Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells.

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL; these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL.

Introduction

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy caused by infection with the retrovirus human T-cell leukemia virus type 1 (HTLV-1) [1]. HTLV-1 transmission requires cell-to-cell contact via a cell-containing body fluid through the following routes: mother-to-child transmission, sexual intercourse, and contaminated blood transfusion or contaminated needle puncture [2]. Currently, it is estimated that 15–20 million people are infected with HTLV-1 worldwide, but the exact number in the general population has remained unclear since most epidemiological studies are limited to endemic regions, such as Japan, Africa, Latin America, and the Caribbean islands [3, 4]. ATLL develops after a 40- to 60-year latency period in approximately 5% of HTLV-1 carriers [5, 6]. ATLL has been subdivided into four classifications: the acute, lymphoma, chronic, and smoldering types. The most common presentation is the acute type, which generally has a poor prognosis with a median survival time of approximately 4–6 months [6]. Although several new therapeutic approaches, such as anti-C-C chemokine receptor 4 (CCR4) antibodies and allogeneic hematopoietic stem cell transplantation (HSCT), have been established [7-9], the overall prognosis of aggressive-type ATLL patients remains unsatisfactory.

Previously, we reported that cell adhesion molecule 1 (CADM1), originally well known as a tumor suppressor in non-small cell lung cancer (NSCLC), is highly expressed in ATLL cells and promotes tumor growth and multiple organ invasion [10-12]. More recently, we discovered that an enhancer element influencing CADM1 expression in the CADM1 promoter region in ATLL cells contains a nuclear factor-κB (NF-κB)-binding sequence and that CADM1 expression is dependent on activation of the canonical NF-κB pathway [13]. The NF-κB pathway is well known for regulating innate immune cells and inflammatory T cells. NF-κB activation occurs via two major signaling pathways, the canonical and noncanonical pathways, which depend on activation of inhibitor of NF-κB α (IκBα) degradation and p100 processing, respectively [14]. In HTLV-1-infected T cells, the HTLV-1-encoded oncoprotein Tax stimulates the activation of both the canonical and noncanonical NF-κB pathways [15]. On the other hand, ATLL cells frequently lose Tax expression but acquire the ability to activate the canonical NF-κB pathway without Tax through genetic and epigenetic alterations involving the T-cell receptor/NF-κB signaling pathway, miR-31 silencing, and IL-17RB overexpression [16]. Along with persistent activation signals, the downregulation of the p97/NSFL1 cofactor p47, a negative regulator of the NF-κB pathway, was found to be essential for the constitutive activation of the NF-κB pathway in ATLL cells [17]. p47 is a major adaptor molecule of the cytosolic AAA ATPase p97 and was recently reported to bind to NEMO with Lys63-linked and linear polyubiquitin chains, leading to lysosome-dependent NEMO degradation and suppression of IκB kinase (IKK) activation. We recently identified that CADM1 overexpression is dependent on NF-κB activation through p47 downregulation by lysosomal-autophagic degradation in ATLL cells [13].

In this manuscript, we further analyzed the mechanism of autophagy activation in ATLL cells and determined whether autophagy suppression in ATLL cells is a potential therapeutic approach. Autophagy, roughly translated as “self-eating,” is a lysosome-dependent multistep process that supports cell survival in a hypoxic, starvation, or stress environment. Autophagy allows cells to recycle altered, damaged, or unused organelles and cellular components to prevent cancer development under normal circumstances. By contrast, during tumor progression, autophagy exerts protumoral activity by increasing resistance to anticancer drugs and providing nutrients for cancer cells [18]. Chloroquine (CQ) and hydroxychloroquine (HCQ), a less toxic metabolite of CQ, are widely used to treat malaria and inflammatory diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [19]. Recently, it has been reported that CQ inhibits autophagy by impairing autophagosome-lysosome fusion [20].

In the present study, we investigated the anticancer effects of CQ and HCQ as monotherapies on ATLL cells in vitro and in vivo. ATLL cells exhibited high autophagic flux to maintain cell proliferation. CQ and HCQ were shown to induce apoptosis and inhibit ATLL cell growth both in vitro and in vivo. Autophagy inhibition by CQ or HCQ promoted p47 protein recovery along with loss of CADM1 expression and NF-κB pathway inhibition, leading to caspase-3-related apoptosis. Our findings suggest that inhibition of autophagy by CQ or HCQ may be a potential approach for ATLL treatment.

Materials and methods

Patient samples

Peripheral blood samples were collected at the Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, in collaboration with the Miyazaki University Hospital. Informed consent was obtained from all the patients and the healthy donor. The Institutional Review Board approved this study at the Faculty of Medicine, University of Miyazaki, following the Declaration of Helsinki, the Ethical Guidelines for Medical and Health Research Involving Human Subjects, and the Ethics Guidelines for Human Genomic/Genetic Analysis Research. The diagnosis of ATLL was based on clinical features, hematological characteristics, and monoclonal integration of the HTLV-1 provirus according to Southern blot analysis results. PBMCs were isolated from the peripheral blood of ATLL patients and a healthy volunteer by Ficoll-Paque density gradient centrifugation (GE Healthcare Bioscience AB, Uppsala, Sweden) according to the manufacturer instructions. Primary cells were cultured in AIM-V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS), 10 μM 2-mercaptoethanol (Thermo Fisher Scientific), and 0.75 μg/ml recombinant human IL2 (PeproTech, Rocky Hill, NJ, USA).

Cell lines

Human interleukin-2 (IL-2)-independent ATLL cell lines (Su9T01, S1T, and MT1), HTLV-1-negative human T-cell acute lymphoblastic leukemia (T-ALL) cell lines (MOLT4 and JURKAT), and HTLV-1-infected T cell lines derived from HAM-TSP patients (HCT1, HCT4, and HCT5) [21, 22] were cultured in complete RPMI-1640 medium (Nacalai Tesque Inc, Kyoto, Japan) supplemented with 10% FBS and 50 μg/ml penicillin (10 U/ml)-streptomycin (10 μg/ml) (Fisher Scientific). The IL2-dependent ATLL cell lines KK1, KOB, and ST1 were cultured in complete RPMI-1640 medium supplemented with 0.75 μg/ml recombinant human IL-2. Both primary cells and cell lines were cultured at 37 °C in a humidified 5% CO2 incubator.

In vivo animal experiments

NOD/Shi-scid/IL-2Rγnull (NOG) mice were purchased from CLEA-Japan Inc. (Tokyo, Japan) and maintained under specific pathogen-free (SPF) conditions. Six- to eight-week-old male mice were xenografted subcutaneously (s.c.) in the right flank with 1 x 107 MT2 cells or Su9T01 cells suspended in PBS in a final volume of 100 μl. Body weight and tumor volume were measured every 3 days starting on the day of xenografting. Tumor volume was estimated using caliper measurement of the longest and shortest diameters of the tumor and then calculated using the following equation: shortest diameter2 × longest diameter × 0.5. When the tumor volume reached 100–150 mm3, mice were randomly divided into 2 (water or 50 mg/kg bw/day CQ, injected intraperitoneally (i.p.), n = 5 per group) or 3 groups (water, 6.5 mg/kg bw/day HCQ, or 60 mg/kg bw/day HCQ; per oral (p.o.), n = 7 per group), and treatment was started immediately. The mice were sacrificed after 21 days’ daily treatment (CQ treated model) or seven days after 21 days’ daily treatment (HCQ treated model). All mice were sacrificed with an inhalation overdose of isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan).

For a survival model, NOG mice (minimum body weight: 22 grams) were intravenously injected with 1 x 106 Su9T01 cells suspended in PBS in a final volume of 100 μl. After 3 days, the mice were randomly divided into 2 groups (water or 60 mg/kg bw/day HCQ, p.o, n = 5 per group), and treatment was started immediately. HCQ or a control vehicle (water) was orally given for a 16-day period. Starting on the day of xenografting, body weight was measured every 3 days, and the general conditions were observed daily for a maximum period of 100 days [23]. In both the subcutaneous tumor model and survival model, humanitarian endpoints were based on the following criteria: decreased activity, decreased water intake, decreased appetite, hair growth, dirt on the coat, abnormal posture, or rapid weight loss (25% in 7 days). When the tumor was observed under the skin, the endpoint was a marked increase in tumor size (tumor diameter is 2 cm or 10% or more of body weight), and at that point, the mice were comforted by over-anesthesia for anesthesia. All animal experiments were approved by the Animal Experiment Review Board of the University of Miyazaki (2017-503-6).

Results

CQ and HCQ inhibit ATLL cell growth in vitro

To assess the effects of CQ or HCQ treatment on ATLL cell growth, four ATLL-derived cell lines (Su9T01, KK1, S1T, and ST1), two T-ALL cell lines (MOLT4 and JURKAT), and healthy human peripheral blood mononuclear cells (PBMCs) as a control were treated with different concentrations of CQ or HCQ (0, 1, 5, 10, 50, 100, and 200 μM) for 48 hours. Both CQ and HCQ were found to inhibit growth in all four ATLL cell lines in a dose-dependent manner (Fig 1A and S1A Fig). The IC50 values of CQ and HCQ were 40 +/- 10.2 and 25.9 +/- 15.1 μM, respectively (S1 Table in S1 File). Dose-dependent decreases in cell growth were also observed in primary ATLL cells from patients with acute-type ATLL after treatment with CQ or HCQ (IC50 = 19.5 +/-1.9 and 13.4 +/- 2.8 μM, respectively) (Fig 1B and S2 Table in S1 File). Interestingly, CQ or HCQ treatment also inhibited cell growth in HTLV-1-infected T cell lines derived from HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) patients in a dose-dependent manner (IC50 CQ = 34.73 +/- 24.89 and IC50 HCQ = 30.37 +/- 12.46) (S1B Fig).

Fig 1

CQ and HCQ inhibit ATLL cell growth in vitro.

(A) Four ATLL cell lines (Su9T01, KK1, S1T, and ST1), two T-ALL cell lines (MOLT4 and JURKAT), and PBMCs from a healthy donor were treated with varying concentrations of CQ or HCQ for 48 hours. Cell viability was determined by an MTT assay. Relative cell viability was calculated based on the percentage of untreated cells (0 μM). Data are presented as the mean ± SD (n = 3). *, #, +, ‡, $, or ¥ p < 0.05 for Su9T01, KK1, S1T, ST1, MOLT4, or JURKAT cells compared to healthy PBMCs, respectively. (B) Primary ATLL cells from four acute-type ATLL patients were treated with varying concentrations of CQ or HCQ for 48 hours. Cell viability was determined with the MTT assay. Relative cell viability was calculated based on the percentage of untreated cells (0 μM). Data are presented as the mean ± SD (n = 3). *, #, or + p < 0.05 for Pt #1, Pt #2, or Pt #3 compared to healthy PBMCs, respectively. (C) S1T and KK1 cell lines were treated with 50 μM CQ or 25 μM HCQ for 6, 12, 18, or 24 hours. The expression of cleaved caspase-3 was examined by Western blot analysis. β-actin was used as a loading control. (D) S1T and KK1 cell lines were treated with 50 μM CQ or 25 μM HCQ for 24 hours, double stained with Annexin-V and DAPI, and then analyzed by flow cytometry.

To determine the mechanism of apoptosis in CQ- or HCQ-treated ATLL cells, we evaluated the expression of cleaved caspase-3 as a marker of apoptosis by Western blot analysis. The results showed that the cleaved caspase-3 level was distinctly increased by CQ treatment in a time-dependent manner (Fig 1C). Moreover, an apoptosis assay using Annexin-V/DAPI double staining revealed a significant increase in apoptotic cells in both KK1 cells and S1T cells after 24 hours of exposure to CQ. Similar results were observed in HCQ-treated KK1 and S1T cells (Fig 1D). These results suggest that CQ and HCQ are potential drug candidates for ATLL treatment.

CQ and HCQ inhibited autophagy and the canonical NF-κB pathway in ATLL cell lines

To determine whether CQ-mediated inhibition of ATLL cell growth is associated with suppression of NF-kB through blockade of p47 degradation via autophagic flux, we initially assayed the turnover of LC3-phospholipid conjugate (LC3-II) as an autophagic marker protein in ATLL cell lines (Su9T01, KOB, KK1, and S1T) that were either untreated or treated with various doses of CQ. CQ treatment caused the accumulation of autophagosomes, as indicated by the increased levels of LC3-II in all four ATLL cell lines, suggesting that CQ inhibits autophagic flux in ATLL cells (S2A Fig). Moreover, by immunofluorescence, we observed a significant accumulation of LC3 puncta in S1T and KK1 treated with CQ/HCQ compared to the untreated group (Fig 2A and S2B Fig).

Fig 2

CQ and HCQ inhibited autophagy and the canonical NF-κB pathway in ATLL cell lines.

(A) Representative confocal immunofluorescence of LC3 in KK1 and S1T cells after treatment with either 50 μM CQ or 25 μM HCQ for 24 hours. Scale bar 10μm. (B) Western blot analysis of CADM1, p47, and the indicated NF-κB and autophagic signaling proteins in S1T cell lines was performed after treatment with either 50 μM CQ or 25 μM HCQ for 6, 12, 18, or 24 hours. β-actin was used as a loading control. The cropped gels/blots are shown in the figure, and the full-length gels/blots are presented in S1 Raw images. (C) LC3-II turnover is presented as the mean ± SD (n = 2 independent experiments, representative blot was shown at Fig 2A). *p < 0.05, **p<0.01 compared to the control.

We next determined whether inhibition of autophagy by CQ/HCQ treatment is associated with the accumulation of p47, resulting in the suppression of the activated NF-κB pathway, in ATLL cells. Along with the accumulation of LC3-II, p47 protein levels increased after treatment of ATLL cell lines with CQ/HCQ (Fig 2B and 2C, S2C and S2D Fig). The upregulation of p47 induced increased NEMO degradation with accumulation of IκBα, resulting in reduced expression of CADM1 (Fig 2B and 2C, S2C and S2D Fig). Therefore, autophagy inhibition by CQ/HCQ treatment restores the expression of p47 and sequentially inhibits the activation of the NF-κB pathway, leading to apoptosis in ATLL cells.

CQ and HCQ inhibit tumor growth and extend the lifespan of ATLL cells xenografted mice

To evaluate the in vivo therapeutic effect of CQ/HCQ on ATLL cells, we first established a subcutaneous xenograft model using immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice. Starting seven days after subcutaneous implantation of Su9T01/ATLL cells, CQ or a control vehicle (water) was intraperitoneally injected at a dose of 50 mg/kg bw daily, and tumor growth was monitored every 3 days for a 16-day period. CQ treatment significantly reduced the tumor volume of Su9T01 subcutaneous xenografts compared with control treatment (Fig 3A and S3A Fig). Then, HCQ was orally administered at a dose of 0, 6.5 or 60 mg/kg bw daily to NOG mice subcutaneously transplanted with Su9T01 or MT2 cells. HCQ treatment at 6.5 mg/kg bw or 60 mg/kg bw effectively inhibited tumor growth in Su9T01- and MT2-transplanted mice (Fig 3B and 3C, S3B and S3C Fig). H&E (Fig 3D) and immunohistochemical staining with an anti-cleaved caspase 3 antibody (Fig 3E) demonstrated that the number of apoptotic cells in tumor xenografts was significantly increased by treatment with HCQ (Fig 3F). The degeneration and necrosis of tumor cells can be seen in the HCQ treated groups (Fig 3D, arrows), which showed condensed hyperchromatic nuclei or fragmented nuclei with shrunk cytoplasm. Some broke down necrotic cells released the cellular debris to the extracellular area. The necrotic areas in the 6.5 mg/kg bw HCQ treated group were moderately observed in the tumor sheet and the center of the mass. The 60 mg/kg bw HCQ treated group showed hypereosinophilic with a large volume cytoplasm and highest intercellular spaces among groups (Fig 3D), along with predominantly observed necrotic area compared to 6.5 mg/kg bw HCQ group. In addition, mouse body weights were unchanged by CQ or HCQ treatment (S4 Fig). Finally, HCQ was administered orally at a dose of 0 or 60 mg/kg bw to NOG mice transplanted with Su9T01 cells via tail vein injection, and mouse survival was monitored. The HCQ-treated group showed a remarkably extended lifespan compared with the control group (Fig 3G). Collectively, these results suggest that CQ/HCQ generates a vigorous antileukemic effect through ATLL cell apoptosis.

Fig 3

CQ/HCQ inhibited tumor growth and extended the lifespan of ATLL cells-xenografted mice.

(A) Tumor volume of Su9T01 xenograft tumors in NOG mice intraperitoneally administered water (control) or 50 mg/kg bw CQ daily for 16 days. (B, C) Tumor volume of MT2 (B) or Su9T01 (C) xenograft tumors in NOG mice orally administered water (control), 6.5 mg/kg bw HCQ, or 60 mg/kg bw HCQ daily for 16 days. (D, E) Representative H&E (D) and IHC images of Caspase-3 (E) in tumor tissues from NOG mice orally administered water (1), 6.5 mg/kg bw HCQ (2), or 60 mg/kg bw HCQ (3) daily for 16 days. Scale bars: 300 μm (D), 30 μm (E). Higher magnification images of the boxed areas are shown on the right (Scale bars: 30 μm). The arrows indicate dead tumor cells. (F) Quantification Caspase3 stained cells is presented as the mean ± SD (n = 10 HPF, representative image was shown at Fig 3E). (G) Kaplan-Meier survival curves of the intravenous Su9T01 leukemia model orally treated with HCQ or water daily for 16 days. *p < 0.05, **p<0.01 compared to the control.

Discussion

In this study, we provided evidence that CQ and HCQ inhibit ATLL cell growth both in assays performed in vitro and in an in vivo mouse model of ATLL. ATLL cells exhibited high autophagic flux, and autophagy inhibition by CQ or HCQ promoted p47 protein recovery and inhibition of NF-κB activation, leading to apoptosis in ATLL cells.

Recently, we demonstrated that p47 degradation by the autophagic process enhances CADM1 expression in ATLL cells [13]. Interestingly, enforced expression of p47 in ATLL cells produced significant cell growth inhibition and downregulation of CADM1 [13]. These findings suggested that the inhibition of autophagy might be a potentially useful approach for ATLL treatment. Therefore, in the present study, we investigated the molecular effects of CQ and HCQ, which are recently described autophagy inhibitors [20]. We found that CQ/HCQ monotherapy inhibited the autophagic process, thus restoring p47 expression and inhibiting the NF-κB pathway with downregulation of CADM1 expression and that this NF-κB pathway inhibition ultimately led to caspase-3-related apoptosis induction.

CQ and HCQ, well-known antimalarial drugs, have been shown to inhibit the autophagic process and are recognized as new promising agents in cancer therapy. In in vitro experiments or in vivo mouse models, CQ/HCQ treatment was shown to induce apoptosis in solid cancer cells including melanoma [24] and pancreatic adenocarcinoma [25] cells, primary effusion lymphoma cells [26], and hematopoietic cancer cells, including acute myeloid leukemia (AML) cells [27, 28]. We observed that CQ/HCQ effectively inhibited ATLL cell proliferation and induced caspase-related apoptosis in a dose- and time-dependent manner. Moreover, our in vivo study demonstrated significant tumor growth inhibition, a high number of apoptotic cells in tumor xenografts, and survival rate improvement by treatment with CQ/HCQ.

Autophagy is a multistage degradation process and can be inhibited at any autophagic flux stage; however, principally, autophagy inhibitors are divided into those acting on the early stage (initiation stage, such as PI3K inhibitors) and those acting on the late stage (fusion and degradation stage, such as autophagolysosome fusion, protease, and lysosomal inhibitors) [29, 30]. Interestingly, CQ and HCQ were initially considered lysosomal inhibitors, with the same mechanism as the late-stage autophagy inhibitor bafilomycin, but recently, it was revealed that CQ inhibits autophagolysosome fusion instead of impairing the lysosomal degradation capacity [20]. In line with these findings, we found that CQ effectively inhibited autophagy, which was accompanied by downregulation of NEMO, an NF-κB modulator that is degraded through the lysosomal pathway. In addition, CQ/HCQ treatment also prevented IκBα degradation, consistent with a previous report showing that the inhibition of autophagy by CQ blocks bortezomib-induced NF-κB activation in diffuse large B-cell lymphoma (DLBCL) cells [31]. Thus, CQ and HCQ may represent a new class of NF-κB inhibitors that affect the stability of important NF-κB signaling molecules, including p47 and IκBα, through autophagy inhibition.

CQ, together with HCQ, a less toxic derivative of CQ, is also useful in the treatment of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE) [32]. Clinically relevant doses of HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model, and no signs of adverse effects were observed. Approximately 50% of ATLL patients exhibit skin manifestations, including nodules, plaques, ulcers, erythroderma and purpura. Although these manifestations are often treated with various skin-directed therapies, such as phototherapy and radiation therapy, the overall survival of eruption-bearing patients is poorer than that of ATLL patients without an eruption [33, 34]. Therefore, CQ and HCQ could be promising drugs for ATLL therapy, which offers innovative insight for drug repositioning.

To sum up, we propose an anti-tumor model of CQ/HCQ in ATLL cells in Fig 4. CQ/HCQ treatment inhibited the high autophagic flux of ATLL cells. At this point, CQ/HCQ rescues p47 protein from the autophagy-lysosomal degradation pathway. The abundant p47 inhibited NEMO-IKK complex interaction, leading to downregulation of NEMO and preventing IKba degradation. Finally, the inhibition of NF-kB activation induced downregulation of CADM1 and caspase-related apoptosis.

Fig 4

Model illustrating the mechanism of CQ/HCQ-induced ATLL cell apoptosis by autophagy and NF-κB pathway inhibition.

In ATLL cells, p47 is degraded by the autophagy-lysosome pathway, leading to increased NEMO protein level and activation of IKK, which result in the constitutive activation of the NF-κB pathways. CQ and HCQ inhibit autophagy and increase p47 protein level, which inhibit NF-κB pathway activation along with loss of CADM1 expression, leading to caspase-3 related apoptosis.

In conclusion, this study showed that autophagy has an essential function in ATLL proliferation and that autophagy inhibition by CQ/HCQ might be a promising therapeutic strategy. Our study provides the rationale for a clinical trial evaluating HCQ in the future.

CQ and HCQ treatment inhibit the growth of ATLL and HTLV-1-infected T-cell lines derived from HAM/TSP patients in vitro.

(A) Four ATLL cell lines (Su9T01, KK1, S1T, and MT2), two T-ALL cell lines (MOLT4 and JURKAT), and PBMCs from a healthy donor were treated with 50 μM CQ or 25 μM HCQ for 48 hours. Cell viability was determined by trypan blue. Relative cell viability was calculated based on the percentage of untreated cells (0 μM). Data are presented as the mean ± SD (n = 3). *p < 0.05 for Su9T01, KK1, S1T, MT2, MOLT4, or JURKAT cells compared to healthy PBMCs, respectively. (B) HCT1, HCT4, and HCT5 cell lines were treated with varying concentrations of CQ or HCQ for 48 hours. The relative cell viability was calculated as the percentage of untreated cells (0 μM). Data are presented as the mean ± SD (n = 3). *, #, or + P < 0.05, HCT1, HCT4, or HCT5 compared to Healthy PBMC, respectively.

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ATLL cells exhibit high autophagic flux levels and CQ/HCQ inhibit autophagy and canonical NF-κB pathway.

(A) Su9T01, KOB, KK1 and S1T cell lines were treated with varying concentrations of CQ for 24 hours. The expression of LC-3 was examined by Western blot analysis. (B) Quantification of LC3 puncta/cell is presented as the mean ± SD (n = 3, representative image was shown at Fig 2A). (C) Western blot analysis of CADM1, p47, and the indicated NF-κB and autophagy signaling proteins was performed in KK1 cell lines after treatment with either 50 μM CQ or 25 μM HCQ for 6, 12, 18, 24 hours. β-actin was used as a loading control. The cropped gels/blots are used in the figure, and the full-length gels/blots are presented in S1 Raw images. (D) LC3-II turnover are presented as the mean ± SD (n = 2 independent experiments, representative blot was shown at S2B Fig). *p < 0.05, **p<0.01 compared to control.

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CQ/HCQ inhibit tumor growth in ATLL cell xenograft mice.

Photograph of a tumor isolated from mice bearing the indicated cell lines after treatment with CQ, HCQ, or water.

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CQ/HCQ treatment does not affect the mice’s body weight.

Records of weight variations of mice at every 3 days. Arrow indicates the time of treatment.

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Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

15 Jun 2021

PONE-D-21-15148

Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells

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6. Please cite Supporting Information Figure S5 in your main manuscript.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. The authors should replace most of the references to the proper ones in the introduction section. I attached revised ref lists for their conveniences.

2. No description of the PBL used in Fig.1A&B. in the Methods section. They should describe how they collected from healthy volunteers.

3. In clinical use, HCQ is less toxic than CQ. In Fig1A&B however, PBL viability seems dropping with less amount of HCG than CQ. Please rationalize this discordance.

4. Cmax of CQ is around 500nM. The viability of PBL and ATL cells look quite similar at this concentration. How the authors improve the efficacy of CQ/HCQ in clinical practice? It seems difficult to have sufficient efficacy with single drug treatment.

Reviewer #2: The study presented by Yanuar Rahmat Fauzi et al. entitled “Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells” shows that CQ and HCQ inhibit ATLL cell growth in in vitro assays as well as in a mouse model for ATLL. The autophagy inhibition mediated by CQ or HCQ promoted p47 protein recovery and inhibition of NF-κB activation, leading to apoptosis in ATLL cell lines.

The manuscript presents interesting data, it is novel, and conclusions are supported by the experimental data. However, the issues listed below should be addressed.

Major Points:

- Page 9. Authors claim that CQ and HCQ impair ATLL cell growth. However, they perform a MTT assay that assays metabolically active cells, then MTT is an indirect parameter of viability instead of cell growth. There are many situations where cells might not divide and be metabolically active. A measurement with trypan blue, or a supravital stain is recommended.

- Figure 2. Treatment with different CQ concentration and LC3 western blot for ST1 cell line should be depicted.

- The authors state that CQ treatment causes accumulation of autophagosomes. They support that state by the increased level of LC3-II in all ATLL cell lines in WB (figure S2A). It would be nice to show the increased LC3 positive dots by immunofluorescence.

- Page 11. Text says that the number of apoptotic cells in tumor xenografts was significantly increased by treatment with HCQ. However, authors do not show any statistical analysis to support such a thing. It would be necessary to quantify the caspase 3 positive cells or alternatively use another methodology such as TUNEL assay in xenografts and apply the appropriate statistical analysis.

- The authors used a Su9To1 cell injection model in the tail vein of mice, then survival was monitored. The authors must clarify this model better in Methods section. Additionally, they should give an explanation that justify the use of this model and/or insert a citation where the model is described.

Minor Points:

- Clarify in the text that autophagy refers to macroautophagy.

- In the title “CQ and HCQ inhibited autophagy and the canonical NF-κB pathway” it must be disclosed that it is in ATLL cells lines, so, it should be “CQ and HCQ inhibited autophagy and the canonical NF-κB pathway in ATLL cells lines”.

- Figure S2C, authors should clarify if LC3-II turnover calculation is from S2B WB.

- To enhance an easy reading, the results from figure S1 B and C must be included in Figure 1.

- How do authors explain the behavior of HTLV-1-infected T cell lines derived from HAM-TSP patients (HCT1, HCT4 and HCT5) in figure S1A?

- Figure S3. Please, clarify which cell line corresponds to each picture.

- In Figure 3, authors have to clarify images’ labels.

- In the title "CQ and HCQ inhibit tumor growth and extend the lifespan of ATLL cells in xenograft mice", Do CQ and HCQ extend the lifespan of ATLL cells or the mice?

- It would be nice to transfer to the discussion section the proposed model in Figure S5.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Maria Noé Garcia

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: PONE-D-21-15148 REF revised.docx

Click here for additional data file.

30 Jun 2021

Response to Reviewers:

June 24th, 2021

Dr. Guillermo Velasco

Academic Editor at PLOS ONE

Reference: Manuscript Number: PONE-D-21-15148

“Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells”

Dear Dr. Guillermo Velasco:

We have revised the manuscript according to the editorial requests. We believe that we have answered all points raised during the review process and hope that the revised manuscript is now acceptable for publication.

Yours sincerely,

Kazuhiro Morishita MD., PhD

Director, HTLV-1/ATL Research, Education and Medical Facility, Faculty of Medicine, University of Miyazaki

Professor, Project for Advanced Medical Research and Development,

Project Research Division, Frontier Science Research Center, University of Miyazaki,

5200 Kihara, Kiyotake, Miyazaki, Miyazaki, JAPAN 889-1692

Phone: +81-985-85-9610 Fax: +81-985-85-0609

E-mail: kmorishi@med.miyazaki-u.ac.jp

Editor’s notes:

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

In response to the editor’s comment, we have changed the writing style to follow PLOS ONE's style requirements. Please kindly let us know if we have missed something.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"The authors are grateful to Dr. Yasuaki Yamada (Nagasaki University, Japan) and Dr. Naomichi Arima (Kagoshima University, Japan) for providing cell lines. This work was supported in part by Grants-in-Aid for Scientific Research (B) (25293081 and 17H03581) (KM) from the Japan Society for the Promotion of Science (JSPS) and by the Takeda Science Foundation (KM)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"This work was supported in part by Grants-in-Aid for Scientific Research (B) (25293081 and 17H03581) (KM) from the Japan Society for the Promotion of Science (JSPS) and by the Takeda Science Foundation (KM)."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

In response to the editor’s comment, we have corrected the sentences, “"The authors are grateful to Dr. Yasuaki Yamada (Nagasaki University, Japan) and Dr. Naomichi Arima (Kagoshima University, Japan) for providing cell lines” in the Acknowledge section (page 16)

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

In response to the editor’s comment, we will provide the repository information.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

In response to the editor’s comment, we have created a separate file, named S1_raw_images.pdf

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

In response to the editor’s comment, we have authenticated the corresponding author’s ORCID iD

6. Please cite Supporting Information Figure S5 in your main manuscript.

In response to the reviewer’s comment, we moved Figure S5 to Figure 4 and added paragraph to the Discussion section

Reviewer #1:

1. The authors should replace most of the references to the proper ones in the introduction section. I attached revised ref lists for their conveniences.

In response to the reviewer’s comment, we have replaced the suggested references (References number 3, 4, 5, 7, 8, 9, and 12).

2. No description of the PBL used in Fig.1A&B. in the Methods section. They should describe how they collected from healthy volunteers.

In response to the reviewer’s comment, we added the sentences “Informed consent was obtained from all the patients and the healthy donor” in the Methods-Patient samples section (page 7).

3. In clinical use, HCQ is less toxic than CQ. In Fig1A&B however, PBL viability seems dropping with less amount of HCQ than CQ. Please rationalize this discordance.

Our study showed that IC50 of HCQ was lower than CQ. Consistent results with other cells (ATLL, T-ALL and HAM-TSP cells), indicating that HCQ is more potent than CQ. So, to reach a similar effect, HCQ needs a lower dose than CQ. Regarding toxicity, our in vivo studies using a recommended maintenance dose of HCQ for SLE in humans, and we did not observe any particular side effects during experiments.

4. Cmax of CQ is around 500nM. The viability of PBL and ATL cells look quite similar at this concentration. How the authors improve the efficacy of CQ/HCQ in clinical practice? It seems difficult to have sufficient efficacy with single drug treatment.

We understand that at 500nM our in vitro data does not show the desired effect. However, our in vivo study used a clinical maintenance recommended dose for SLE, and it showed a significant tumor growth inhibition and extended the survival. Currently, we do not have any data to explain this in vitro-in vivo discrepancy.

Reviewer #2: The study presented by Yanuar Rahmat Fauzi et al. entitled “Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells” shows that CQ and HCQ inhibit ATLL cell growth in in vitro assays as well as in a mouse model for ATLL. The autophagy inhibition mediated by CQ or HCQ promoted p47 protein recovery and inhibition of NF-κB activation, leading to apoptosis in ATLL cell lines.

The manuscript presents interesting data, it is novel, and conclusions are supported by the experimental data. However, the issues listed below should be addressed.

Major Points:

1. Page 9. Authors claim that CQ and HCQ impair ATLL cell growth. However, they perform a MTT assay that assays metabolically active cells, then MTT is an indirect parameter of viability instead of cell growth. There are many situations where cells might not divide and be metabolically active. A measurement with trypan blue, or a supravital stain is recommended.

In response to the reviewer’s comment, we performed a growth assay measured by trypan blue. The results showed that there was significance difference cell growth between KK1, S1T, MT2, MOLT4 and JURKAT compared to healthy PBMC (Fig S1 B). We added citation, sentences and figures in the Result and Supplementary methods section (page 9, Figure S1B)

2. Figure 2. Treatment with different CQ concentration and LC3 western blot for ST1 cell line should be depicted.

We think the reviewer mistyping S1T as ST1, as the correlated cell line in the figure 2 is S1T.

In response to the reviewer’s comment, we assayed the LC3 turnover of S1T that were either untreated or treated with various doses of CQ. The results showed increased levels of LC3-II in a dose dependent pattern (Fig S2A). We added sentences and figures in the Result section (page 11, Figure S1B)

3. The authors state that CQ treatment causes accumulation of autophagosomes. They support that state by the increased level of LC3-II in all ATLL cell lines in WB (figure S2A). It would be nice to show the increased LC3 positive dots by immunofluorescence.

In response to the reviewer’s comment, we performed an immunofluorescence assay and quantified the puncta. The results showed that CQ/HCQ treatment significantly increased the LC3 expression. We added sentences and figures in the Result section (page 11, Figure 2C and S2D)

4. Page 11. Text says that the number of apoptotic cells in tumor xenografts was significantly increased by treatment with HCQ. However, authors do not show any statistical analysis to support such a thing. It would be necessary to quantify the caspase 3 positive cells or alternatively use another methodology such as TUNEL assay in xenografts and apply the appropriate statistical analysis.

In response to the reviewer’s comment, we quantified the caspase 3 stained cells and analyzed the data. The results showed that the number of caspase 3 stained cells in tumor xenografts was significantly increased by treatment with HCQ. We added the sentences and figures in the results section (page 12, Figure 3G)

5. The authors used a Su9To1 cell injection model in the tail vein of mice, then survival was monitored. The authors must clarify this model better in Methods section. Additionally, they should give an explanation that justify the use of this model and/or insert a citation where the model is described.

In response to the reviewer’s comment, we added sentences, “After 3 days, the mice were randomly divided into 2 groups (water or 60 mg/kg bw/day HCQ, p.o.), and treatment was started immediately. HCQ or a control vehicle (water) was orally given for a 16-day period”, and insert the citation (page 9)

Minor Points:

1. Clarify in the text that autophagy refers to macroautophagy.

As the reviewer noted, we added the sentences “An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy(herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway” in the Abstract section (Page 3).

2. In the title “CQ and HCQ inhibited autophagy and the canonical NF-κB pathway” it must be disclosed that it is in ATLL cells lines, so, it should be “CQ and HCQ inhibited autophagy and the canonical NF-κB pathway in ATLL cells lines”.

As the reviewer noted, we corrected the title “CQ and HCQ inhibited autophagy and the canonical NF-κB pathway” to “CQ and HCQ inhibited autophagy and the canonical NF-κB pathway in ATLL cells lines” in the Results section (page 10) .

3. Figure S2C, authors should clarify if LC3-II turnover calculation is from S2B WB.

As the reviewer noted, we changed the sentences “LC3-II turnover is presented as the mean ± SD (n = 2). *p < 0.05, **p<0.01 compared to the control” to “LC3-II turnover is presented as the mean ± SD (n = 2 independent experiments, representative blot was shown at Fig 2 A). *p < 0.05, **p<0.01 compared to the control” in the figure legend

4. To enhance an easy reading, the results from figure S1 B and C must be included in Figure 1.

As the reviewer noted, we moved the “Figure S1 B and C” to the “Figure 1 C and D.

5. How do authors explain the behavior of HTLV-1-infected T cell lines derived from HAM-TSP patients (HCT1, HCT4 and HCT5) in figure S1A?

As the reviewer noted, we edited the sentences “Interestingly, CQ or HCQ treatment also inhibited cell growth in HTLV-1-infected T cell lines derived from HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) patients (Fig S1A)” to “Interestingly, CQ or HCQ treatment also inhibited cell growth in HTLV-1-infected T cell lines derived from HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) patients in a dose-dependent manner (IC50 CQ = 34.73 +/- 24.89 and IC50 HCQ = 30.37 +/- 12.46) (Fig S1A)” in the Results section (page 9).

6. Figure S3. Please, clarify which cell line corresponds to each picture.

As the reviewer noted, we added the label to each group

(A. Su9T1 cell line xenografted mice, B. MT2 cell line xenografted mice and C. Su9T1 cell line xenografted mice)

7. In Figure 3, authors have to clarify images’ labels.

As the reviewer noted, we added the label to each group

(A. Su9T01 cell line xenografted mice, B. MT2 cell line xenografted mice and C. Su9T01 cell line xenografted mice)

8. In the title "CQ and HCQ inhibit tumor growth and extend the lifespan of ATLL cells in xenograft mice", Do CQ and HCQ extend the lifespan of ATLL cells or the mice?

As the reviewer noted, we corrected the title "CQ and HCQ inhibit tumor growth and extend the lifespan of ATLL cells in xenograft mice" to “CQ and HCQ inhibit tumor growth and extend the lifespan of ATLL cells xenografted mice” in the Results Section (page 11)

9. It would be nice to transfer to the discussion section the proposed model in Figure S5.

As the reviewer noted, we moved the proposed model in figure S5 to main figure 4, and added the paragraph “To sum up, we propose an anti-tumor model of CQ/HCQ in ATLL cells in Fig 4. CQ/HCQ treatment inhibited the high autophagic flux of ATLL cells. At this point, CQ/HCQ rescues p47 protein from the autophagy-lysosomal degradation pathway. The abundant p47 inhibited NEMO-IKK complex interaction, leading to downregulation of NEMO and preventing IKba degradation. Finally, the inhibition of NF-kB activation induced downregulation of CADM1 and caspase-related apoptosis” in the Discussion section (page 14)

Submitted filename: Reviewers comments Letter.docx

Click here for additional data file.

21 Jul 2021

PONE-D-21-15148R1

Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells

PLOS ONE

Dear Dr. Morishita,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Specifically please address the modifications indicated by reviewer 2.

Please submit your revised manuscript by Sep 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Guillermo Velasco, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: They nicely reorganized their first manuscript to the revised one including some additional data and texts.

Reviewer #2: The revised manuscript by Yanuar Rahmat Fauzi et al. has been improved and authors have responded to my questions and concerns. However, I still see necessary to clarify the following three points:

1. Please, explain in the materials and methods section, how LC3 positive dots were quantified in immunofluorescence (Figure S2D), and mainly the used criteria.

2. On the same line, with the previous point, please explain in the materials and methods section, quantification criteria for caspase 3 in Figure 3G.

3. The quality of figures 3 D and E should be improved.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Maria Noé Garcia

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Jul 2021

Response to Reviewers:

July 22nd, 2021

Dr. Guillermo Velasco

Academic Editor at PLOS ONE

Reference: Manuscript Number: PONE-D-21-15148R1

“Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells”

Dear Dr. Guillermo Velasco:

We have revised the manuscript according to the editorial requests. We believe that we have answered all points raised during the review process and hope that the revised manuscript is now acceptable for publication.

Yours sincerely,

Kazuhiro Morishita MD., PhD

Director, HTLV-1/ATL Research, Education and Medical Facility, Faculty of Medicine, University of Miyazaki

Professor, Project for Advanced Medical Research and Development,

Project Research Division, Frontier Science Research Center, University of Miyazaki,

5200 Kihara, Kiyotake, Miyazaki, Miyazaki, JAPAN 889-1692

Phone: +81-985-85-9610 Fax: +81-985-85-0609

E-mail: kmorishi@med.miyazaki-u.ac.jp

Editor’s notes:

Journal Requirements:

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Reviewer #1: They nicely reorganized their first manuscript to the revised one including some additional data and texts.

Reviewer #2: The revised manuscript by Yanuar Rahmat Fauzi et al. has been improved and authors have responded to my questions and concerns. However, I still see necessary to clarify the following three points:

1. Please, explain in the materials and methods section, how LC3 positive dots were quantified in immunofluorescence (Figure S2D), and mainly the used criteria.

In response to the reviewer’s comment, we added the sentences “Average numbers of LC3 puncta/cell were quantified by blinded manual counting of puncta, with at least 30 cells counted per group.” in the Supplementary Methods section (page 6).

2. On the same line, with the previous point, please explain in the materials and methods section, quantification criteria for caspase 3 in Figure 3G.

In response to the reviewer’s comment, we added the sentences “Quantification of anti-cleaved caspase-3 positive cells was performed by blinded manual counting the number of positive cells/High Power Field (HPF).” in the Supplementary Methods section (page 7).

3. The quality of figures 3 D and E should be improved.

In response to the reviewer’s comment, we have changed figures 3 D and E.

Submitted filename: Response to Reviewers-20210722.docx

Click here for additional data file.

4 Aug 2021

Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells

PONE-D-21-15148R2

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6 Aug 2021

PONE-D-21-15148R2

Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells

Dear Dr. Morishita:

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PLOS ONE