AMOT Suppresses Tumor Progression Via Regulating DNA Damage Response Signaling in Diffuse Large B-Cell Lymphoma

Cancer Gene Therapy https://doi.org/10.1038/s41417-020-00258-5

ARTICLE

AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell lymphoma

1,2,3 1,2 1,2 1,2 1,2 1,2,4,5,6 1,2,4,5,6 Tan Sang ● Juan Yang ● Jiarui Liu ● Yang Han ● Ying Li ● Xiangxiang Zhou ● Xin Wang

Received: 7 June 2020 / Revised: 22 October 2020 / Accepted: 4 November 2020 © The Author(s), under exclusive licence to Springer Nature America, Inc. 2021

Abstract Angiomotin (AMOT) is a membrane protein that is aberrantly expressed in a variety of solid tumors. Accumulating evidence support that AMOT is involved in the pathological processes of tumor proliferation, apoptosis, and invasion. However, the potential role of AMOT in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains elusive. In the present study, we investigated the expression level and biological function of AMOT in DLBCL. AMOT expression was signiﬁcantly reduced in DLBCL biopsy section, and low AMOT expression was associated with poor clinical prognosis. Overexpression of AMOT by lentivirus in human DLBCL cells induced cell viability inhibition concomitant with an increased percentage of cells in G1 phase and decreased percentage in S phase. Moreover, AMOT upregulation increased the

1234567890();,: 1234567890();,: sensitivity of DLBCL cells to doxorubicin. Furthermore, overexpression of AMOT led to reduced activation of key kinases for the DNA damage response (DDR). The above results indicated that AMOT acts as a tumor suppressor via inhibition of the DDR, thus reducing the viability while increasing the chemosensitivity in DLBCL. In summary, AMOT may be a novel potential target for DLBCL therapeutic intervention.

Introduction

These authors contributed equally: Xiangxiang Zhou, Xin Wang Diffuse large B-cell lymphoma (DLBCL) is the most Supplementary information The online version of this article (https:// common type of highly aggressive non-Hodgkin’s lym- doi.org/10.1038/s41417-020-00258-5) contains supplementary phoma. Though the long-term survival rate of DLBCL is material, which is available to authorized users. ~60% after immunochemotherapy with R-CHOP [1], * Xiangxiang Zhou 30–40% of patients will present refractory or relapsed [email protected] process after an initial response to therapy [2, 3]. Gene * Xin Wang heterogeneity accounts for different clinical outcomes [4], [email protected] therefore, the research of individualized treatment schemes based on new molecular targets and oncogenic pathways is 1 Department of Hematology, Shandong Provincial Hospital, the main research strategy at current. Cheeloo College of Medicine, Shandong University, Angiomotin (AMOT) was initially identiﬁed in 2001 as a Jinan, Shandong 250021, China medium that binding to angiostatin and regulating angio- 2 School of Medicine, Shandong University, Jinan, Shandong genesis or endothelial cell migration [5–7]. Human AMOT 250012, China gene is located in chromosome Xq23 with a 2025-bp open 3 Department of Hematology, Jinan Central Hospital, Cheeloo reading frame. AMOT-p130 and AMOT-p80 are two clas- College of Medicine, Shandong University, Jinan, Shandong 250012, China sic isoforms which are nearly identical at the C-terminal, while there is an extended glutamine-rich domain at the N- 4 Department of Hematology, Shandong Provincial Hospital Afﬁliated to Shandong First Medical University, Jinan, Shandong terminal of AMOT-p130 [7]. These two splicing formations 250021, China have different functions in regulating polarity rearrange- – 5 Shandong Provincial Engineering Research Center of Lymphoma, ments related to cell shaping and migration [8 10]. In Jinan, Shandong 250021, China murine, inactivation of AMOT led to failure in cell growth 6 National Clinical Research Center for Hematologic Diseases, and migration in the anterior visceral endoderm, causing Jinan, Shandong 250021, China early embryonic lethal [11]. Consistently, knockout of T. Sang et al.

AMOT in zebrafish inhibited the proliferation rate of epi- (TBD Science, Tianjin, China). The study was approved by thelial cells [12, 13] and led to defects in endothelial the Medical Ethics Committee of Jinan Central Hospital migration [14]. affiliated to Shandong University. In accordance with the At present, the role of AMOT in several solid tumors is Declaration of Helsinki, the informed consent was obtained still controversial. AMOT expression was decreased in lung before all samples were collected. cancer, breast cancer, undifferentiated pleomorphic sar- coma, ovarian serous carcinoma, and clear cell renal cell Cell lines carcinoma [15–19], and was related to poor prognosis of these diseases. Nevertheless, in epithelial ovarian cancer, Human DLBCL cell lines LY1 and LY8 were obtained prostate cancer, liver cancer, osteosarcoma, and sinonasal from Dr. B. Hilda Ye (Albert Einstein College of Medicine, tumors, AMOT was overexpressed and promoted tumor cell NY, USA), and human DLBCL cell line Val was provided proliferation or invasion [10, 20–23]. AMOT participated in by Pro. Wing C. Chan (City of Hope National Medical the regulation of several signaling pathways, including Center, CA, USA). Cells were maintained in IMDM med- Hippo, Wnt, ERK1/2, and VEGFR-2 pathways [12, 24–29]. ium (Gibco, Carlsbad, USA) containing 10% fetal bovine However, the effect of AMOT in hematopoietic malig- serum (HyClone, Logan, USA) and 1% penicillin/strepto- nancies has not been reported. mycin mixture. All cells were incubated at 37 °C in a Increased replication pressure and alteration of the DNA humidified atmosphere with 5% CO2. All human cell lines damage response (DDR) are crucial features of genetic were examined for mycoplasma infection periodically and instability and are closely related to tumorigenesis [30]. authenticated using small tandem repeat profiling conducted DDR senses different types of damage and initiates DNA by LGC standards and ATCC. repair, involving transcriptional activation, cell cycle, senescence, and apoptosis [31]. The ataxia-telangiectasia Immunohistochemistry (IHC) mutated (ATM) and ATM and RAD3-related (ATR) protein kinases are the main regulators of the DDR program. Formalin-fixed and paraffin-embedded tissue sections Despite their functional similarity, ATM is activated pri- obtained from DLBCL and RLH patients were used to marily during DNA double-strand breaks, phosphorylates perform IHC. The RLH tissues were used as control, as checkpoint kinases 1 (Chk1) at S317 and S345, while ATR previously described [34, 35]. After deparaffinized and primarily regulates damaged replication forks, phosphor- hydrated, the antigen was retrieved in 10 mM sodium citrate ylates checkpoint kinases 2 (Chk2) at Thr68 [32, 33]. In buffer (pH = 6.0) under high-pressure for 10 min. Three some cases, AMOT was considered a substrate for ATM percent solution of H2O2 was used at 37 °C for 30 min to and ATR, but this finding has not been confirmed by further block the samples from endogenous peroxidases. After that, experiments [31]. sections were incubated with primary anti-AMOT (1:200, In this study, we evaluated the expression level of Abcam, 85143, Cambridge, UK) at 4 °C overnight, then AMOT in DLBCL. AMOT expression was upregulated to rinsed by 1× PBS for 15 min, and treated with second assess its effect on cell growth with or without doxorubicin antibody from (Golden Bridge, Beijing, China) at 37 °C for treatment. The results showed that AMOT could act as a 30 min. After incubation with SABC from SP reagent kit, negative regulator of DDR signaling in DLBCL. As far as the sections were stained with DAB (Golden Bridge, Beij- we know, this is the first report to confirm the regulatory ing, China), counterstained with hematoxylin and mounted effect of AMOT on the key enzymes of DDR in DLBCL. with neutral gum. The criterion for a positive result is that the positive cells rate exceeds 10%. Two independent researchers scored the IHC staining at different time points. Materials and methods Real-time quantitative polymerase chain reaction Patients and samples (qRT-PCR)

Fifty-two specimens of newly diagnosed DLBCL and Total RNA was isolated from peripheral blood mononuclear twenty-five specimens of reactive lymphoid hyperplasia cells (PBMCs) and cultured cells by RNAiso Plus (TaKaRa, (RLH) were collected at Jinan Central Hospital affiliated to Dalian, China) and assessed by UV spectrophotometry for Shandong University. According to the WHO classification, the concentrations and purity. After reverse transcription of all patients were independently diagnosed by two experi- total RNA into cDNA using the reagents kit (TaKaRa, enced pathologists. Peripheral venous blood was collected Dalian, China), the amplification reactions were carried out from three healthy volunteers (N1, N2, and N3) and with specific primers by means of SYBR Green Premix Ex mononuclear cells were isolated by Ficoll centrifugation Taq II kit (Takara, Dalian, China) on a Light Cycler 480 AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell. . . real-time PCR system (Roche, Basel, CH). AMOT-specific efficiencies were evaluated by GFP and then validated by primers were the following: forward, 5′-AGCTCCAGG qRT-PCR and western blot assays. CAGCATGTGAA-3′; reverse, 5′-CTGAAACGTTGGT GGGCTGA-3′. Primers of GAPDH and DDR members are Cell viability assay listed in Table S1. GAPDH was used as an endogenous control. The relative quantification was calculated by the LY1 and LY8 cells with designed treatment were seeded at 2−ΔΔCt method. a density of 8000 cells per well into 96-well plates. At the designated time, 10 μl/well of CCK-8 (Dojindo, Kumamoto, Western blot Japan) was incubated with the cells at 37 °C for 2 h, then followed by absorbance values detection at 450 nm by a Total protein from the PBMCs and cultured cells was microplate reader (Thermo Scientifc, Rockford, IL, USA). extracted using RIPA Lysis buffer (Shenergy, Shanghai, China) containing protease inhibitor cocktail tablets (She- Cell cycle analysis nergy, Shanghai, China) and phosphatase inhibitor cocktail (Bosterbio, Wuhan, China), according to the operating Cells with designed treatment were harvested and fixed in instructions. Protein concentrations were measured using ice-cold 70% ethanol for 30 min, then rinsed by 1×PBS, and a BCA assay (Shenergy, Shanghai, China), then equal resuspended in 50 μg/ml PI staining (BD Biosciences, amounts of total protein were separated by electrophoresis Bedford, USA) according to the manufacturer’s instruc- on a 7.5 or 10% polyacrylamide gel (Bio-Rad, USA). The tions. After incubation in the dark room for 15 min, the cells protein extracts were transferred onto PVDF membranes were passed through a strainer to prepare single cell sus- (Millipore, Billerica, USA), and the membranes were pension. Then cell cycle distribution was detected by on blocked in tris-buffered saline (contained 0.05% Tween) Navios Flow Cytometer (Beckman Coulter, CA, USA) and with 5% defatted milk or 1% bovine serum album for 1 h. analyzed by ModFit LT (Verity Software House, USA). Then the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies were used Cell apoptosis analysis with the concentration of 1:1000 as follows: anti-AMOT (Abcam, 85143, Cambridge, UK), anti-p-ATM, anti-p- Cells with designed treatment were washed in ice-cold ATR, anti-p-Chk1, anti-p-Chk2, and anti-p-H2A.X (Cell PBS and resuspended in 100 µl binding buffer, then Signaling Technology, 9947, MA, USA). Anti-GAPDH incubated with 5 µl 7AAD and 5 µl Annexin V-PE (BD (1:1000, Golden Bridge, TA-08, Beijing, China) was used Biosciences, Bedford, USA) in a dark room for 15 min. as the endogenous control. Goat anti-rabbit or goat anti- Afterward, another 400 µl of binding buffer was added to mouse secondary antibodies labeled with HRP (1:5000, each sample and mix well, then the cells were subjected to Golden bridge, ZB-2301, ZB-2305, Beijing, China) were the Navios Flow Cytometer, and distinguished into four hybrid binding at room temperature for 1 h, and the final groups: Annexin V-PE-/7AAD- (viable cells), Annexin signals were detected with the AI600 Imaging System V-PE-/7AAD + (dead cells), Annexin V-PE+/7AAD-(early- (General Electric, Boston, USA) using an ECL kit (Milli- stage apoptosis cells), and Annexin V-PE+/7AAD+ (late- pore, Billerica, MA, USA). Finally, Image J software was stage apoptosis cells). The ratio of early-stage apoptotic cells used to quantified the intensities of protein bands. was used to perform statistical analysis.

Cell transfection Statistical analysis

LY1 and LY8 cells were respectively seeded into 96-well All experiments were independently repeated at least in plates at a concentration of 1 × 105 cells per well on the day triplicate. GraphPad Prism 7.0 (GraphPad Software, CA, before transfection. According to the manufacturer’s pro- USA) and SPSS17.0 (IBM, NY, USA) were used for tocol, appropriate doses of lentivirus (GeneChem, Shang- statistical analysis, and data were presented as mean ± hai, China) and transfection enhancer were co-cultivation standard deviation (mean ± SD). The correlation between with the cells. Both the Empty vector lentivirus (LV-Con) the expression of AMOT in tissue sections and the clinical and AMOT overexpression lentivirus (LV-AMOT) parameters of DLBCL patients was analyzed by Chi-square expressed green ﬂuorescent protein (GFP) and puromycin test. Kaplan–Meier analysis was performed for subsistence resistance genes. After 48 h, 5 μg/ml puromycin (Gene- analysis. Differences between groups were analyzed by Chem, Shanghai, China) was added to the culture medium ANOVA or unpaired t-tests. P < 0.05 was considered as to screen stably transfected viable cells. The infection statistically signiﬁcant. T. Sang et al.

Fig. 1 AMOT was downregulated in DLBCL and related to poor those with negative expression. C The mRNA expression levels of prognosis. A Compared with RLH, the expression of AMOT was AMOT in DLBCL cell lines and controls were detected by qRT-PCR significantly reduced in DLBCL tissues (original magnification, ×100, assays. ***P < 0.001. D The expression of AMOT protein was con- upper panel and ×400, lower panel). B Kaplan–Meier curves showed firmed to be decreased in DLBCL groups by western blot assays. patients with AMOT positive expression had longer survival than ***P < 0.001.

Results that AMOT may be a biomarker to assess the risk and clinical prognosis in DLBCL. Next, we verified the AMOT was downregulated in DLBCL and related to expression of AMOT in human DLBCL cell lines. As poor tumor prognosis expected, DLBCL cells exhibited remarkably lower mRNA as well as protein levels of AMOT, compared to the control IHC staining was carried out to check the expression level groups (Fig. 1C, D). of AMOT in DLBCL. The percentage of DLBCL samples positive for AMOT was 40.4% (21/52), lower than the AMOT restrained DLBCL cell proliferation percentage of 80% (20/25) of RLH tissues (Fig. 1A). The clinical data from DLBCL patients was collected and To investigate the biological function of AMOT in vitro, the relationship between decreased AMOT expression and LY1 and LY8 cells were stably transfected with LV-Con or clinical parameters of DLBCL were analyzed. As presented LV-AMOT. The GFP expression ratio indicated that more in Table 1, the expression of AMOT was negatively asso- than 80% of cells were successfully transfected with the ciated with serum lactate dehydrogenase (LDH, P = 0.023) lentivirus, as indicated by qRT-PCR and western blot assay and international prognostic index (IPI) score (P = 0.017). (Fig. 2A, B). Compared with LV-Con groups, cell viability Furthermore, Kaplan-Meier analysis indicated that the was significantly suppressed in LV-AMOT groups median survival of patients was 5.04 years in AMOT- (Fig. 2C). We then used XAV939, a tankyrase inhibitor that positive group, significantly longer than 1.94 years in stabilizes the concentration of AMOT protein, to treat the AMOT-negative group (Fig. 1B). The results above suggest DLBCL cell lines. XAV939 inhibited the viability of AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell. . .

Table 1 Correlation between AMOT expression and clinical activating phosphorylation of several key checkpoint pro- characteristics of DLBCL patients. teins was examined by western blot assay. AMOT upre- Characteristics No. of patients AMOT expression P value gulation attenuated the activating phosphorylation of ATM Positive (%) Negative (Ser1981), ATR (Ser428), Chk1 (Ser345), Chk2 (Thr68), and H2AX (Ser139) (Fig. 3C). However, the mRNA Age (years) expression level of these genes did not change (Fig. 3D). <60 19 12 (63.1%) 7 0.219 ≥60 33 15 (45.5%) 18 AMOT overexpression increased the response of Gender DLBCL cells to doxorubicin Male 25 13 (52%) 12 0.991 Female 27 14 (51.9%) 13 The regulation of AMOT to the DDR pathway led us to Ann Arbor stage hypothesize that AMOT upregulation might sensitize I or II 11 8 (72.7%) 3 0.12 DLBCL cells to DNA damaging reagents. To confirm our III or IV 41 19 (46.3%) 22 hypothesis, we incubated LV-AMOT and LV-Con cells B symptoms with different concentrations of doxorubicin (80, 160, and Absent 41 23 (56.1%) 18 0.245 240 nM) for 48 h. Then the cell viability was evaluated by Present 11 4 (36.4%) 7 CCK-8 assay. The LV-AMOT group showed increased Subtype sensitivity to doxorubicin compared to the LV-Con group GCB 21 8 (38.1%) 13 0.1 (Fig. 4A). Next, we incubated the cells with doxorubicin Non-GCB 31 19 (61.3%) 12 (160 nM) for 24, 48, and 72 h, and similarly, observed an Serum LDH increase in drug sensitivity in the LV-AMOT group (Fig. 4B). At last, we incubated the cells with doxorubicin Normal 23 16 (69.6%) 7 0.023 (160 nM) for 24 h and performed cell cycle and apoptosis Elevated 29 11 (37.9%) 18 detection. Compared with the control, the LV-AMOT group Extranodal involvement showed an increased accumulation of cells in the G1 phase Absent 15 9 (60%) 6 0.458 (Fig. 4C). As expected, under the treatment of doxorubicin, Present 37 18 (48.6%) 19 the apoptosis rate of cells in LV-AMOT group was statis- IPI score tically different from the control group (Fig. 4D). 0–2 19 14 (73.7%) 5 0.017 3–5 33 13 (39.4%) 20 AMOT upregulation attenuated DNA repair GCB germinal center B-cell-like, LDH lactate dehydrogenase, IPI potential in response to doxorubicin in DLBCL cells international prognostic index. To investigate the potential mechanism by which AMOT DLBCL cells in a time-and concentration-dependent man- enhances the sensitivity of DLBCL cells to chemotherapy, ner (Fig. 2D). Enhancement of the AMOT protein con- we incubated LV-AMOT and control cells with doxorubicin centration were detected in LY1 and LY8 cells following (160 nM) for 24 h, respectively, then checked the phos- incubation with XAV939 for 24 h (Fig. 2E). phorylation of ATM, ATR, Chk1, and H2AX proteins by western blot. The LV-AMOT groups were noted to have Roles of AMOT on cell cycle, apoptosis, and DNA decreased levels of phosphorylated ATM, ATR, Chk1, damage response proteins Chk2, and H2AX (Fig. 5). We speculated that AMOT reduced the phosphorylation level of these key checkpoint We then sought to study whether AMOT can regulate the proteins, thereby attenuating their response to doxorubicin- cell cycle and apoptosis of DLBCL cells using PI staining induced DNA damage. and annexin-V-based apoptotic assays. The results revealed that overexpressed AMOT increased the proportion of cells in the G1 phase in both LY1 (31.03 ± 1.0% in LV-Con vs. Discussion 36.31 ± 0.74% in LV-AMOT group, P = 0.002) and LY8 (29.69 ± 1.1% in LV-Con vs. 49.94 ± 0.64% in LV-AMOT Aberrant AMOT expression has been reported in solid group, P = 0.000) cells, with a concomitant decrease in the tumors, but never in hematopoietic malignancy. For the first S phase (Fig. 3A). However, no significant change in the time, we elucidated in this study that AMOT may work as a apoptosis rate after AMOT upregulation was observed in potential tumor suppressor in the development of DLBCL. both LY1 and LY8 cells (Fig. 3B). Since the DDR was AMOT was suppressed during the pathogenesis of DLBCL, related to cell cycle arrest, the effect of AMOT on the while overexpression of AMOT inhibited the progression of T. Sang et al.

Fig. 2 Role of AMOT in DLBCL cell proliferation. A, B Increased tankyrase inhibitor XAV939 on the viability of DLBCL cells. LY1 and expression of AMOT was conﬁrmed by qRT-PCR and western blot in LY8 cells were incubated with different concentrations (40, 80, and DLBCL cells stably transfected with AMOT overexpressing lentivirus 120 μM) of XAV939, and cell viability was measured at 24, 48, (***P < 0.001). C Overexpression of AMOT markedly decreased cell and 72 h. E After incubated with XAV939 (80 μM) for 24 h, AMOT viability assayed by CCK8 (**P < 0.01, ***P < 0.001). D Effect of protein concentration was increased.

DLBCL in vitro. Furthermore, upregulation of AMOT led evaluation of DLBCL in the context of individualized to increased sensitivity of DLBCL cells to DNA damaging treatment. In the previous study, epigenetic mechanisms reagents including doxorubicin. In terms of biological like promoter methylation or histone deacetylation may mechanism, AMOT could regulate the cell cycle by inhi- decrease the expression of AMOT in angiogenesis and biting DDR signalings in DLBCL cells. These encouraging sarcomagenesis [15, 38], so we speculate that an analogous findings may help to develop new treatment strategies and mechanism may be involved in DLBCL. The specific provide individualized governance in DLBCL. mechanism requires further investigation. In the present study, we identified for the first time that We next explored the potential functions of AMOT in the expression of AMOT was remarkably repressed in the pathogenesis of DLBCL by gain-of-function assay. The DLBCL tissues and cell lines compared to reactive hyper- results indicated that AMOT could inhibit cell viability, plasia and PBMCs from healthy donors. Through the cor- induce cell cycle arrest, and increase the sensitivity of cells relation analysis, we found that the reduction of AMOT was to chemotherapeutics, instead of directly induce apoptosis associated with high LDH and IPI scores, which were in DLBCL. Tankyrases belonged to the poly (ADP-ribose) indicators of poor prognosis in patients with DLBCL. In the polymerase family which recognized its substrate through previous studies, AMOT had been reported as tumor sup- the tankyrase-binding domain [39] and induced the sub- pressor in several solid malignancies [36]. In advanced cellular localization changes or degradation of its substrate gastric cancer, lower AMOT-p130 was associated with [40]. Tankyrase inhibitors could stabilize intracellular higher TNM stage, venous invasion, shorter overall, and AMOT protein levels and consequently inhibited tumor disease-free survival [37]. The results demonstrate that cell proliferation by regulating downstream mechanisms AMOT may be a potential biomarker for prognostic [15, 41, 42]. In this study, XAV939 inhibited the AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell. . .

Fig. 3 AMOT induced cell cycle arrest and attenuated DDR C Declined expression levels of phosphorylated ATM (Ser1981), ATR checkpoint protein activation. A AMOT-overexpression increased (Ser428), Chk1 (Ser345), Chk2 (Thr68), and H2AX (Ser139) were the proportion of cells in the G1 phase in LY1 and LY8 cells. **P < observed in LV-AMOT transfected DLBCL cells. D Compared with 0.01, ***P < 0.001. B No signiﬁcant difference of apoptosis ratio was the control group, LV-AMOT cells had no signiﬁcant difference in observed in LV-AMOT groups compared to control groups. mRNA levels of ATM, ATR, Chk1, Chk2, and H2AX. proliferation of DLBCL cells, which may be related to the carcinogenic mutations cause spontaneous DNA damage, increase of AMOT protein concentration in the cytoplasm. which inhibits the progression of incipient cancer cells [43]. As is widely known, the ability of self-repair after DNA The DNA damage checkpoints are important mediators of damage is closely related to the development of tumors. The the tumorigenesis barrier, which is activated early in tumor T. Sang et al.

Fig. 4 AMOT increases chemosensitivity to doxorubicin in doxorubicin (160 nM) for different times were measure by CCK-8. DLBCL cell lines. A The relative viability of cells treated with dif- C The cell cycle distribution was determined after treatment with ferent doses of doxorubicin for 48 h in LV-AMOT and control cells doxorubicin (160 nM) for 24 h. D Cell apoptotic rate of cells treated was detected by CCK-8. B The relative viability of cells treated with with doxorubicin (160 nM) for 24 h was measured. AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell. . .

Fig. 5 AMOT attenuated the key checkpoint kinase activity in phosphorylation of ATM (Ser1981), ATR (Ser428), Chk1 (Ser345), doxorubicin-induced DNA damage response. Western blotting Chk2 (Thr68), and H2AX (Ser139) after doxorubicin (160 nM) treat- showed that AMOT upregulation attenuated the activating ment for 24 h. development and later dysregulated. The cancer cells may repair potential. This current study showed that AMOT rely on the DDR pathway to maintain genomic instability could attenuate the phosphorylation activation of DNA [30, 44]. ATM and ATR are essential components of DDR damage checkpoint proteins, thereby inhibiting the ability that are activated when cells are subjected to DNA repli- of DLBCL cells to repair DNA damage. In this study, we cation stress or DNA double-strand breaks [45]. Under observed that AMOT overexpression resulted in a shortened these conditions, checkpoint kinase Chk1 and Chk2 are S phase of the cell cycle, but no significant change in the G2 phosphorylated and inhibit the phosphorylation of cell phase, indicating that there may be other offsetting factors division cycle (CDC) 25A protein, which subsequently that require further study. regulates downstream kinases and blocks the cell cycle in S Many studies had demonstrated that ATM-Chk2 or and G2 phases. This mechanism allows tumor cells to make ATR-Chk1 pathway inhibitors could enhance the sensitivity adequate repairs and thereby promote their survival [46]. of various tumors, including lymphoma and leukemia, to The activated Chk1 transfers into the nucleus to prevent radiation or chemotherapy drugs [43, 50–52]. Doxorubicin new source excitation [47]. For hematopoietic tumors, is an anti-mitotic cytotoxic drug that works primarily by Miroslav et al. had shown that Chk1 was a key protein in interfering with DNA synthesis and disrupting DNA tertiary the development of B cell lymphoma [48]. As a member of structure. In the current study, AMOT protein exhibits anti- the histone H2A family, H2AX is a key promoter that cancer effect, and a decrease in the activity of key kinases in amplifies signals in the early stages of DNA damage and the DDR pathway has been observed. When doxorubicin continuously recruits repair factors to nuclear foci [49]. The was applied to AMOT-overexpressed DLBCL cells, an suppression of DNA damage checkpoints decreases DNA increased proportion of apoptosis was observed, suggesting T. Sang et al. an increased sensitivity to doxorubicin. Thus, AMOT may 4. Sehn LH, Gascoyne RD. 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