Targeting Snail2 improve sensitivity to parthenolide in T-cell acute lymphoblastic leukemia through PUMA activation

Targeting Snail2 improve sensitivity to parthenolide in T-cell acute lymphoblastic leukemia through PUMA activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Targeting Snail2 improve sensitivity to parthenolide in T-cell acute lymphoblastic leukemia through PUMA activation cuiming cao, YAWEI ZHOU, WEICHEN WANG This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5749923/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Objective: Current therapies for childhood T-cell acute lymphoblastic leukemia (T-ALL) have increased survival rates to about 85%. The standard of care is chemotherapy, but approximately 20% patients exhibit primary or secondary resistance to current therapies. Parthenolide (PTL) has been shown to have excellent anti-cancer activity in pediatric leukemia xenografts, with minimal effects on normal hemopoietic cells. Some leukemia initiating cell populations remain resistant to PTL. This study examined mechanisms for this resistance and how to overcome the resistance. Methods: Snail2 protein expression was detected in 9 T-ALL patients, 9 normal bone marrow (NBM) cells and 4 T-ALL cell lines. We investigated the effects of loss of-function and gain-of-function of Snail2 or P53 up-regulated modulator of apoptosis on survival, colony formation, apoptosis and chemosensitivity of T-ALL cells to PTL in vitro and in vivo . Results: Snail2 protein expression was elevated in primary T-ALL patients and cell lines. CEM cells with rich Snail2 expression is resistant to PTL. JURKAT cells with poor Snail2 expression sensitizes to PTL. In vitro, Snail2 inhibition by siRNA, sensitized CEM cells to apoptosis by PTL, resulting in PUMA upregulation and caspase-3 cleavage. Inhibition of PUMA by RNA interference in Snail2-knockdown CEM cells rescued the resistance of CEM cells to PTL treatment. Snail2 re-expression in JURKAT cells is resistant to PTL. The pro-survival effect of Snail2 was found to be caused by direct repression of PTL-induced PUMA expression by Snail2 in JURKAT cells. Additionally, an orthotopic allograft in vivo model demonstrated that the Snail2 inhibitor enhanced responses to PTL in CEM cells by inducing PUMA-dependent cell apoptosis. Conclusion: This study demonstrates a pivotal role for Snail2/PUMA signals in T-ALL cell survival. It may be possible to achieve greater toxicity to childhood T-ALL by combining PTL with Snail2 knockdown. Parthenolide acute lymphoblastic leukemia Snail2 P53 up-regulated modulator of apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T-cell progenitors representing about 15% of pediatric and 25% of adult acute lymphoblastic leukemia cases [1]. Despite significant advances in treatment, approximately one out of five patients exhibit primary or secondary resistance to current therapies [2], which include glucocorticoids as a key component; indeed, the overall clinical outcome depends on the initial response to glucocorticoids [3]. Consequently, understanding the pathological mechanisms that lead to the appearance of this malignancy and developing novel and more effective targeted therapies is an urgent need. Biologically T-ALL is hallmarked by genomic/genetic lesions impacting on a number of targetable pathways, comprising Notch, JAK/STAT, PI3K/Akt/mTOR, and MAPK signaling pathway [4]. As more than 60% of T-ALL patients harbor activating mutations of NOTCH1[5], therapeutic strategies aimed at blocking Notch signaling have been proposed. Snail2 (Slug) is a transcriptional repressor, which promotes EMT, a process that accompanies cancer cell invasion and metastasis [6]. Snail2 upregulation was observed in high-grade invasive duct carcinomas, which exhibit high rate of recurrence, metastasis, and drug resistance [7]. Snail2 was also found to protect tumor cells from apoptosis induced by radiation, which is reminiscent of the protective effect of Snail2 against DNA damage observed in hematopoietic progenitor cells by repressing P53 up-regulated modulator of apoptosis (PUMA)[8]. Shao et al. has reported that Notch signaling positively regulates the EMT, invasion, and growth of breast cancer cells by inducing Snail2 expression and inhibits PUMA expression [9,10]. Hence disrupting the Snail2-Puma axis in cancer cells may impinge on the survival of metastatic cells. Belle et al has reported that repression of PUMA expression is essential for multipotent progenitor (MPP) survival, and partly contributes to maintaining hematopoietic stem cell (HSC) function [11]. Guirguis et al. reported that the absence of PUMA reduced apoptosis and expanded the numbers of myelodysplastic syndrome (MDS) repopulating cells without a detrimental effect on leukemic progression [12]. In lymphoid malignancies, the dual mTORC1/mTORC2 inhibition OSI-027 induces transactivation of the PUMA, leading to increase of cell apoptosis. Targeting PUMA protected cells from OSI-027 cytotoxicity [13].Therefore, activation of PUMA may be an effective methods for the T-ALL treatment, Parthenolide (PTL) is a sesquiterpene lactone found in feverfew, which is currently considered to be responsible for the herb’s therapeutical potential.The patent application for tumor suppression was approved in 2005 [14]. Additionally, the in vitro and in vivo antitumor potential of PTL in multiple cancer types has been confirmed by numerous researches, which mainly resulted from its cytotoxicity to the bulk population of cancer cells as well as from selectively targeting cancer stem cells (CSCs); it is a subpopulation currently believed to be responsible for chemotherapy resistance and tumor relapse [15-17]. The mechanisms of PTL-induced anticancer activity are not entirely clear. Parthenolide (PN) has been shown to inhibit NF-κB signaling [18], NOTCH1 signaling [5], PI3K/Akt signaling [19] and other pro-survival signaling pathways, induce apoptosis and reduce a subpopulation of cancer stem-like cells [20,21]. Multimodal therapies that include PTL or its derivatives seem to be promising approaches enhancing sensitivity of cancer cells to therapy and diminishing development of resistance [22-25]. Recent research has found that PTL may have therapeutic potential in childhood ALL by eradicating all LIC populations to prevent disease progression and reduce relapse [26]. Benjamin C et al. reported that mesenchymal stem cells released thiols and protected leukemia cells from reactive oxygen species stress, which is associated with parthenolide cytotoxicity. Blocking cystine uptake by mesenchymal stem cell prevented thiol release and significantly reduced leukemia cell resistance to PTL, suggesting that PTL may be possible to achieve greater toxicity to childhood T-cell acute lymphoblastic leukemia [27]. However, the mechanism of cytotoxicity in PTL in T-ALL cells has not been previously investigated. In this study, we define a novel molecular mechanism under-lying PTL blocks Snail2 expression and induced Snail2-dependent PUMA activation, leading to cellular apoptosis and growth inhibition in T-ALL cells. Targeting Snail2 upregulated PUMA expression and increased PTL sensitivity in T-ALL cells. Materials and Methods Patients From 2022.6 to 2023.8, hospitalized children diagnosed with T-ALL based on Chinese Children’s Leukemia Group-ALL 2008 (CCLG-ALL 2008) protocol at The central hospital of Jinan, Shandong. China were included in this study. The diagnosis of T-ALL was based on evaluation of bone marrow smears according to morphologic and cytochemical criteria of French-American-British (FAB) and immunophenotypic criteria. This study protocol was approved by the Ethical Review Board of Investigation in Human Beings of Jinan central hospital. Informed consents were obtained orally from the patients or guardians of each patient. Cell line and culture CCRF-CEM and Jurkat cells were obtained from ATCC (Shanghai, China). They were all cultured in RPMI 1640 (Invitrogen, Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C under 5% CO2. Small-interfering RNAs (siRNAs) and in vitro siRNA transfection The small-interfering RNAs (siRNAs) and Silencer negative control (si-control) were purchased from Invitrogen (Shanghai, China). The siRNA-targeting Snail2 (si-Snail2) or siRNA-targeting PUMA (si-PUMA) and si-control were transfected into cells with lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions. After 24h and 48 h of transfection with the siRNAs (0.1 nmol/2 × 10 5 cells), the cells were harvested for further experiments. Snail2 Plasmids and transfection The cell expression construct (pcDNA3.1- human Snail2, pSnail2) and pcDNA3.1 were obtained from Invitrogen (Shanghai, China). 1.5 × 10 5 cells plated in six-well plates were transiently transfected with a pSnail2 expression vector or pcDNA3. 24 hours after plating, transfection was performed as recommended by the manufacturer by adding, in each well, a mixture containing 200 μl of 150 mmol/L NaCl, 9 μl of Exgene and 2 μg of the Snail2 expression vector. As a control, cells were transfected with the corresponding empty vector pcDNA3. At 24, 48 hours after transfection, cells were collected for Western blotting analyses. All experiments were set up to obtain at least 60% of transfected cells. For stable transfected cells, the infected cells were allowed to recover for 48 hs and were cultured for 14 days with puromycin-containing medium (1 µg/mL). Short hairpin RNA (shRNA) and lentiviral infection Lentiviral transduction particles carrying short hairpin RNA (shRNA) sequence against human Snail2 and control non-target sequence (Sigma-Aldrich; NM_003068/TRCN0000284362 and SHC002V) were used to knockdown Snail2 expression in CEM cells according to the manufacturer’s protocol. Briefly, cells were incubated with lentiviral particles in the presence of hexadimethrine bromide (8 µg/mL) for 36 hs. Infected cells were allowed to recover for 48 hs and were cultured for 14 days with puromycin-containing medium (1 µg/mL). The stable knockdown cells were identified by Western blotting using anti-Snail2 antibody (Cell Signaling Technology, Beverly, MA; 9585) and were cultured in puromycin-free RPMI 1640 medium. Determination of apoptosis and clonogenic survival T-ALL cells (10 6 /ml) were treated with PTL (5μM) in RPMI 1640 complete medium for 48 h. After drug treatment, apoptosis was analyzed by annexin V staining and flow cytometry using the FACSCalibur cytometer (BD Biosciences). For clonogenic survival assays, the cells were cultured on plastic or on Matrigel for 4 h and then treated with PTL for 24 h. The cells were recovered, washed and then seeded at 1 × 10 4 cells/ml in complete medium with 1% methylcellulose (StemCell Technologies, Vancouver, BC). After 14 days, colonies with >50 cells were counted. Immunofluorescence Observations Immunofluorescence observations were performed to investigate cleaved caspase-3 expression in the T-ALL cell according to the manufacturer's instructions, and analyzed using the FACSCalibur cytometer (BD Biosciences). Briefly, cells stained with indicated anti-cleaved caspase-3 antibodies were resuspended in phosphate-buffered saline. The images were captured by a Zeiss LSM 710 Confocal Microscope (Carl Zeiss, Oberkochen, Germany). Growth inhibition assay Cells were cultured in 96-well plates at a density of 5000 cells/well and left to recover. The quantity of viable cells was estimated by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cells were treated with PTL (5 µg). After 48 h incubation at 37 °C, 20 µl of MTT solution (5 mg/ml in PBS) (Sigma, USA, A101161) was added into each well and cells were incubated at 37 °C for 4 hours. After adding DMSO to wells to dissolve the formazan crystals, plates were read using a plate reader at 570 nm against 630 nm. The experiment was performed three times. The percentage of viable cells was determined by comparison to untreated control cells. Western blot analysis Cell lysates were prepared and separated on 10% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes. Membranes were blotted with the primary antibodies and developed after secondary antibody incubation using the ECL Kit (Amersham International, Amersham, UK) according to the manufacturer's protocols. The primary antibodies were below：anti-Snail2, anti-PUMA, anti-Bim, anti-Noxa, anti-cleaved caspase-3 and a-Turbulin. In vivo experiments To evaluate the in vivo proliferative effect of Snail2, the CEM/sh-control and CEM/sh-Snail2 cells (1 × 10 6 cells/mouse) were injected subcutaneously into the flanks of the nude mice. When tumors reached a mean volume of 50-100 mm 3 , mice were treated with 30mg/kg/per 3 days parthenolide [28] or with equivalent volume of 10% DMSO through intraperitoneal injections for 21 days. Mice were weighed weekly and tumors were measured every 72 hours. At the conclusion of the experiment, mice were euthanized, and tumors were excised, measured, and then fixed in 10% formalin for IHC staining. For western blotting, unfixed tumors were homogenized with a tissue tearer and cell lysates were assessed. The study protocol was approved by Medicine Institutional Animal Care & Use Committee, the Jinan Central Hospital, Jinan, Shandong, China. TUNEL assay Formalin-fixed-embedded subcutaneous tumor samples from nude mice were first cut into 5-μm-thick sections. TUNEL was used to detected cell apoptosis as the manufactured methods. Statistical Analyses. All calculations and statistical analyses were carried out using SPSS25 software The data are expressed as mean ± standard deviation of n = 3 determinations. In short-term in vivo and in vitro assays, we used the analysis of variance one-way ANOVA with Bonferroni’s multiple comparison test or the Student t test. We considered results with P < 0.05 as statistically significant. Results Snail2 is highly expressed in T-cell acute lymphoblastic leukemiain (T-ALL) To determine the expression of Snail2 in T-ALL, we first analyzed Snail2 expression by Western blot in 9 independent T-ALL patient cohorts and 9 normal bone marrow (NBM) cells. The results showed that Snail2 protein expression was elevated in primary T-ALL as compared to normal bone marrow (BM) cells (Figure 1A). To further correlate the level of Snail2 protein expression, we next detects the Snail2 protein expression in T-ALL cell lines (JURKAT, CCRF-CEM, MOLT-3, and MOLT-4), also find enhanced Snail2 expression was in these cells. JURKAT has lowest Snail2 expression and CCRF-CEM(CEM) has highest Snail2 expression (Figure 1B), so we used JURKAT and CEM cells for further study. Targeting Snail2 inhibits the growth and improves apoptosis of CEM cells in vitro CEM and JURKAT cells were transfected with Snail2 siRNA for 48 h. Snail2 protein was inhibited in both cells by western blot assay (Figure 1A). No change was found in control siRNA transfected cells (Figure 2A). The growth inhibitory effect of Snail2 siRNA or control siRNA transfected CEM and JURKAT was evaluated by the MTT assay in both cells. Snail2 siRNA had a significant growth inhibitory effect on CEM cells, but not for JURKAT cells (Figure 2B). Snail2 siRNA also increased the Annexin V-FITC stained apoptotic population of CEM cells compared to control (Figure 2C). No Annexin V-FITC stained apoptotic population of Snail2 siRNA transfected JURKAT cells was found compared to controls (Figure 2C). The mismatched control siRNA did not inhibit cell viability and apoptosis. Immunofluorescence analysis of cleaved Caspase-3 in si-Snail2 transfected CEM cells manifested robust apoptosis upon si-control transfected CEM cells (Figure 2D). No significant observsion was fond in JURKAT cells (data not show). By colony formation assay, targeting snail2 in Snail shRNA transfected CEM cells inhibited colony formation, but not in Snail shRNA transfected JURKAT cells (Figure 2E). This results indicated that targeting Snail2 inhibits growth and improves apoptosis in Snail2-overexpressed T-ALL cells. Targeting Snail2 enhances parthenolide (PTL) cytotoxicity in CEM cells in vitro We first examined the cytotoxic activity of PTL, as single agents and in combination with Snail2 siRNA in CEM and JURKAT cells. Both of the cells were transfected with Snail2 siRNA, control siRNA or without siRNA or incubated with PTL (5 μM) for 48 h, harvested, and then cell viability analyzed by an MTT assay. As shown in Figure 3A, PTL (5 μM) treatment alone for 48 h, cell viability was decreased in both of the cells, but significant difference was shown in JURKAT cells. However, the combination of PTL (5 μM) and Snail2 siRNA significantly decreased the cell viability in CEM cells compared to PTL alone. The combination of Snail2 siRNA with PTL did not significantly reduce JURKAT cell viability compared to PTL alone (Figure 3A). By colony formation assay, PTL (10 μM) treatment alone significantly inhibited colony formation in JURKAT cells but not CEM cells (Figure 3B). However, the combination of PTL (10 μM) and Snail2 shRNA significantly inhibited colony formation in CEM cells, but not in JURKAT cells compared to PTL alone (Figure 3B). PTL (5 μM) treatment alone increased the Annexin V-FITC stained apoptotic population of JURKAT cells, but not CEM cells (Figure 3B). However, the combination of PTL (5 μM) and Snail2 siRNA significantly increased the Annexin V-FITC stained apoptotic population of CEM cells, but not JURKAT cells compared to PTL alone (Figure 3C). Immunofluorescence analysis of cleaved Caspase-3 in PTL alone treated JURKAT cells, the PTL (5 μM) in combination with Snail2 siRNA treated CEM cells manifested robust apoptosis (Figure 3D). No significant observation was fond in JURKAT cells (data not show). Enhanced Snail2 decreases PTL cytotoxicity in JURKAT cells in vitro By transiently transfecting pcDNA3.1-Snail2, we established a Snail2-overexpressed Jurkat and CEM cell clone (Jurkat-Snail2, CEM-Snail2), and empty vector transfected cell (Jurkat-Emp, CEM-Emp) was used as control. Snail2 was overexpressed in Jurkat-Snail2 cells compared to Jurkat-Emp cells. Rich Snail2 protein expression was also detected in CEM-Snail2 cells, but no significant increase was found compared to the CEM-Emp cells or untreated CEM cells (Fig. 4A). In the Jurkat cells, Snail2 overexpression inhibits PTL (5 μM)-induced cell apoptosis (Fig. 4B) and reversed PTL-induced cytotoxicity by MTT assay (Fig. 4C). In Snail2-overexpressed CEM cells, following PTL (5 μM) treatment for 48 h, Snail2 overexpression did not affect cell apoptosis or cell viability induced by PTL treatment (Fig.4B-4C). Immunofluorescence analysis showed that enhanced Snail2 expression inhibits PTL-induced cleaved Caspase-3 activation in JURKAT cells (data not shown). Colony formation assay has the similar results as MTT (data not shown). It was most likely that CEM cell expressed Snail2 at high levels and elevated endogenous Snail2 levels may not be sufficient to promote CEM cell growth and inhibits apoptosis. Targeting Snail2 enhances PTL-induced apoptosis through inducing PUMA expression We examined the effect of Snail2 knockdown on PTL-induced PUMA expression in the CEM cells. Moderate PUMA expression was observed in the untreated CEM cells (Fig. 5A). si-Snail transfection blocked Snail2 expression, but improved PUMA expression (Fig. 5A). PTL induced less PUMA expression, but combined PTL and si-Snail2 treatment significantly promoted PUMA expression (Fig. 5A). The shRNA Snail2 transfected CEM cells were transiently transfected with PUMA siRNA, then treated with PTL (5 μM) for 48 h, and cell viability was analyzed by an MTT. As shown in Figure 5B, targeting PUMA reversed Snail2 shRNA/PTL induced cell growth inhibition. PUMA siRNA transfection also reversed shRNA Snail2/PTL induced cell apoptosis (Figure 5C). Immunofluorescence analysis of cleaved Caspase-3 indicated cleaved Caspase-3 was significantly reduced in the PUMA siRNA transfected CEM cells (Figure 5C). In the JURKAT cells, PTL (5 μM) treatment alone for 48 h improved PUMA protein expression, but reversed PUMA expression in the pcDNA3-Snail2 transfected cells following PTL (5 μM) treatment for 48 h (Figure 5D). MTT assay showed that pcDNA3-Snail2 transfection reversed PTL-induced cell growth inhibition (Fig. 4B). pcDNA3-Snail2 transfection also reversed PTL induced cell apoptosis (Figure 4C). Immunofluorescence analysis of cleaved Caspase-3 indicated cleaved Caspase-3 was significantly reduced in the pcDNA3-Snail2 transfected JURKAT cells (Figure 5E).Thus, repression of PUMA by Snail2 in JURKAT cells contributes to survival by reducing apoptosis. We also detected the levels of the p53 target gene Noxa, the non-p53 regulated Bim gene, or the anti-apoptotic Bcl-2 and Bcl-xL proteins in the CEM and JURKAT cells. The results showed that they were not affected by Snail2 siRNA and pSnail2 (data not shown). Thus, Snail2 protects T-ALL cells from apoptosis triggered by PTL. Effective therapeutic combination of PTL with the Snail2 inhibitor in vivo We assessed whether the Snail2 silencing was effective on PTL induced tumor inhibition in vivo. Nude mice bearing CEM/shRNA-Snails tumors were injected i.p. with PTL as the methods reported. The result shows that Snail2 shRNA plus PTL exhibited significant antitumor activity in the CEM xenograft mode compared to the effects of PTL or sh-control/PTL applied. As shown in Fig. 6A, the tumor size of mice was significantly reduced in the presence of SNAIL2 shRNA. Then, all mice in the groups were killed, and the tumor tissues were removed for TUNEL and Western blot assay. Compared to the vehicle group or the single treatment group with PTL, the combined administration of the PTL and Snail2 shRNA transfected tumor displayed increased apoptosis (Fig. 6B) and PUMA expression (Fig. 6C). Discussion Understanding the mechanisms underlying cancer chemoresistance is likely to lead to more effective therapies and to a better control of patient relapse. In this study, we report that blockade of the Snail2 improves the sensitivity of PTL to T-ALL by activating PUMA signals. Our results showed that Snail2 is more commonly present in the T-ALL patients and T-ALL cell lines. We did not, however, observe increased Snail2 expression in non-leukemic bone marrow, suggesting that enhanced Snail2 expression might be related to the occurrence or biological behavior of T-ALL. To date, PTL is the only drug that has been shown to be capable of completely eradicating childhood ALL in NSG xenografts, as a single agent [26]. Most studies, using such models, report reduction in leukemia burden but levels often increase on cessation of treatment. A recent in vitro study reported [27] that PTL treatment only reduced viability of T-ALL cells to less than 30%, suggesting some T-ALL cells may be resistant to PTL. In our study, PTL treatment reduced viability of poor-Snail2-expressed JURKAT cells to 35% which was similar to the report. In the rich-Snail2-expressed CEM cells, PTL treatment reduced viability of T-ALL cells to less than 10%. To confirm the effect of Snail2 on cell survival, we re-expressed Snail2 in the JURKAT cells and Snail2-knockdown CEM cells. It is notable that Snail2 re-expression reversed JURKAT cell resistance to PTL and Snail2 knowdown significantly reduced CEM cell resistance to PTL. We conducted clonogenic survival assays to evaluate long-term survival, which was consistent with the MTT results above. This is the first report demonstrating Snail2 provide a protective effect to T-ALL cells against PTL. Moreover, we have shown that targeting Snail2 can overcome this effect. Programmed cell death or apoptosis of white blood cells is a common feature, and is thought to contribute to the cytopenias particularly in early stages of the disease [3,4]. Since Snail2 attenuation sensitized cells to apoptosis, we determined whether it affected the expression of the pro-apoptotic gene Puma, which was shown to be repressed by Snail2 [8]. Targeting Snail2 in CEM cells increased PTL-induced apoptosis. Knockdown of Snail2 by siRNA in CEM cells induced caspase-3 cleavage and expression of known Snail2-repressed pro-apoptotic protein PUMA, indicating a Snail2 role in anti-apoptosis signaling. To verify whether PUMA contributes to apoptosis in Snail2-knockdown CEM cells, PUMA was silenced in Snail2-sh CEM cells using a pool of PUMA siRNAs. Treatment with PTL reduced the number of active-caspase-3 positive cells by 80% in PUMA-siRNA treated Snail2-sh CEM cells relative to control cells, followed by reduced cell apoptosis. In vivo, CEM tumors from Snail2-sh cells also displayed higher PUMA levels. In the JURKAT cells, PTL treatment induced cell apoptosis, followed by PUMA expression and caspase-3 cleavage. However, Snail2 re-expression repressed PTL-induced PUMA expression and caspase-3 cleavage. These findings suggest that Snail2 contributes to cell survival by inhibiting PUMA signals. Whether PTL directly blocked Snail2 or directly activating PUMA was beyond the scope of this study. Otherwise, whether PTL activated p53- dependent PUMA or p53- independent PUMA need further investigate. However, it is evident that Snail2 levels decreased and PUMA levels increased, putting the cells under higher levels of caspase-3 cleavage, which is likely to drive PTL- induced apoptosis. Furthermore, Snail2 levels increased and PUMA levels dropped, putting the cells under lower levels of caspase-3 cleavage, which is likely to inhibit PTL-induced apoptosis. In support of a role for Snail2 in suppressing PTL-induced apoptosis, Snail2 was found to protect tumor cells from apoptosis induced by radiation, which is reminiscent of the protective effect of Snail2 against DNA damage observed in hematopoietic progenitor cells [8,29]. Our results showed that Snail2 targets PUMA, and no other pro-apoptotic gene, including the p53-response gene, Noxa, or the non-p53 target gene Bim to suppress apoptosis. The involvement of Puma in apoptosis is underscored by that Puma siRNA lowered the threshold for induction of apoptosis by PTL. Importantly, the Snail2-PUMA axis was found to be similar in vivo to in vitro as shown by that PTL in Snail2-knockdown CEM cells suppressed tumor growth by inducing cell apoptosis. In sum, our study points to a pivotal function for Snail2 in survival, which allows tumor cells to overcome apoptosis and survive, achieving by repression of the pro-apoptotic gene PUMA. PTL could inhibit Snail2 and upregulate PUMA, and increase the cytotoxicity of PTL to T-ALL. Hence PTL could be a novel promising approach to treat T-ALL in the future. Declarations Conflicts of Interest : The authors declare no conflict of interest Authors’ contributions Zhou conceptualised and designed the study. Zhou and Cao performed the In vitro experiments. Wang and Cao performed the in Vivo experiments. Cao analyzed the data. Zhou edited the manuscript. 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Parthenolide attenuates 7,12-dimethylbenz[a]anthracene induced hamster buccal pouch carcinogenesis. Mol Cell Biochem. 2018;440(1-2):11-22. Diamanti P, Cox CV, Moppett JP, Blair A. Parthenolide eliminates leukemia-initiating cell populations and improves survival in xenografts of childhood acute lymphoblastic leukemia. Blood. 2013;121(8):1384-93. Ede BC, Asmaro RR, Moppett JP, Diamanti P, Blair A. Investigating chemoresistance to improve sensitivity of childhood T-cell acute lymphoblastic leukemia to parthenolide. Haematologica. 2018;103(9):1493-1501. Provance OK, Geanes ES, Lui AJ, Roy A, Holloran SM, Gunewardena S, Hagan CR, Weir S, Lewis-Wambi J. Disrupting interferon-alpha and NF-kappaB crosstalk suppresses IFITM1 expression attenuating triple-negative breast cancer progression. Cancer Lett. 2021;514:12-29. Perez-Losada J, Sanchez-Martin M, Perez-Caro M, Perez-Mancera PA, Sanchez-Garcia I. The radioresistance biological function of the SCF/kit signaling pathway is mediated by the zinc-finger transcription factor Slug. Oncogene. 2003;22:4205–11 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5749923","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":396768264,"identity":"398fb1ec-f73d-4ae9-9c8a-f0dd37f2c11d","order_by":0,"name":"cuiming cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYNACAwYeIJnAwNjAwMDPzHzwAWlaJNvZkg2Itw2kxeA8j5kAPkX87YcPfuYp2CZjzr/g2YOfO2zsjQ8zmDEw1NhE49IicSYtWZrH4DaP5YwH6Ya9Z9IStx1mSHvAcCwttwGnL3LMmEFaDG4cSJNmbDucYHaY4bgBY8Nh3Fr436BqsTduZmyTwKtFAmbL+QawFsYNzMxseLVI3HiWLDkHbAtDmmRvW1rijMNszAYJePzC35988MObP7ftDc6fSZP42WZjz99//uODDzU2OLUg2ZeTgOAk4FKFat/xA0SpGwWjYBSMgpEHALm3WPz+eCLzAAAAAElFTkSuQmCC","orcid":"","institution":"Jinan Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"cuiming","middleName":"","lastName":"cao","suffix":""},{"id":396768265,"identity":"65a679db-fc46-4c69-ab67-9e0fa0a9e4f5","order_by":1,"name":"YAWEI ZHOU","email":"","orcid":"","institution":"Jinan Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"YAWEI","middleName":"","lastName":"ZHOU","suffix":""},{"id":396768266,"identity":"177b59bf-4a5e-4796-bbf4-e5947dda78ae","order_by":2,"name":"WEICHEN WANG","email":"","orcid":"","institution":"Jinan Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"WEICHEN","middleName":"","lastName":"WANG","suffix":""}],"badges":[],"createdAt":"2025-01-02 08:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5749923/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5749923/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73245766,"identity":"386b79d4-611f-476c-bee9-839aa990d663","added_by":"auto","created_at":"2025-01-08 07:06:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh Snail2 expression in T-cell acute lymphoblastic leukemia (T-ALL).\u003c/strong\u003eA. Snail2 expression was analyzed in nine primary T-ALL and nine normal bone marrow (NBM) by western blot assay. B, Snail2 expression was analyzed in T-ALL cell lines (JURKAT, CCRF-CEM, MOLT-3, and MOLT-4) by western blot assay.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/89c14403382c55a34c48bf77.jpg"},{"id":73245296,"identity":"61719699-034b-4e6a-b588-ac25fdb29d52","added_by":"auto","created_at":"2025-01-08 06:58:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":507762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting Snail2 inhibits cell survival and induces cell apoptosis\u003c/strong\u003e. A, CEM and JURKAT cells were transfected with Snail2 siRNA or si-control for 48 h. Snail2 protein was inhibited in both cells by western blot assay. B, CEM and JURKAT cells were transfected with Snail2 siRNA or si-control for 24, 48 and 72 h, cell viability was detected by MTT assay. C, CEM and JURKAT cells were transfected with Snail2 siRNA for 24, 48 48 h. Cell apoptotic was detected by Flow cytometry analysis. Mean values and standard deviations (error bars) of Annexin V-FITC positive cells are shown for triplicate samples assayed over two separate experiments. D, Immunofluorescence images of cleaved Caspase-3 (c-caspase 3, red) and DAPI (blue) in CEM cells undergoing DMSO, si-control or si-Snail2 transfection for 48 h. Scale bar, 50 μm. (Right) Quantifications of fluorescence signals. E, Colony formation assay for CEM and JURKAT knockdown cells. Asterisks indicate significant difference from the non-targeting shRNA control (P \u0026lt; 0.05). Data are presented as mean + standard deviation.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/596ca29a54598f7c1dc5a4d4.jpg"},{"id":73245300,"identity":"c2f52994-78e1-4d0e-9a0a-1a4f5c29d0bf","added_by":"auto","created_at":"2025-01-08 06:58:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":516335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting Snail2 improves T-cell acute lymphoblastic leukemia (T-ALL) cells from parthenolide (PTL)-induced cell death.\u003c/strong\u003e A, CEM and JURKAT cells were transfected with Snail2 siRNA or si-control and then treated with PTL(5 μM) for 48 h. The cell viability was detected by MTT assay. B, Colony formation assay for CEM and JURKAT knockdown or/and in combination with PTL treatment cells. Asterisks indicate significant difference from the non-targeting shRNA control (P \u0026lt; 0.05). Data are presented as mean + standard deviation.C, The Snail2 shRNA transfected CEM and JURKAT cells were treated with PTL for 48 h. Cell apoptotic was detected by Flow cytometry analysis. Mean values and standard deviations (error bars) of Annexin V-FITC positive cells are shown for triplicate samples assayed over two separate experiments. D, Immunofluorescence images of cleaved Caspase-3 (c-caspase 3, red) and DAPI (blue) in both cells undergoing DMSO, si-control or si-Snail2 transfection in combination with PTL treatment for 48 h. Scale bar, 50 μm. Quantifications of fluorescence signals.\u003c/p\u003e","description":"","filename":"fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/6440f7b1fa03c11273f64253.jpg"},{"id":73245298,"identity":"708b1637-1118-4178-a910-b68769446de4","added_by":"auto","created_at":"2025-01-08 06:58:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSnail2 protect T-cell acute lymphoblastic leukemia (T-ALL) cells from parthenolide (PTL)-induced cell death.\u003c/strong\u003e A, CEM and JURKAT cells were transfected with pcDNA3-Snail2 or pcDNA3 for 48 h. Snail2 protein expression was detected by western blot assay. B, CEM and JURKAT cells were transfected with pcDNA3-Snail2 siRNA or pcDNA3 for 24 h, then treated with PTL (5 μM) for 48 h. Cell apoptotic was detected by Flow cytometry analysis. C, B, CEM and JURKAT cells were transfected with pcDNA3-Snail2 siRNA or pcDNA3 for 24 h, then treated with PTL (5 μM) for 48 h. The cell viability was detected by MTT assay.*P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"FIG.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/6ccb10b8645fd4c25d851b5b.jpg"},{"id":73245301,"identity":"67bc148d-e542-4e8e-8e9f-5077aedee583","added_by":"auto","created_at":"2025-01-08 06:58:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":284774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSnail2 inhibition overcomes protective effects to PTL in T-ALL cells and increased PUMA expression. \u003c/strong\u003eA, CEM cells were transfected with Snail2 siRNA or si-control for 24 h, or co-transfection with PUMA siRNA or si-control, then treated with PTL for 48 h. Snail2 and PUMA protein expression was detected by western blot assay. B, the stable Snail2 siRNA transfected CEM cells were transiently transfected with PUMA si RNA or si-control 24 h, then treated with PTL (5 μM) for 48 h. The cell viability was detected by MTT assay.*P\u0026lt;0.05.C, Immunofluorescence images of cleaved Caspase-3 (c-caspase 3, red) and DAPI (blue) in CEM cells undergoing PTL treatment in Snail2 siRNA/PUMA siRNA (control siRNA) transfected CEM cells. Scale bar, 50 μm. Quantifications of fluorescence signals. D, JURKAT cells were transfected with pcDNA3-Snail2 or pcDNA3 for 24 h, then treated with PTL for 48 h. Snail2 and PUMA protein expression was detected by western blot assay. E, Immunofluorescence images of cleaved Caspase-3 (c-caspase 3, red) and DAPI (blue) in JURKAT cells undergoing PTL treatment in pcDNA3-Snail2 (pcDNA3) transfected JURKAT cells. Scale bar, 50 μm. Quantifications of fluorescence signals.\u003c/p\u003e","description":"","filename":"FIG.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/541a81e12339dddd0e875d08.jpg"},{"id":73245767,"identity":"2531f6f0-0e5a-4efb-9991-81b2048ad3a8","added_by":"auto","created_at":"2025-01-08 07:06:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":274119,"visible":true,"origin":"","legend":"\u003cp\u003ePTL inhibited the growth of CEM xenografts. A total of 12 nude mice were inoculated with shRNA Snail2 or shRNA control CEM cells (N=6 per groups). A total of 12 nude mice were inoculated with CEM cells. When tumors reached a homogeneous size (50–10 mm3), the mice were treated with PTL for 21 days. A, Tumor growth results after 21 days of treatment with PTL, shRNA Snail2, shRNA and the combination of PTL and shRNA. Day 0 indicates the day animals received the treatments. All values indicate mean values ± SEM. **p \u0026lt; 0.01, n =6. B, Immunohistochemistry staining for PUMA in tissue isolated from mice following 24 days of treatment. Brown indicates positive signal within tissue. * P\u0026lt;0.05, student’s t test.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/cec318db07955083b01d7e0d.jpg"},{"id":73247319,"identity":"0f5a9a19-49ee-4377-8934-1cd642610621","added_by":"auto","created_at":"2025-01-08 07:14:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2312732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5749923/v1/2305c782-014d-4dcf-bd73-4b2c40c59635.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting Snail2 improve sensitivity to parthenolide in T-cell acute lymphoblastic leukemia through PUMA activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T-cell progenitors representing about 15% of pediatric and 25% of adult acute lymphoblastic leukemia cases [1]. Despite significant advances in treatment, approximately one out of five patients exhibit primary or secondary resistance to current therapies [2], which include glucocorticoids as a key component; indeed, the overall clinical outcome depends on the initial response to glucocorticoids [3]. Consequently, understanding the pathological mechanisms that lead to the appearance of this malignancy and developing novel and more effective targeted therapies is an urgent need.\u003c/p\u003e\n\u003cp\u003eBiologically T-ALL is hallmarked by genomic/genetic lesions impacting on a number of targetable pathways, comprising Notch, JAK/STAT, PI3K/Akt/mTOR, and MAPK signaling pathway [4]. As more than 60% of T-ALL patients harbor activating mutations of NOTCH1[5], therapeutic strategies aimed at blocking Notch signaling have been proposed. Snail2 (Slug) is a transcriptional repressor, which promotes EMT, a process that accompanies cancer cell invasion and metastasis [6]. Snail2 upregulation was observed in high-grade invasive duct carcinomas, which exhibit high rate of recurrence, metastasis, and drug resistance [7]. Snail2 was also found to protect tumor cells from apoptosis induced by radiation, which is reminiscent of the protective effect of Snail2 against DNA damage observed in hematopoietic progenitor cells by repressing P53 up-regulated modulator of apoptosis (PUMA)[8]. Shao et al. has reported that Notch signaling positively regulates the EMT, invasion, and growth of breast cancer cells by inducing Snail2 expression and inhibits PUMA expression [9,10]. Hence disrupting the Snail2-Puma axis in cancer cells may impinge on the survival of metastatic cells. Belle et al has reported that repression of PUMA expression is essential for multipotent progenitor (MPP) survival, and partly contributes to maintaining hematopoietic stem cell (HSC) function [11]. Guirguis et al. reported that the absence of PUMA reduced apoptosis and expanded the numbers of myelodysplastic syndrome (MDS) repopulating cells without a detrimental effect on leukemic progression [12]. In lymphoid malignancies, the dual mTORC1/mTORC2 inhibition OSI-027 induces transactivation of the PUMA, leading to increase of cell apoptosis. Targeting PUMA protected cells from OSI-027 cytotoxicity [13].Therefore, activation of PUMA may be an effective methods for the T-ALL treatment,\u003c/p\u003e\n\u003cp\u003eParthenolide (PTL) is a sesquiterpene lactone found in feverfew, which is currently considered to be responsible for the herb\u0026rsquo;s therapeutical potential.The patent application for tumor suppression was approved in 2005 [14]. Additionally, the in vitro and in vivo antitumor potential of PTL in multiple cancer types has been confirmed by numerous researches, which mainly resulted from its cytotoxicity to the bulk population of cancer cells as well as from selectively targeting cancer stem cells (CSCs); it is a subpopulation currently believed to be responsible for chemotherapy resistance and tumor relapse [15-17]. The mechanisms of PTL-induced anticancer activity are not entirely clear. Parthenolide (PN) has been shown to inhibit NF-\u0026kappa;B signaling [18], NOTCH1 signaling [5], PI3K/Akt signaling [19] and other pro-survival signaling pathways, induce apoptosis and reduce a subpopulation of cancer stem-like cells [20,21]. Multimodal therapies that include PTL or its derivatives seem to be promising approaches enhancing sensitivity of cancer cells to therapy and diminishing development of resistance [22-25]. Recent research has found that PTL may have therapeutic potential in childhood ALL by eradicating all LIC populations to prevent disease progression and reduce relapse [26]. Benjamin C et al. reported that mesenchymal stem cells released thiols and protected leukemia cells from reactive oxygen species stress, which is associated with parthenolide cytotoxicity. Blocking cystine uptake by mesenchymal stem cell prevented thiol release and significantly reduced leukemia cell resistance to PTL, suggesting that PTL may be possible to achieve greater toxicity to childhood T-cell acute lymphoblastic leukemia [27]. However, the mechanism of cytotoxicity in PTL in T-ALL cells has not been previously investigated. In this study, we define a novel molecular mechanism under-lying PTL blocks Snail2 expression and induced Snail2-dependent PUMA activation, leading to cellular apoptosis and growth inhibition in T-ALL cells. Targeting Snail2 upregulated PUMA expression and increased PTL sensitivity in T-ALL cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom 2022.6 to 2023.8, hospitalized children diagnosed with T-ALL based on Chinese Children\u0026rsquo;s Leukemia Group-ALL 2008 (CCLG-ALL 2008) protocol at The central hospital of Jinan, Shandong. China were included in this study. The diagnosis of T-ALL was based on evaluation of bone marrow smears according to morphologic and cytochemical criteria of French-American-British (FAB) and immunophenotypic criteria. This study protocol was approved by the Ethical Review Board of Investigation in Human Beings of Jinan central hospital. Informed consents were obtained orally from the patients or guardians of each patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell line and culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCCRF-CEM and Jurkat cells were obtained from ATCC (Shanghai, China). They were all cultured in RPMI 1640 (Invitrogen, Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100\u0026thinsp;U/ml), and streptomycin (100\u0026thinsp;\u0026mu;g/ml) at 37\u0026thinsp;\u0026deg;C under 5% CO2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall-interfering RNAs (siRNAs) and \u003cem\u003ein vitro\u003c/em\u003e siRNA transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe small-interfering RNAs (siRNAs) and Silencer negative control (si-control) were purchased from Invitrogen (Shanghai, China). The siRNA-targeting Snail2 (si-Snail2) or siRNA-targeting PUMA (si-PUMA) and si-control were transfected into cells with lipofectamine 2000 reagent (Invitrogen) following the manufacturer\u0026rsquo;s instructions. After 24h and 48\u0026thinsp;h of transfection with the siRNAs (0.1\u0026thinsp;nmol/2\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e cells), the cells were harvested for further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSnail2 Plasmids and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell expression construct (pcDNA3.1- human Snail2, pSnail2) and pcDNA3.1 were obtained from Invitrogen (Shanghai, China). 1.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells plated in six-well plates were transiently transfected with a pSnail2 expression vector or pcDNA3. 24 hours after plating, transfection was performed as recommended by the manufacturer by adding, in each well, a mixture containing 200 \u0026mu;l of 150 mmol/L NaCl, 9 \u0026mu;l of Exgene and 2 \u0026mu;g of the Snail2 expression vector. As a control, cells were transfected with the corresponding empty vector pcDNA3. At 24, 48 hours after transfection, cells were collected for Western blotting analyses. All experiments were set up to obtain at least 60% of transfected cells. For stable transfected cells, the infected cells were allowed to recover for 48 hs and were cultured for 14 days with puromycin-containing medium (1 \u0026micro;g/mL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShort hairpin RNA (shRNA) and lentiviral infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentiviral transduction particles carrying short hairpin RNA (shRNA) sequence against human Snail2 and control non-target sequence (Sigma-Aldrich; NM_003068/TRCN0000284362 and SHC002V) were used to knockdown Snail2 expression in CEM cells according to the manufacturer\u0026rsquo;s protocol. Briefly, cells were incubated with lentiviral particles in the presence of hexadimethrine bromide (8 \u0026micro;g/mL) for 36 hs. Infected cells were allowed to recover for 48 hs and were cultured for 14 days with puromycin-containing medium (1 \u0026micro;g/mL). The stable knockdown cells were identified by Western blotting using anti-Snail2 antibody (Cell Signaling Technology, Beverly, MA; 9585) and were cultured in puromycin-free RPMI 1640 medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of apoptosis and clonogenic survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT-ALL cells (10\u003csup\u003e6\u003c/sup\u003e/ml) were treated with PTL (5\u0026mu;M) in RPMI 1640 complete medium for 48\u0026thinsp;h. After drug treatment, apoptosis was analyzed by annexin V staining and flow cytometry using the FACSCalibur cytometer (BD Biosciences). For clonogenic survival assays, the cells were cultured on plastic or on Matrigel for 4\u0026thinsp;h and then treated with PTL for 24\u0026thinsp;h. The cells were recovered, washed and then seeded at 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e cells/ml in complete medium with 1% methylcellulose (StemCell Technologies, Vancouver, BC). After 14 days, colonies with \u0026gt;50 cells were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Observations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence observations were performed to investigate cleaved caspase-3 expression in the T-ALL cell according to the manufacturer\u0026apos;s instructions, and analyzed using the FACSCalibur cytometer (BD Biosciences). Briefly, cells stained with indicated anti-cleaved caspase-3 antibodies were resuspended in phosphate-buffered saline. The images were captured by a Zeiss LSM 710 Confocal Microscope (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth inhibition assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were cultured in 96-well plates at a density of 5000 cells/well and left to recover. The quantity of viable cells was estimated by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The cells were treated with PTL (5 \u0026micro;g). After 48 h incubation at 37 \u0026deg;C, 20 \u0026micro;l of MTT solution (5 mg/ml in PBS) (Sigma, USA, A101161) was added into each well and cells were incubated at 37 \u0026deg;C for 4 hours. After adding DMSO to wells to dissolve the formazan crystals, plates were read using a plate reader at 570 nm against 630 nm. The experiment was performed three times. The percentage of viable cells was determined by comparison to untreated control cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lysates were prepared and separated on 10% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes. Membranes were blotted with the primary antibodies and developed after secondary antibody incubation using the ECL Kit (Amersham International, Amersham, UK) according to the manufacturer\u0026apos;s protocols. The primary antibodies were below：anti-Snail2, anti-PUMA, anti-Bim, anti-Noxa, anti-cleaved caspase-3 and a-Turbulin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the in vivo proliferative effect of Snail2, the CEM/sh-control and CEM/sh-Snail2 cells (1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e cells/mouse) were injected subcutaneously into the flanks of the nude mice. When tumors reached a mean volume of 50-100 mm\u003csup\u003e3\u003c/sup\u003e, mice were treated with 30mg/kg/per 3 days parthenolide [28] or with equivalent volume of 10% DMSO through intraperitoneal injections for 21 days. Mice were weighed weekly and tumors were measured every 72 hours. At the conclusion of the experiment, mice were euthanized, and tumors were excised, measured, and then fixed in 10% formalin for IHC staining. For western blotting, unfixed tumors were homogenized with a tissue tearer and cell lysates were assessed. The study protocol was approved by Medicine Institutional Animal Care \u0026amp; Use Committee, the Jinan Central Hospital, Jinan, Shandong, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFormalin-fixed-embedded subcutaneous tumor samples from nude mice were first cut into 5-\u0026mu;m-thick sections. TUNEL was used to detected cell apoptosis as the manufactured methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analyses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll calculations and statistical analyses were carried out using SPSS25 software The data are expressed as mean \u0026plusmn; standard deviation of n = 3 determinations. In short-term\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e assays, we used the analysis of variance one-way ANOVA with Bonferroni\u0026rsquo;s multiple comparison test or the Student t test. We considered results with P \u0026lt; 0.05 as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSnail2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;is highly expressed in T-cell acute lymphoblastic leukemiain (T-ALL)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the expression of Snail2 in T-ALL, we first analyzed\u0026nbsp;Snail2 expression by Western blot in 9 independent T-ALL patient cohorts and 9 normal bone marrow (NBM) cells. The results showed that Snail2 protein expression was elevated in primary T-ALL as compared to normal bone marrow (BM) cells (Figure 1A). To further correlate the level of Snail2 protein expression, we next detects the Snail2 protein expression in T-ALL cell lines (JURKAT, CCRF-CEM, MOLT-3, and MOLT-4), also find enhanced Snail2 expression was in these cells. JURKAT has lowest Snail2 expression and\u0026nbsp;CCRF-CEM(CEM) has highest Snail2 expression (Figure 1B), so we used JURKAT and CEM cells for further study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSnail2 inhibits the growth and improves apoptosis of CEM cells in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCEM and JURKAT cells were transfected with Snail2 siRNA for 48 h. Snail2 protein was inhibited in both cells by western blot assay (Figure 1A).\u0026nbsp;No change was found in control siRNA transfected cells (Figure 2A). The growth inhibitory effect of Snail2 siRNA or control siRNA transfected CEM and JURKAT was evaluated by the MTT assay in both cells. Snail2 siRNA had a significant growth inhibitory effect on CEM cells, but not for JURKAT cells (Figure 2B). Snail2 siRNA also increased the Annexin V-FITC stained apoptotic population of\u0026nbsp;CEM cells\u0026nbsp;compared to control\u0026nbsp;(Figure 2C). No Annexin V-FITC stained apoptotic population of Snail2 siRNA transfected JURKAT cells was found compared to controls (Figure 2C). The mismatched control siRNA did not inhibit cell viability and apoptosis. Immunofluorescence analysis of cleaved Caspase-3 in si-Snail2 transfected CEM cells manifested robust apoptosis upon si-control transfected CEM cells (Figure 2D). No significant observsion was fond in JURKAT cells (data not show). By colony formation assay, targeting snail2 in Snail shRNA transfected CEM cells inhibited colony formation, but not in Snail shRNA transfected JURKAT cells (Figure 2E). This results indicated that targeting Snail2 inhibits growth and improves apoptosis in Snail2-overexpressed T-ALL cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeting Snail2 enhances parthenolide (PTL) cytotoxicity in CEM cells in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first examined the cytotoxic activity of PTL, as single agents and in combination with Snail2 siRNA in CEM and JURKAT cells. Both of the cells were transfected with Snail2 siRNA, control siRNA or without siRNA or incubated with PTL (5 \u0026mu;M) for 48 h, harvested, and then cell viability analyzed by an MTT assay. As shown in Figure 3A,\u0026nbsp;PTL (5 \u0026mu;M) treatment alone for 48 h, cell viability was decreased in both of the cells, but significant difference was shown in JURKAT cells.\u0026nbsp;However, the combination of PTL (5 \u0026mu;M) and Snail2 siRNA significantly decreased the cell viability in CEM cells compared to PTL alone. The combination of Snail2 siRNA with PTL did not significantly reduce JURKAT cell viability compared to PTL alone (Figure 3A). By colony formation assay, PTL (10 \u0026mu;M) treatment alone significantly inhibited colony formation in JURKAT cells but not CEM cells (Figure 3B). However, the combination of PTL (10 \u0026mu;M) and Snail2 shRNA significantly inhibited colony formation in CEM cells, but not in JURKAT cells compared to PTL alone (Figure 3B). PTL (5 \u0026mu;M) treatment alone increased the Annexin V-FITC stained apoptotic population of JURKAT cells, but not CEM cells (Figure 3B).\u0026nbsp;However, the combination of PTL (5 \u0026mu;M) and Snail2 siRNA significantly increased the Annexin V-FITC stained apoptotic population of CEM cells, but not JURKAT cells compared to PTL alone (Figure 3C). Immunofluorescence analysis of cleaved Caspase-3 in PTL alone treated JURKAT cells, the PTL (5 \u0026mu;M) in combination with Snail2 siRNA treated CEM cells manifested robust apoptosis (Figure 3D). No significant observation was fond in JURKAT cells (data not show).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnhanced Snail2 decreases PTL cytotoxicity in JURKAT cells\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy transiently transfecting pcDNA3.1-Snail2, we established a Snail2-overexpressed Jurkat and CEM cell\u0026nbsp;clone (Jurkat-Snail2, CEM-Snail2), and empty vector transfected cell (Jurkat-Emp, CEM-Emp) was used as control. Snail2 was overexpressed in Jurkat-Snail2 cells compared to Jurkat-Emp cells. Rich Snail2 protein expression was also detected in CEM-Snail2 cells, but no significant increase was found compared to the CEM-Emp cells or untreated CEM cells (Fig. 4A).\u003c/p\u003e\n\u003cp\u003eIn the Jurkat cells, Snail2 overexpression inhibits PTL (5 \u0026mu;M)-induced cell apoptosis (Fig. 4B) and reversed PTL-induced cytotoxicity by MTT assay (Fig. 4C). In Snail2-overexpressed CEM cells, following PTL (5 \u0026mu;M) treatment for 48 h, Snail2 overexpression did not affect cell apoptosis or cell viability induced by PTL treatment (Fig.4B-4C). Immunofluorescence analysis showed that enhanced Snail2 expression inhibits PTL-induced cleaved Caspase-3 activation in JURKAT cells (data not shown). Colony formation assay has the similar results as MTT (data not shown). It was most likely that CEM cell expressed Snail2 at high levels and elevated endogenous Snail2 levels may not be sufficient to promote CEM cell growth and inhibits apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeting Snail2 enhances PTL-induced apoptosis through inducing PUMA expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe examined the effect of Snail2 knockdown on PTL-induced PUMA expression in the\u0026nbsp;CEM\u0026nbsp;cells. Moderate PUMA expression was observed in the untreated CEM cells (Fig. 5A). si-Snail transfection blocked Snail2 expression, but improved PUMA expression (Fig. 5A). PTL induced less PUMA expression, but combined PTL and si-Snail2 treatment significantly promoted PUMA expression (Fig. 5A). The shRNA Snail2 transfected CEM cells were transiently transfected with PUMA siRNA, then treated with PTL (5 \u0026mu;M) for 48 h, and cell viability was analyzed by an MTT. As shown in Figure 5B, targeting PUMA reversed Snail2 shRNA/PTL induced cell growth inhibition. PUMA siRNA transfection also reversed shRNA Snail2/PTL induced cell apoptosis (Figure 5C). Immunofluorescence analysis of cleaved Caspase-3 indicated cleaved Caspase-3 was significantly reduced in the PUMA siRNA transfected CEM cells (Figure 5C).\u003c/p\u003e\n\u003cp\u003eIn the JURKAT cells, PTL (5 \u0026mu;M) treatment alone for 48 h improved PUMA protein expression, but reversed PUMA expression in the pcDNA3-Snail2 transfected cells following PTL (5 \u0026mu;M) treatment for 48 h (Figure 5D).\u003c/p\u003e\n\u003cp\u003eMTT assay showed that pcDNA3-Snail2 transfection reversed PTL-induced cell growth inhibition (Fig. 4B). pcDNA3-Snail2 transfection also reversed PTL induced cell apoptosis (Figure 4C). Immunofluorescence analysis of cleaved Caspase-3 indicated cleaved Caspase-3 was significantly reduced in the pcDNA3-Snail2 transfected JURKAT cells (Figure 5E).Thus, repression of PUMA by Snail2 in JURKAT cells contributes to survival by reducing apoptosis.\u003c/p\u003e\n\u003cp\u003eWe also detected the levels of the p53 target gene Noxa, the non-p53 regulated Bim gene, or the anti-apoptotic Bcl-2 and Bcl-xL proteins in the CEM and JURKAT cells. The results showed that they were not affected by Snail2 siRNA and pSnail2 (data not shown). Thus, Snail2 protects T-ALL cells from apoptosis triggered by PTL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffective therapeutic combination of PTL with the Snail2 inhibitor in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed whether the Snail2 silencing was effective on PTL induced tumor inhibition in vivo. Nude mice bearing CEM/shRNA-Snails tumors were injected i.p. with PTL as the methods reported. The result shows that Snail2 shRNA plus PTL exhibited significant antitumor activity in the CEM xenograft mode compared to the effects of PTL or sh-control/PTL applied. As shown in Fig. 6A, the tumor size of mice was significantly reduced in the presence of SNAIL2 shRNA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThen, all mice in the groups were killed, and the tumor tissues were removed for TUNEL and Western blot assay. Compared to the vehicle group or the single treatment group with PTL, the combined administration of the PTL and Snail2 shRNA transfected tumor displayed increased apoptosis (Fig. 6B) and PUMA expression (Fig. 6C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding the mechanisms underlying cancer chemoresistance is likely to lead to more effective therapies and to a better control of patient relapse. In this study, we report that blockade of the Snail2 improves the sensitivity of PTL to T-ALL by activating PUMA signals. Our results showed that Snail2 is more commonly present in the T-ALL patients and T-ALL cell lines. We did not, however, observe increased Snail2 expression in non-leukemic bone marrow, suggesting that enhanced Snail2 expression might be related to the occurrence or biological behavior of T-ALL.\u003c/p\u003e\n\u003cp\u003eTo date, PTL is the only drug that has been shown to be capable of completely eradicating childhood ALL in NSG xenografts, as a single agent [26]. Most studies, using such models, report reduction in leukemia burden but levels often increase on cessation of treatment. A recent in vitro study reported [27] that PTL treatment only\u0026nbsp;reduced viability of T-ALL cells to less than 30%, suggesting some T-ALL cells may be resistant to PTL. In our study, PTL treatment reduced viability of poor-Snail2-expressed JURKAT cells to 35% which was similar to the report. In the rich-Snail2-expressed CEM cells, PTL treatment reduced viability of T-ALL cells to less than 10%. To confirm the effect of Snail2 on cell survival, we re-expressed Snail2 in the JURKAT cells and Snail2-knockdown CEM cells.\u003c/p\u003e\n\u003cp\u003eIt is notable that Snail2 re-expression reversed JURKAT cell resistance to PTL and Snail2 knowdown\u003c/p\u003e\n\u003cp\u003esignificantly reduced CEM cell resistance to PTL. We conducted clonogenic survival assays to evaluate long-term survival, which was consistent with the MTT results above. This is the first report demonstrating Snail2 provide a protective effect to T-ALL cells against PTL. Moreover, we have shown that targeting Snail2 can overcome this effect.\u003c/p\u003e\n\u003cp\u003eProgrammed cell death or apoptosis of white blood cells is a common feature, and is thought to contribute to the cytopenias particularly in early stages of the disease [3,4]. Since Snail2 attenuation sensitized cells to apoptosis, we determined whether it affected the expression of the pro-apoptotic gene Puma, which was shown to be repressed by Snail2 [8]. Targeting Snail2 in CEM cells increased PTL-induced apoptosis. Knockdown of Snail2 by siRNA in CEM cells induced caspase-3 cleavage and expression of known Snail2-repressed pro-apoptotic protein PUMA, indicating a Snail2 role in anti-apoptosis signaling. To verify whether PUMA contributes to apoptosis in Snail2-knockdown CEM cells, PUMA was silenced in Snail2-sh CEM cells using a pool of PUMA siRNAs. Treatment with PTL reduced the number of active-caspase-3 positive cells by 80% in PUMA-siRNA treated Snail2-sh CEM cells relative to control cells, followed by reduced cell apoptosis. In vivo, CEM tumors from Snail2-sh cells also displayed higher PUMA levels. In the JURKAT cells, PTL treatment induced cell apoptosis, followed by PUMA expression and\u0026nbsp;caspase-3 cleavage.\u0026nbsp;However, Snail2 re-expression repressed PTL-induced PUMA expression and caspase-3 cleavage. These findings suggest that Snail2 contributes to cell survival by inhibiting PUMA signals. Whether PTL directly blocked Snail2 or directly activating PUMA was beyond the scope of this study. Otherwise, whether PTL activated p53-\u0026nbsp;dependent PUMA\u0026nbsp;or p53- independent PUMA need further investigate. However, it is evident that Snail2 levels decreased and PUMA levels increased, putting the cells under higher levels of caspase-3 cleavage, which is likely to drive PTL- induced apoptosis. Furthermore, Snail2 levels increased and PUMA levels dropped, putting the cells under lower levels of caspase-3 cleavage, which is likely to inhibit PTL-induced apoptosis.\u003c/p\u003e\n\u003cp\u003eIn support of a role for Snail2 in suppressing PTL-induced apoptosis, Snail2 was found to protect tumor cells from apoptosis induced by radiation, which is reminiscent of the protective effect of Snail2 against DNA damage observed in hematopoietic progenitor cells [8,29]. Our results showed that Snail2 targets PUMA, and no other pro-apoptotic gene, including the p53-response gene, Noxa, or the non-p53 target gene Bim to suppress apoptosis. The involvement of Puma in apoptosis is underscored by that Puma siRNA lowered the threshold for induction of apoptosis by PTL. Importantly, the Snail2-PUMA axis was found to be similar in vivo to in vitro as shown by that PTL in Snail2-knockdown CEM cells suppressed tumor growth by inducing cell apoptosis. In sum, our study points to a pivotal function for Snail2 in survival, which allows tumor cells to overcome apoptosis and survive, achieving by repression of the pro-apoptotic gene PUMA. PTL could inhibit Snail2 and upregulate PUMA, and increase the cytotoxicity of PTL to T-ALL. Hence PTL could be a novel promising approach to treat T-ALL in the future.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhou conceptualised and designed the study. Zhou and Cao performed the In vitro experiments. Wang and Cao performed the in Vivo experiments. Cao analyzed the data. Zhou edited the manuscript. All have read and approved this version of the manuscript, and confirm that this is the case.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371(9617):1030\u0026ndash;1043.\u003c/li\u003e\n\u003cli\u003eHunger SP, Mullighan CG. Acute lymphoblastic leukemia in children. N. Engl. J. Med. 2015;373:1541-1552.\u003c/li\u003e\n\u003cli\u003eDordelmann M, et al. Prednisone response is the strongest predictor of treatment outcome in infant acute lymphoblastic leukemia. Blood. 1999;94:1209-1217.\u003c/li\u003e\n\u003cli\u003eCordo V, van der Zwet JCG, Cante-Barrett K, Pieters R, Meijerink JPP. T-cell acute lymphoblastic leukemia: a roadmap to targeted therapies. Blood Cancer Discov. 2021;2(1):19-31.\u003c/li\u003e\n\u003cli\u003eIndraccolo S, Minuzzo S, Masiero M, Amadori A. Ligand-driven activation of the notch pathway in T-ALL and solid tumors: why Not(ch)? 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Indomethacin and juglone inhibit inflammatory molecules to induce apoptosis in colon cancer cells. J Biochem Mol Toxicol. 2020 Feb;34(2):e22433.\u003c/li\u003e\n\u003cli\u003eBelle JI, Petrov JC, Langlais D, Robert F, Cencic R, Shen S, Pelletier J, Gros P, Nijnik A. Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance. Cell Death Differ. 2016;23(5):759-75.\u003c/li\u003e\n\u003cli\u003eGuirguis AA, Slape CI, Failla LM, Saw J, Tremblay CS, Powell DR, Rossello F, Wei A, Strasser A, Curtis DJ. PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia. Cell Death Differ. 2016;23(6):1049-59.\u003c/li\u003e\n\u003cli\u003eGupta M, Hendrickson AE, Yun SS, Han JJ, Schneider PA, Koh BD, Stenson MJ, Wellik LE, Shing JC, Peterson KL, Flatten KS, Hess AD, Smith BD, Karp JE, Barr S, Witzig TE, Kaufmann SH. 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Targeting acute myeloid leukemia stem cell signaling by natural products. Mol Cancer. 2017;16(1):13.\u003c/li\u003e\n\u003cli\u003eDai Y, Guzman ML, Chen S, Wang L, Yeung SK, Pei XY, Dent P, Jordan CT, Grant S. The NF (Nuclear factor)-\u0026kappa;B inhibitor parthenolide interacts with histone deacetylase inhibitors to induce MKK7/JNK1-dependent apoptosis in human acute myeloid leukaemia cells. Br J Haematol. 2010;151(1):70-83.\u003c/li\u003e\n\u003cli\u003eJeyamohan S, Moorthy RK, Kannan MK, Arockiam AJ. Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer. Biotechnol Lett. 2016;38(8):1251-60.\u003c/li\u003e\n\u003cli\u003eTalib WH, Al Kury LT. Parthenolide inhibits tumor-promoting effects of nicotine in lung cancer by inducing P53 - dependent apoptosis and inhibiting VEGF expression. 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Mol Cell Biochem. 2018;440(1-2):11-22.\u003c/li\u003e\n\u003cli\u003eDiamanti P, Cox CV, Moppett JP, Blair A. Parthenolide eliminates leukemia-initiating cell populations and improves survival in xenografts of childhood acute lymphoblastic leukemia. Blood. 2013;121(8):1384-93.\u003c/li\u003e\n\u003cli\u003eEde BC, Asmaro RR, Moppett JP, Diamanti P, Blair A. Investigating chemoresistance to improve sensitivity of childhood T-cell acute lymphoblastic leukemia to parthenolide. Haematologica. 2018;103(9):1493-1501.\u003c/li\u003e\n\u003cli\u003eProvance OK, Geanes ES, Lui AJ, Roy A, Holloran SM, Gunewardena S, Hagan CR, Weir S, Lewis-Wambi J. Disrupting interferon-alpha and NF-kappaB crosstalk suppresses IFITM1 expression attenuating triple-negative breast cancer progression. Cancer Lett. 2021;514:12-29.\u003c/li\u003e\n\u003cli\u003ePerez-Losada J, Sanchez-Martin M, Perez-Caro M, Perez-Mancera PA, Sanchez-Garcia I. The radioresistance biological function of the SCF/kit signaling pathway is mediated by the zinc-finger transcription factor Slug. Oncogene. 2003;22:4205\u0026ndash;11\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Parthenolide, acute lymphoblastic leukemia, Snail2, P53 up-regulated modulator of apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-5749923/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5749923/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Objective:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCurrent therapies for childhood T-cell acute lymphoblastic leukemia (T-ALL) have increased survival rates to about 85%. The standard of care is chemotherapy, but approximately 20% patients exhibit primary or secondary resistance to current therapies. Parthenolide (PTL) has been shown to have excellent anti-cancer activity in pediatric leukemia xenografts, with minimal effects on normal hemopoietic cells. Some leukemia initiating cell populations remain resistant to PTL. This study examined mechanisms for this resistance and how to overcome the resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSnail2 protein expression was detected in 9 T-ALL patients, 9 normal bone marrow (NBM) cells and 4 T-ALL cell lines. We investigated the effects of loss of-function and gain-of-function of Snail2 or P53 up-regulated modulator of apoptosis on survival, colony formation, apoptosis and chemosensitivity of T-ALL cells to PTL \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSnail2 protein expression was elevated in primary T-ALL patients and cell lines. CEM cells with rich Snail2 expression is resistant to PTL. JURKAT cells with poor Snail2 expression sensitizes to PTL. In vitro, Snail2 inhibition by siRNA, sensitized CEM cells to apoptosis by PTL, resulting in PUMA upregulation and caspase-3 cleavage. Inhibition of PUMA by RNA interference in Snail2-knockdown CEM cells rescued the resistance of CEM cells to PTL treatment. Snail2 re-expression in JURKAT cells is resistant to PTL. The pro-survival effect of Snail2 was found to be caused by direct repression of PTL-induced PUMA expression by Snail2 in JURKAT cells. Additionally, an orthotopic allograft in vivo model demonstrated that the Snail2 inhibitor enhanced responses to PTL in CEM cells by inducing PUMA-dependent cell apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study demonstrates a pivotal role for Snail2/PUMA signals in T-ALL cell survival. It may be possible to achieve greater toxicity to childhood T-ALL by combining PTL with Snail2 knockdown.\u003c/p\u003e","manuscriptTitle":"Targeting Snail2 improve sensitivity to parthenolide in T-cell acute lymphoblastic leukemia through PUMA activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 06:58:18","doi":"10.21203/rs.3.rs-5749923/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":"9b74fdf2-a67f-46fc-af57-521a664866cc","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-08T06:58:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 06:58:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5749923","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5749923","identity":"rs-5749923","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}