c/h2\u003e \u003cp\u003eFlow cytometry (FCM) was employed to assess cell apoptosis and cell-cycle dynamics. Following a 48h treatment with BIBR1532 or IM, K562 and K562R cells were harvested. Then the cells were subsequently washed twice with cold phosphate-buffered saline (PBS), resuspended in 300 \u0026micro;L of cold PBS, and treated with 7-AAD (7-amino-actinomycin D) and Annexin PE. To facilitate cell-cycle analysis, K562 and K562R cells were fixed in 70% ethanol and incubated overnight at 4\u0026deg;C. The results from three independent experiments were analyzed using a fluorescence-activated cell (FAC) sorter.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eColony Formation Assays\u003c/h3\u003e\n\u003cp\u003eSix-well plates were inoculated with 500 K562 and K562R cells. The cells were subjected to analysis and enumeration fourteen days following treatment with hTERT shRNAs.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eK562 and K562R cells, which had been treated with BIBR1532 or small interfering RNA, were harvested 48h post-treatment. The extraction of the cells was performed in accordance with the manufacturer's instructions utilizing RIPA buffer. Protein concentration was determined by BCA assay. And then an equivalent amount of protein was loaded onto 10% SDS-PAGE gels. The proteins were subsequently transferred on to PVDF membranes, which were then probed with a primary antibody after incubation with a secondary antibody, detection was conducted using HRP substrate. Before probing, the membranes were blocked with 5% BSA in TBST.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA purification, reverse transcription, and real-time polymerase chain reaction (PCR) amplification.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal RNA was extracted 24h post-treatment with BIBR1532 using Trizol (Invitrogen Life Technologies). Reverse transcription was conducted utilizing the PrimeScript\u0026trade; RT Reagent Kit with gDNA Eraser (Takara Bio). Quantitative reverse-transcription PCR was performed on the complementary DNA using SYBR Premix Ex Taq technology (Takara Bio) and a light-cycler device (Roche Diagnostics). The protocol included an initial activation step of 30s at 95 ℃, followed by 40 cycles comprising denaturation for 15s at 95℃ and a combined annealing/extension step for 60s at 60 ℃. The specificity of the products was confirmed through melting curve analysis, and the relative quantification values were calculated using the 2\u003csup\u003e\u0026ndash;∆∆Ct\u003c/sup\u003e method. The primers for the target gene were previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe results are presented as the mean accompanied by the standard deviation (SD). Comparisons were conducted using the independent samples Student's t-test, as implemented in GraphPad Prism 5. A p-value of less than 0.05 was considered indicative of statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eK562R cells with higher TERT expression exhibit Bcr-Abl kinase-independent TKI resistance\u003c/p\u003e \u003cp\u003eFor 24h and 48h, K562 cells and their IM-resistant derivative, K562R, were cultivated in the presence and absence of IM. Notably, in the absence of IM, K562R demonstrated superior proliferative capacity over K562 at both 24 and 48 hours. Intriguingly, even when subjected to 1.25 \u0026micro;M of IM, K562R exhibited a notably higher level of viability compared to K562 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The induction of tyrosine kinase-independent resistance has been implicated in the upregulation of Stat5 and c-Myc, both of which also serve as regulators of TERT expression. Our western blotting analysis revealed that K562R cells exhibit elevated levels of phosphorylated Bcr-Abl, phosphorylated Stat5, and phosphorylated c-Myc compared to K562 cells, further corroborating the involvement of these signaling pathways in conferring resistance. Simultaneously, treatment with a low dose of IM (1\u0026micro;M) failed to completely diminish the phosphorylation level in K562R cells, as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Immunofluorescence analysis further underscored this finding, revealing that K562R cells display a higher abundance of Bcr-Abl protein compared to K562 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To delve deeper, we examined whether the presence or absence of IM impacted TERT protein expression in both K562 and K562R cells. The results presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE indicate that TERT expression is upregulated in K562R cells compared to K562 cells, suggesting a potential influence of IM on TERT expression. Collectively, these findings reveal that K562R cells exhibit greater resistance to IM and possess higher viability than K562 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInhibition of TERT potently reduced proliferation activity in K562R and K562 cells through activation of p73, p21, and FOXO3a\u003c/p\u003e \u003cp\u003eK562 and K562R cells were treated with si-TERT for 24 hours to examine the impact of TERT knock-down on the growth of CML cell lines. Following this, WB was used to measure TERT expression. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA illustrates how si-TERT-3 dramatically reduced TERT expression in K562R and K562 cells. According to the CCK-8 assay, si-TERT decreases K562 and K562R cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, the colony formation assay indicates that interfering with TERT can dramatically reduce the in vitro proliferation of CML cells (2D, 2E). To further evaluate the proliferation of CML cells after inhibition of hTERT, we examined the molecules that control the cell cycle. Remarkably, a decrease in the mRNA expression of c-Myc and a significant increase in the mRNA expression levels of p73, p21, and FOXO3a were observed following a 24-hour disruption of the TERT-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-I). WB results also demonstrated that TERT inhibition raised the expression of P73, P21, and FOXO3a while decreasing the expression of Stat5, c-Myc, and other proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Taken together, our data revealed that the proliferative effect of TERT in CML cells is probably mediated through the activation of p73 and p21.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBIBR1532 enhanced the IM-induced effect and induced G2 arrest in K562R cells.\u003c/p\u003e \u003cp\u003eBIBR1532 is the most potent non-peptidic, non-nucleoside small molecule inhibitor of telomerase catalytic subunit (hTERT) discovered thus far. Its anticancer value has recently been evaluated in considerable preclinical studies, indicating the potent ability of this inhibitor to repress tumor cell growth in several types of cancers. To determine to what extent the synergistic effect of BIBR1532 would impact the expression of TERT in CML cells, the mRNA and protein expression of TERT were measured by RT-PCR and WB. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, BIBR1532 showed significantly restricted TERT expression. To address whether TERT inhibition could improve the cytotoxic effect of the IM used in CML treatment, we analyzed the effects of both individual and combination treatments of BIBR1532 and IM on the viability and cell count of CML cells. Our results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) revealed that single agents of BIBR1532 exerted dose-dependent growth suppressive and cytotoxic effects in K562 and K562R. Our data also demonstrated that BIBR1532 markedly augmented the sensitivity of cells to IM at 10 \u0026micro;M, as revealed by the decreased viability and number of viable cells. Measuring the effects of BIBR1532 on cell cycle progression displayed that BIBR1532 increased the G2 cell population in K562 and K562R cells than untreated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Moreover, analysis of the cell cycle also exhibited that treatment of the cells with BIBR1532 significantly increased the percentage of cell populations in the G2 phase coupled with a decreased percentage of cells in the G1 and S phases, as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBIBR1532 enhanced IM-induced apoptosis in K562R cells\u003c/p\u003e \u003cp\u003eTo determine whether the cytotoxic effect induced by the drug combination is mediated through the induction of apoptosis, drug-treated cells were subjected to an annexin-V-staining assay. We discovered that simultaneous treatment of cells with BIBR1532 and IM resulted in a marked increase in the percentage of apoptosis compared to the IM-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-E), which indicated that BIBR1532 enhanced IM-induced apoptosis in K562R cells. As illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, BIBR1532 enhanced the mRNA expression level of pro-apoptotic members, such as Bad and Bax. These results recommend that BIBR1532 induces apoptosis of K562 and K562R cells via activation of Bad and Bax.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImatinib, the most typical clinical treatment, promotes the response rate of most chronic myeloid leukemia patients. However, 15\u0026ndash;20% of the patients, acquire resistance to imatinib, so it is urgent to seek novel drug targets or combined treatment therapy. Activation or upregulation of telomerase is considered as a critical role in the advancement of most human malignancies. While the implication of telomerase in the development of imatinib resistance has already been suggested [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], however, there is no direct obvious demonstration of the influence of telomerase inhibition has been given.\u003c/p\u003e \u003cp\u003eIn our study, we explore the consequence of the knockdown of hTERT or BIBR1532, a small molecule inhibitor of telomerase catalytic subunit (TERT), on proliferation and apoptosis in K562 and K562R cells. First of all, we investigated that K562R cells exhibit more potent viability and more resistance to IM than K562 cells. Meanwhile, western blot results indicate that resistance to IM may involve Stat5 and c-Myc. To investigate the effect of TERT knock-down in the proliferation of CML cell lines, K562 and K562R cells were treated with si-TERT for 24h, and then, the expression of TERT was determined by using WB. Interestingly, 24 h of interference with the TERT-3 led to a significant increase in the mRNA expression level of p73, p21, and FOXO3a and a decrease in the mRNA expression of c-Myc. Based on synergistic experiments, we encountered an enhanced reduction in the viability of K562R cells when IM was used in combination with BIBR1532. Our results also delineated that BIBR1532 significantly induces chronic myeloid leukemia cell apoptosis and cell cycle arrest, as evidenced by elevated G2 cell population, and decreased number of inhibitor-treated viable cells.\u003c/p\u003e \u003cp\u003eThe transcription factor p73, which is a potent surrogate for p53, elicits anticancer effects through either activation of programmed cell death or induction of cell cycle arrest by regulating the expression level of a large cohort of target genes. It has been reported that p73 could provide a signal that up-regulates FOXO3a, which in turn induces G1 cell cycle arrest mainly through activation of p21, as a key regulator of cell cycle progression at the G2 phase[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consistent with this, our data showed that knockdown of hTERT not only elevated the transcriptional activity of both FOXO3a and p21 but also induced a robust increase in the cell population of the G2 phase. Moreover, Kartasheva revealed that the mRNA level of c-Myc[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], as a strong regulator of TERT transcription, was reduced by p73. In agreement with the inductive effect on p73, our results showed that si-hTERT significantly suppressed the c-Myc mRNA level. It has been reported that high expression of Stat5 and c-Myc are critical parameters that determine the sensitivity of Bcr-Abl (+) cells against IM. Juin Hsien Chai has reported that the TERT promoter was significantly activated by STAT5a and c-Myc[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We then detected the protein expression of Stat5 and c-Myc after being treated with si-hTERT. Our results showed that inhibition of telomerase by BIBR1532 significantly reduced the protein level of c-Myc, Stat5, and phosphorylation of Stat5. To our surprise, the level of Erk and p-Erk also decreased. These results indicate the complicated mechanism involved in the telomerase-related CML resistance, but the underlying mechanism is still ambiguous.\u003c/p\u003e \u003cp\u003eOur study explores the effect of BIBR1532 in CML cells on proliferation and apoptosis. BIBR1532 enhances imatinib sensibility in K562 and K562R cells by inhibiting telomerase activity. Taken together, BIBR1532 may produce a synergistic anticancer effect in CML cells and telomerase activity may be a novel therapy target for the treatment of CML.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Natural Science Foundation of Chongqing, China (No.cstc2021jcyj-msxmX0289). We would like to thank Mrs. Deng for their valuable contributions to this research. We gratefully acknowledge the Laboratory of the Second Affiliated Hospital of Chongqing Medical University for providing the necessary equipment for this study.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Natural Science Foundation of Chongqing, China (No.cstc2021jcyj-msxmX0289).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eThe data associated with our study are all included in the article/supp material/referenced in the article. No datasets were generated or analyzed during the current study.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTeng Wang conceived and designed the research project. Wen Liu and Deng Fang completed the experiments and wrote the manuscript. Teng Wang analyzed and interpreted the data and supervised the entire study process and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBraun TP, Eide CA, Druker BJ. Response and Resistance to BCR-ABL1-Targeted Therapies. Cancer Cell. 2020 Apr 13;37(4):530-542.\u003c/li\u003e\n\u003cli\u003eHazlehurst LA, Bewry NN, Nair RR, et al. Signaling networks associated with BCR-ABL-dependent transformation. Cancer Control. 2009;16(2):100-7.\u003c/li\u003e\n\u003cli\u003eOsman AEG, Deininger MW. Chronic Myeloid Leukemia: Modern therapies, current challenges, and future directions. Blood Rev. 2021 Sep;49:100825. \u003c/li\u003e\n\u003cli\u003eKantarjian H SC, Hochhaus A, Guilhot F, Schiffer C, GambacortiPasserini C, Niederwieser D, et al. International STI571 CML Study Group: Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002;346:645-52.\u003c/li\u003e\n\u003cli\u003eVetrie D, Helgason GV, Copland M. The leukemia stem cell: similarities, differences and clinical prospects in CML and AML. Nat Rev Cancer. 2020 Mar;20(3):158-173. \u003c/li\u003e\n\u003cli\u003eHoushmand M, Simonetti G, Circosta P, Gaidano V, Cignetti A, Martinelli G, Saglio G, Gale RP. Chronic myeloid leukemia stem cells. Leukemia. 2019 Jul;33(7):1543-1556. \u003c/li\u003e\n\u003cli\u003eNg JJ, Ong ST. Therapy Resistance and Disease Progression in CML: Mechanistic Links and Therapeutic Strategies. Curr Hematol Malig Rep. 2022 Dec;17(6):181-197.\u003c/li\u003e\n\u003cli\u003eHughes TP, Shanmuganathan N. Management of TKI-resistant chronic phase CML.Hematology Am Soc Hematol Educ Program. 2022 Dec 9;2022(1):129-137.\u003c/li\u003e\n\u003cli\u003eMilojkovic D, Apperley J. Mechanisms of resistance to imatinib and second-generation tyrosine inhibitors in chronic myeloid leukemia. Clin Cancer Res 2009;15:7519-29.\u003c/li\u003e\n\u003cli\u003eFr\u0026iacute;as C, Garc\u0026iacute;a-Aranda C, De Juan C, et al. Telomere shortening is associated with poor prognosis and telomerase activity correlates with DNA repair impairment in non-small cell lung cancer. Lung Cancer.2008;60:416-425.\u003c/li\u003e\n\u003cli\u003eOh B-K, Kim H, Park YN, et al. High telomerase activity and long telomeres in advanced hepatocellular carcinomas with poor prognosis. Lab Invest. 2008;88:144-152.\u003c/li\u003e\n\u003cli\u003eMatsuda Y, Yamashita T, Ye J, et al. Phosphorylation of hTERT at threonine 249 is a novel tumor biomarker of aggressive cancer with poor prognosis in multiple organs. J Pathol. 2022 Jun;257(2):172-185.\u003c/li\u003e\n\u003cli\u003eLe\u0026atilde;o R, Apol\u0026oacute;nio JD, Lee D, Figueiredo A, Tabori U, Castelo-Branco P. Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer. J Biomed Sci. 2018 Mar 12;25(1):22.\u003c/li\u003e\n\u003cli\u003eHannen R, Bartsch JW. Essential roles of telomerase reverse transcriptase hTERT in cancer stemness and metastasis. FEBS Lett. 2018 Jun;592(12):2023-2031.\u003c/li\u003e\n\u003cli\u003eYasukawa M, Ando Y, Yamashita T, et al. CDK1-dependent phosphorylation of hTERT contributes to cancer progression. Nat Commun. 2020 Mar 25;11(1):1557.\u003c/li\u003e\n\u003cli\u003eGhaffari S, Shayan-Asl N, Jamialahmadi A, et al. Telomerase activity and telomere length in patients with acute promyelocytic leukemia: indicative of proliferative activity, disease progression, and overall survival. Ann Oncol. 2008;19:1927-1934. \u003c/li\u003e\n\u003cli\u003eVarma N, Anand MS, Varma S, Juneja SS. Role of hTERT and WT1 gene expression in disease progression and imatinib responsiveness of patients with BCR-ABL positive chronic myeloid leukemia. Leuk Lymphoma. 2011 Apr;52(4):687-93.\u003c/li\u003e\n\u003cli\u003eChai JH, Zhang Y, Tan WH, Chng WJ, Li B, Wang X. Regulation of hTERT by BCR-ABL at multiple levels in K562 cells. BMC Cancer. 2011 Dec 9;11:512.\u003c/li\u003e\n\u003cli\u003eGrandjenette C, Schnekenburger M, Gaigneaux A, et al. Human telomerase reverse transcriptase depletion potentiates the growth-inhibitory activity of imatinib in chronic myeloid leukemia stem cells. Cancer Lett. 2020 Jan 28;469:468-480.\u003c/li\u003e\n\u003cli\u003eYamada O, Ozaki K, Furukawa T, et al. Activation of STAT5 confers imatinib resistance on leukemic cells through the transcription of TERT and MDR1. Cell Signal. 2011 Jul;23(7):1119-27.\u003c/li\u003e\n\u003cli\u003ePlyasova AA, Zhdanov DD. Alternative Splicing of Human Telomerase Reverse Transcriptase (hTERT) and Its Implications in Physiological and Pathological Processes. Biomedicines. 2021 May 9;9(5):526.\u003c/li\u003e\n\u003cli\u003eCollins K. The biogenesis and regulation of telomerase holoenzymes. Nat Rev Mol Cell Biol. 2006 Jul;7(7):484-94. \u003c/li\u003e\n\u003cli\u003eZhang H, Hu N. Telomerase reverse transcriptase induced thyroid carcinoma cell proliferation through PTEN/AKT signaling pathway. Mol Med Rep. 2018 Aug;18(2):1345-1352. \u003c/li\u003e\n\u003cli\u003eBashash D, Zareii M, Safaroghli-Azar A, Omrani MD, Ghaffari SH. Inhibition of telomerase using BIBR1532 enhances doxorubicin-induced apoptosis in pre-B acute lymphoblastic leukemia cells. Hematology. 2017 Jul;22(6):330-340. \u003c/li\u003e\n\u003cli\u003eBashash D, Ghaffari S, Zaker F, et al. Direct short-term cytotoxic effects of BIBR 1532 on acute promyelocytic leukemia cells through induction of p21 coupled with downregulation of c-Myc and TERT transcription. Cancer Invest. 2012;30:57-64.\u003c/li\u003e\n\u003cli\u003eBrassat U, Balabanov S, Bali D, et al. Functional p53 is required for the effective execution of telomerase inhibition in BCR-ABL-positive CML cells. Exp Hematol.2011;39:66-76. e62.\u003c/li\u003e\n\u003cli\u003eBakalova R, Ohba H, Zhelev Z, Ishikawa M, Shinohara Y, Baba Y. Cross-talk between Bcr-Abl tyrosine kinase, protein kinase C and telomerase-a potential reason for resistance to Glivec in chronic myelogenous leukemia. Biochem Pharmacol 2003;66:1879-84.\u003c/li\u003e\n\u003cli\u003eYamada O, Kawauchi K, Akiyama M, Ozaki K, Motoji T, Adachi T, et al. Leukemic cells with increased telomerase activity exhibit resistance to imatinib. Leuk Lymphoma 2008;49:1168-77.\u003c/li\u003e\n\u003cli\u003eVossio S, Balsano C, Costanzo A, et al. DN-p73 is activated after DNA damage in a p53-dependent manner to regulate p53-induced cell cycle arrest. Oncogene. 2002;21:3796\u0026ndash;3803.\u003c/li\u003e\n\u003cli\u003eBeitzinger M, Oswald C, Beinoraviciute-Kellner R, et al. Regulation of telomerase activity by the p53 family member p73. Oncogene. 2006;25:813-826.\u003c/li\u003e\n\u003cli\u003eHenderson YC, Breau RL, Liu T-J, et al. Telomerase activity in head and neck tumors after the introduction of wild-type p53, p21, p16, and E2F-1 genes by means of recombinant adenovirus. Head Neck. 2000;22:347-354.\u003c/li\u003e\n\u003cli\u003eKartasheva NN, Lenz-Bauer C, Hartmann O, et al.\u0026Delta;Np73 can modulate the expression of various genes in a p53-independent fashion. Oncogene 2003;22:8246\u0026ndash;8254.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CML, telomerase, hTERT, BIBR1532, p73, p21, Foxo3a","lastPublishedDoi":"10.21203/rs.3.rs-5754607/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5754607/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe targeting of telomerase in cancer therapy elicits significant attention due to recent findings indicating a correlation between high telomerase activity and adverse cancer outcomes, as well as disease resistance. The precise role of telomerase and its catalytic subunit, human telomerase reverse transcriptase (hTERT), in chronic myeloid leukemia (CML) has not been thoroughly elucidated. However, telomerase activity is recognized as a mechanism underlying resistance to imatinib (IM), a tyrosine kinase inhibitor. The loss of telomerase activity in CML has also been associated with acquiring infinite proliferative potential, which is closely linked to maintenance through telomerase reactivation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eK562 cells, along with their IM-resistant derivatives (K562R), were subjected to si-RNA targeting hTERT or a combination treatment involving IM and the hTERT inhibitor BIBR1532. To evaluate the role of hTERT in drug resistance, cell viability following exposure to si-hTERT, IM, and the combination of IM and BIBR1532 was evaluated using the Cell Counting Kit-8 (CCK-8) assay and colony-formation assays. The percentage of apoptosis was quantified using the Annexin V assay. Additionally, the transcriptional and protein expression levels of p73, p21, FOXO3a, c-Myc, hTERT, and other apoptosis-related target genes were analyzed through quantitative polymerase chain reaction (qPCR) and Western blotting (WB).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn K562R and K562 cell lines, telomerase activity was significantly reduced following the pharmacological inhibition of telomerase using the inhibitor BIBR1532, as well as through the knockdown of hTERT. Flow cytometry analysis revealed that the knockdown of hTERT induced apoptosis in chronic myeloid leukemia (CML) cells and results in cell-cycle arrest at the G2 phase. Furthermore, the inhibition of hTERT was found to suppress the transcriptional activity of c-Myc while concurrently reactivating the transcription factors p73, p21, and Foxo3a. The combination of BIBR1532 with imatinib exhibited a synergistic anticancer effect on CML cells.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, the combination of BIBR1532 is a novel therapeutic strategy for CML that may soon be clinically accessible.\u003c/p\u003e","manuscriptTitle":"Inhibition of TERT suppressed proliferation and induced apoptosis in chronic myeloid leukemia cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-06 09:54:40","doi":"10.21203/rs.3.rs-5754607/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"abf754bc-412d-449d-b499-b08dc0b7958b","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-21T07:39:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-06 09:54:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5754607","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5754607","identity":"rs-5754607","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}