Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2022 Nov 22;17(11):e0277893. doi: 10.1371/journal.pone.0277893

A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia

Yi Chen 1, Tianze Wu 1, Chengbin Yang 1, Mingzhu Lu 1, Zhenxia Chen 1, Mingli Deng 1, Yu Jia 1, Yongtai Yang 1, Xiaofeng Liu 1, Hongyan Wang 2, Yun Ling 1,3, Lei Lu 2,*, Yaming Zhou 1,*
Editor: Jianhong Zhou4
PMCID: PMC9681083  PMID: 36413544

Abstract

Aberration of PI3K signaling pathway has been confirmed to be associated with several hematological malignancies including acute myeloid leukemia (AML). FD268, a pyridinesulfonamide derivative characterized by the conjugation of 7-azaindole group, is a newly identified PI3K inhibitor showing high potent enzyme activity at nanomole concentration. In this study, we demonstrated that FD268 dose-dependently inhibits survival of AML cells with the efficacy superior to that of PI-103 (pan-PI3K inhibitor) and CAL-101 (selective PI3Kδ inhibitor) in the tested HL-60, MOLM-16, Mv-4-11, EOL-1 and KG-1 cell lines. Further mechanistic studies focused on HL-60 revealed that FD268 significantly inhibits the PI3K/Akt/mTOR signaling pathway, promotes the activation of pro-apoptotic protein Bad and downregulates the expression of anti-apoptotic protein Mcl-1, thus suppressing the cell proliferation and inducing caspase-3-dependent apoptosis. The bioinformatics analysis of the transcriptome sequencing data also indicated a potential involvement of the PI3K/Akt/mTOR pathway. These studies indicated that FD268 possesses high potent activity toward AML cells via inhibition of PI3K/Akt/mTOR signaling pathway, which sheds some light on the pyridinesulfonamide scaffold for further optimization and investigation.

1 Introduction

Myeloid cells originated from hematopoietic stem cells represent a diverse range of innate leukocytes which are constantly supplied to all tissues via circulation [1]. Their abnormal differentiation may cause an increase of immature malignant cells and fewer differentiated red blood cells, platelets and white blood cells, resulting into acute myeloid leukemia (AML) [2, 3]. Owing to its highly aggressive and fatal feature, targeted therapy is commonly recommended as an important way to treat AML besides the bone marrow/stem cell transplant. With the increased understanding of its pathogenesis and subtype-specific pathology, various targets including vascular endothelial growth factor (VEGF) [4], isocitrate dehydrogenase (IDH) [5], Fms-like tyrosine kinase (FLT3) [6], hedgehog pathway [7, 8], ATR-CHK1 pathway [9], etc. have been revealed and specific inhibitors are thereby progressively developed [10–12]. Phosphoinositide 3-kinases (PI3Ks) are a group of heterodimeric lipid kinases regulating crucial cellular processes [13, 14]. It is observed the constitutive activation of PI3K/Akt and the mammalian target of rapamycin (mTOR) in more than 50% AML cases, which makes this signaling pathway a promising target [15–18].

Considerable PI3K/Akt/mTOR inhibitors have been constantly developed and studied either as a monotherapy or a combined therapy, providing valuable insights in AML [19–21]. BKM120, a morpholinopyrimidine derivative, has been confirmed to abrogate the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest in AML cells [22] and impair the viability of AML cells through inducing p21-mediated G2/M cell cycle arrest and reducing expression of NF-κB anti-apoptotic proteins [23]. BEZ235, an imidazole[4,5-c]quinoline derivative, has been revealed to suppress proliferation and induce apoptosis in AML cells via increasing miR-1-3p and subsequently down-regulating BAG4, EDN1 and ABCB1 [24]. The isoform-selective PI3Kδ inhibitor CAL-101, possessing a purine quinazolinone scaffold with the propeller-type conformation, has been identified to inhibit pre-rRNA synthesis and proliferation of AML cells through totally suppressing Akt phosphorylation [25]. Pan-PI3K inhibitor PI-103, belonging to pyridofuropyrimidine class, has shown antileukemic activity in AML via dephosphorylating Akt and suppressing mTORC1 activation [26]. Despite these encouraging results, AML remains a therapeutic challenge due to the intrinsically chemoresistant feature. Given the broader anti-leukemic effects associated with this pathway, the identification of novel PI3K/Akt/mTOR inhibitors and exploration of underlying mechanisms would greatly benefit to the translation to clinically optimized strategies for AML treatment.

Sulfonamide-containing compounds developed in recent years represent a large number of PI3K/Akt/mTOR inhibitors including pan- and isoform-selective inhibitors [27–31]. However, their efficacies and underlying mechanisms in AML cell lines are rarely been explored. By conjugating pyridinesulfonamide motif with 7-azaindole/azaindazole groups, we recently developed a series of PI3K inhibitors [32]. N-(5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl) benzenesulfonamide (hereafter named as FD268, Fig 1A) is the one showing high potent enzyme activity at nanomole concentration. We herein focus on its anti-proliferation effects on AML cells. Our studies revealed that FD268 significantly inhibits their viabilities with the efficacies superior to that of PI-103 and CAL-101. Mechanistic studies revealed that FD268 suppresses cell proliferation, gets cell cycle arrests and induces apoptosis through the inhibition of PI3K activity, there by downregulating Akt phosphorylation at Ser473/Thr308 sites, inhibiting mTOR phosphorylation and downstream targets. Our studies here demonstrated the potent activity of FD268 and the underlying therapeutic mechanism, suggesting a novel pyridinesulfonamide scaffold worthy of further investigation.

Fig 1. FD268 inhibits cell viability of AML cell lines.

Fig 1

Open in a new tab

(A) Structure of the compound FD268. (B-F) Icreasing concentrations of FD268, PI-103, and CAL-101 were used to treat HL-60, MOLM-16, Mv-4-11, KG-1 and EOL-1 cells for 48 h, and the antiproliferative activity of FD268 against five AML cell lines was determined by the CCK-8 assay in triplicate. Growth inhibitory curves and the IC50 values of FD268, PI-103, and CAL-101 in AML cells were also analyzed. The values were shown as the mean ± standard from three independent experiments. NA means no real available value.

2 Materials and methods

2.1 Chemicals

The compound FD268 was synthesized in our laboratory. Pan-PI3K inhibitor PI-103 and isoform-specific PI3K inhibitor CAL-101 were purchased from Selleckchem (Houston, Texas, USA). Stock solutions of all the inhibitors were prepared in dimethyl sulfoxide (DMSO), and 10 μL aliquots were frozen at -80°C.

2.2 Cell culture and treatment

Human leukemic cell lines, MOLM-16 (established from the peripheral blood of a 20-year-old man with acute myeloid leukemia AML) [33], Mv-4-11 (established from the peripheral blood of a 20-year-old man with acute myeloid leukemia AML) [34], EOL-1 (derived from the peripheral blood of a 33-year-old man with hypereosinophilia case terminating in blastic transformation.) [35] and KG-1 (obtained from a 59-year-old Caucasian male with erythroleukemia that evolved into acute myelogenous leukemia) [36], were purchased from Shanghai Bogoo Biotechnology Co., Ltd (Shanghai, China), and the HL-60 (obtained by leukopheresis from a 36-vear-old Caucasian female with acute promvelocytic leukemia) [37] cell line was from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in RPMI-1640 media containing 10% fetal bovine serum (Gibco Life Technologies, Gaithersburg, MD, USA) and 1% penicillin-streptomycin solution (Gibco Life Technologies, Gaithersburg, MD, USA) at 37°C in a humidified atmosphere containing 5% CO2.

2.3 Cell viability assays

Cell viability assays were investigated by the Cell Counting Kit-8 (CCK-8; Vamzyme, Nanjing, China). Briefly, cells were seeded in triplicates at a density of 1 × 104 cells per well on 96-well plates and allowed to adhere overnight. Cells were incubated with different concentrations of PI-103, CAL-101 and FD268 for 48 h. The controls were incubated with 0.1% DMSO. CCK-8 solution was added to each well and further incubated for 2 h at 37°C. The absorbance of optical density was measured at 450 nm using a multi-well spectrophotometer (BioTek Instruments, Inc., mit Hauptsitz in Winooski, VT, USA). The inhibitory rate was calculated using the following formula: 1 –(experimental group OD value/control group OD value) ×100% and the IC50 value was calculated by fitting the data to a logistic curve using GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA, USA). At least three independent experiments were carried out in each cell line.

2.4 Cell cycle and apoptosis analysis

Cells were seeded in triplicates at a density of 5 × 105 cells (cell cycle assays) and 4 × 105 cells (apoptosis assays) per well in 6-well plates and treated with FD268 (0.125, 0.25 and 0.5μM) or DMSO control for 24 h and 48 h, respectively. Cells were harvested after treatment and washed with PBS. For cell-cycle distribution analysis, cells were fixed in ice-cold 70% ethanol, stained by the Propidium Iodide (PI) staining solution (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then incubated at room-temperature in dark for 15 min. For cell apoptosis assays, cells were stained using FITC Annexin V/propidium iodide (PI) Apoptosis Detection Kit (Vamzyme, Nanjing, China) according to the manufacturer’s protocol. The cell cycle analysis and early apoptosis analysis were conducted using the Flow Cytometer system (Beckman Coulter, Brea, CA, USA). The percentage of cells in each phase was calculated using ModFit LT software (Verity Software House, Inc., ME, USA), the cell apoptosis data analysis was performed using FlowJo software (BD Biosciences, San Jose, CA, USA). At least three independent experiments were carried out in cell apoptosis assay.

2.5 Western blot analysis

Cells were seeded in triplicates at a density of 1 × 106 cells per well in 6-well plates and treated with FD268 (0.063, 0.125, 0.25, 0.5 and 1 μM, respectively) or DMSO control for 24 h and then lysed with ice-cold radioimmunoprecipitation (RIPA) buffer (Thermo Fisher Scientific, Inc, Waltham, MA, USA) containing protease and phosphatase inhibitor cocktail tablet (Roche, Mannheim, Germany). Total protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Equal amounts of protein were separated on SDS-polyacrylamide gels (10, 12%), and electro‑transferred to PVDF membranes. Nonspecific protein binding was blocked with 5% bovine serum albumin (BSA) for 1.5 h at room temperature and probed with the primary antibodies overnight at a 1:1000 dilution at 4°C. The primary antibodies: anti-β-actin polyclonal antibody (Cat#10494-1-AP), Phospho-Akt (Ser473) (Cat#4060), Phospho-Akt (T308) (Cat#13038), Akt (Cat#4685), Phospho-mTOR (Ser2448) (Cat#5536), mTOR (Cat#2983), Phospho-4EBP1 (Ser65) (Cat#9451), 4EBP-1 (Cat#9644), Phospho-p70 S6 Kinase (Thr389) (Cat#9205), caspase-3 (Cat#14220), cleaved caspase-3 (Cat#9661), cleaved PARP (Cat#5625), Bad (Cat#9239), Bax (Cat#5023), Bcl-2 (Cat#15071), Mcl-1 (Cat#94296), and Bcl-XL (Cat#2764) were from Cell Signaling Technology (Beverly, MA, USA). Blots were incubated with 1:3000 dilution sheep anti-rabbit IgG (Cat#7074, Cell Signaling Technology Inc., Beverly, MA, USA) at room temperature for 1 h, and immunoreactive protein bands were visualized using Pierce ECL Western blotting substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA). The blot density was determined by FUSION FX software (Vilber Lourmat Inc., France). At least two independent experiments were carried out in western blot assay.

2.6 Immunofluorescence

HL-60 cells were seeded in triplicates at a density of 1 × 106 cells per well in 6-well plates and treated with FD268 (0.125 and 0.5 μM, respectively) or DMSO control for 24 h. Immunofluorescence staining was performed according to the standard operating procedure. Cells were washed with PBS and fixed in pre-cooled methanol for 30 min. Methanol was removed and cells were washed twice with PBS, followed by permeation with 0.2% Triton X-100 for 30 min at room temperature. Cells were washed twice with PBS and blocked with 2% BSA for 1 h at 37°C. Cells were then incubated with primary antibody against p-Akt (Ser473) (1:200, Cell Signaling Technology Inc., Beverly, MA, USA) in 1% BSA at 4°C overnight. Cells were washed twice with PBS and then incubated with secondary antibodies CoraLite594-conjugated Goat-Anti-Rabbit IgG(H+L) (1:200, Cell Signaling Technology Inc., Beverly, MA, USA) in PBS for 1 h at 37°C. Cells were washed twice with PBS, and nuclei were stained with DAPI for 3 min at room temperature. Cells were washed twice with PBS and visualized. Images were obtained by a fluorescence microscopy.

2.7 RNA extraction and real-time RT-PCR

Cells were seeded in triplicates at a density of 1 × 106 cells per well in 6-well plates and treated with FD268 or DMSO as control for 24 h. Total RNA was prepared using the TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Briefly, one microgram of isolated total RNA was reverse transcribed using SuperScript III Reverse Transcriptase kit (Vamzyme, Nanjing, China) to complementary DNA (cDNA). Expression of relative genes of FD268-treated cells was measured by SYBR green based real-time quantitative PCR using Fast SYBR Green Master Mix (Vamzyme, Nanjing, China) at the StepOne RealTime PCR System. All real-time PCR reactions were performed in triplicate. A melting curve analysis was applied to verify the specificity of the products, and the values for relative quantification were calculated based on 2−ΔΔCt relative expression formula and normalized against the geometric average expression HPRT1 housekeeping genes. Nucleotide sequences of the primers (Life Technologies) used for qRT-PCR were listed in S1 Table.

2.8 RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis

HL-60 cells were seeded in triplicates at a density of 1 × 106 cells per well in 6-well plates and treated with FD268 (0.125, 0.5 μM) or DMSO for 24 h. Cells were harvested and the total RNA was extracted for later use. 1000 ng high-quality digested RNA was employed using the VAHTS mRNA-seq v2 Library Prep kit for Illumina (Vamzyme, Nanjing, China), and sequencing libraries were created according to the manufacturer’s protocol. Briefly, first- and second-strand cDNA were synthesized using random hexamer primers. The End-It DNA End Repair kit was used to repair the cDNA fragments. An “A” nucleotide was added to the 30 ends of the fragments, followed by adapter ligation. The ligated cDNA products were subjected to PCR amplification. The library quality was determined using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The Illumina Nova600 platform (Illumina, San Diego, CA, USA) was used for RNA-seq. The quality of RNA Sequencing (RNA-seq) data was evaluated using FastQC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was applied to identify the significant pathway of the differentially expressed mRNAs. Fisher’s exact test was used to identify the significant results, and the false discovery rate (FDR) was applied to correct the p-values (p value < 0.05; fold change > 1.5).

2.9 Statistical analysis

All experiments were analyzed using one-way ANOVA in Graphpad Prism 8.0. (GraphPad Software, Inc., La Jolla, CA, USA). Experiments were presented as the means ± standard error in three independent experiments. Statistical analysis was performed using One-way ANOVA to determine the significance between groups. Differences were considered statistically significant when the P value was less than 0.05 (*).

3 Results

3.1 FD268 exhibits antiproliferative activity against AML cells

Conjugation of pyridinesulfonamide motif with 7-azaindazole group led to the screen out of FD268. It has been identified as a potent pan-PI3K inhibitor with the IC50 value of ~1, ~4, ~7 and ~0.7 nM for PI3K α, β, γ and δ isomer respectively [32]. Since the identified association of the poor prognosis and chemoresistance in AMLs with the PI3K/Akt/mTOR activation, we herein investigated the antiproliferative activity of FD268 against five hematological cancer cell lines: HL-60, MOLM-16, Mv-4-11, KG-1 and EOL-1. As shown in Fig 1, FD268 exhibited a clear dose-dependent inhibitory activity with the 50% inhibitory concentration (IC50) values of 0.027 ± 0.003, 0.072 ± 0.019, 0.043 ± 0.004, 1.191 ± 0.013 and 0.837 ± 0.118 μM respectively. The inhibition activity was comparable to that of pan-PI3K inhibitor PI-103, among which a ~40 fold enhancement was observed in HL-60. On the other hand, the inhibitory activity of FD268 was superior to that of clinical available PI3Kδ selective inhibitor CAL-101 in all tested lines (0.479 ± 0.189, 13.25 ± 2.52, 4.43 ± 1.01μM for HL-60, MOLM-16 and Mv-4-11), especially in MOLM-16 with a ~100 fold enhancement. These results demonstrated that FD268 possessed strong antiproliferative activity against the tested AML cells, especially in HL-60 and MOLM-16.

3.2 FD268 induces cell cycle arrest and apoptosis in AML cells

Given the high potent activity of FD268 against HL-60 and MOLM-16 as well as their corresponding morphological changes (observable cell shrinkage, cytoplasmic condensation and nuclear fragmentation) (S1 Fig.), the cell cycle and apoptosis were consequently analyzed. As shown in Fig 2, the cell population distributed in G0/G1, S and G2/M phase was 35.5 ± 3.7%, 45.8 ± 2.2% and 18.8 ± 3.7% respectively for the untreated HL-60. When incubated with FD268, the proportion of G0/G1 phase cells increased to 52.9 ± 2.4%, meanwhile S and G2/M phase cells decreased to 34.2 ± 4.4% and 12.9 ± 3.6% respectively, suggesting that the cells were arrested in the G1 phase. In contrast, treatment of FD268 induced the increase of G2/M phase cells from 12.3 ± 2.1% to 24.1 ± 6.8% in MOLM-16, indicating the G2 phase cycle arrest. The cell-line-dependent cell-cycle arrest and the observable cell morphological changes raised our interest whether FD268 exerted distinct modes of cell death. Thus, FD268-treated HL-60 and MOLM-16 were analyzed by flow cytometry with annexin V/propidium iodide double staining. The flow cytometric data showed that the total proportion of AnnexinV-FITC positive and AnnexinV-FITC/PI positive cells dose-dependently increased from 1.8 ± 0.3% (control group) to 28.8 ± 4.3% (0.5μM-FD268-treated groups) in HL-60 and from 2.5 ± 0.1% to 10.0 ± 2.1% in MOLM-16 (Fig 3A and 3B). Considering the cascade processes of caspase-3 and its substrate poly (ADPribose) polymerase (PARP) in inducing cell programmed death, the cleavages of these two proteins were consequently examined by western blot assay, which showed a dose dependent increase of both caspase-3 and PARP cleavage. (Fig 3C). These results demonstrated that FD268 induces apoptosis of the tested two cell lines.

Fig 2. FD268 induces cell cycle arrest in HL-60 and MOLM-16 cells.

Fig 2

Open in a new tab

(A) Induction of cell cycle arrest by FD268 at the increasing concentrations for 24 h in HL-60 and MOLM-16. Cell cycle phase analysis was performed by flow cytometry. (B) Quantitation of the G0/G1, S, and G2 phase cells of HL-60 and MOLM-16. The data shown are the mean ± standard from two independent experiments. Statistical differences compared to control group were analyzed with one-way ANOVA. Statistical significance is represented with asterisks *P < 0.05 and ***P < 0.001.

Fig 3. FD268 induces cell apoptosis in HL-60 and MOLM-16 cells.

Fig 3

Open in a new tab

(A) Induction of cell apoptosis by FD268 at the increasing concentrations for 48h. Apoptotic cells were quantified by flow cytometry after staining with PI and FITC-Annexin V. (B) The summary of populations stained with only Annexin V (early apoptosis) and with both Annexin V and PI (late apoptosis). The data shown are the mean ± standard from three independent experiments. Statistical differences compared to control group were analyzed with one-way ANOVA. Statistical significance is represented with asterisks ***P < 0.001, ****P < 0.0001. (C) Western blot was applied to detect cleaved PARP, cleaved caspase-3 and caspase-3 as indicators of apoptotic cell death. Intensity of the bands was measured, and the fold change of the intensity was compared with that of the control and indicated below the bands. β-actin was used as a loading control.

To take mechanistic insights into the cell-line-dependent cell-cycle arrest and apoptosis, the expression of cyclin-dependent kinase 2 (CDK2) and CDKs inhibitor (p21Cip1, p27Kip1) mRNA were firstly measured. Treatment of 0.25 μM FD268 resulted into a decrease in the expression level of p21Cip1, p27Kip1 and CDK2 in HL-60, but an increase of p27Kip1 and CDK2 in MOLM-16 (Fig 4A and 4B). Since that CDK2 mRNA appeared late in G1 or in early S phase, the differences in the expression may lead to the cell-line-dependent cell-cycle arrest. Nevertheless, on protein level, CDK2 showed decreases to 40 and 60% of the initial level in HL-60 and MOLM-16 by FD268 (Fig 4C). To investigate the underlying mechanism of the cell apoptosis, the expression of Bcl-2 family members, Bad, Bcl-2 and Mcl-1, were consequently measured on the transcriptional and protein level for the tested two cell lines respectively. At the transcriptional level, the anti-apoptosis gene Bcl-2 and Mcl-1 were both dose-dependently downregulated in HL-60, while the pro-apoptotic gene Bad was dose-independent (Fig 5A). In contrast, the gene Bad, Bcl-2 and Mcl-1 was respectively upregulated in MOLM-16 induced by FD268 (Fig 5B). At protein level, FD268 induced a 50% increase in the expression level of pro-apoptotic protein Bad, meanwhile inhibited the anti-apoptotic protein Mcl-1 in both HL-60 and MOLM-16 dose-dependently (Fig 5C). While, the protein levels of other markers of apoptosis, Bcl-2, Bax and Bcl-XL, did not change significantly in FD268-treated cells. These results demonstrated that FD268 could induce the cell cycle arrest and apoptosis in HL-60 and MOLM-16 while the associated inhibition mechanisms are slightly different.

Fig 4. FD268 regulates the expressions of cell cycle related protein in HL-60 and MOLM-16 cells.

Fig 4

Open in a new tab

Cells were treated with increasing concentrations of FD268 for 24 h. Transcriptional levels of cell cycle related genes p21Cip1, p27Kip1 and CDK2 were determined using qRT-PCR in HL-60 (A) and MOLM-16 (B) cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance is represented with asterisks *P < 0.05, **P < 0.01, ****P < 0.0001. (C) The levels of apoptosis-related protein CDK2 in control and FD268-treated cells were determined by western blot. Intensity of the bands was measured, and the fold change of the intensity was compared with that of the control and indicated below the bands. β-actin was used as a loading control.

Fig 5. FD268 regulates Bcl-2 family apoptotic proteins in HL-60 and MOLM-16 cells.

Fig 5

Open in a new tab

Cells were treated with increasing concentrations of FD268 for 24 h. Transcriptional levels of apoptosis-related genes Bcl-2, Bad and Mcl-1 were determined using qRT-PCR in HL-60 (A) and MOLM-16 (B) cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance is represented with asterisks *P < 0.05, **P < 0.01, ***P < 0.001. (C) The levels of apoptosis-related proteins including Bad, Mcl-1, Bcl-2, Bax, Bcl-XL, and in control and FD268-treated cells were determined by western blot. Intensity of the bands was measured, and the fold change of the intensity was compared with that of the control and indicated below the bands. β-actin was used as a loading control.

3.3 FD268 inhibits the activation of PI3K/Akt/mTOR signaling in AML cells

Considering the differences of cell cycle arrest and apoptosis at gene level, we examined the phosphorylation status of AKT, a marker for cellular PI3K activity, and downstream substrates of PI3K signaling in HL-60 and MOLM-16 induced by FD268. As shown in Fig 6, FD268 dose-dependently inhibited the expression of phosphorylated Akt (Ser473 and Thr308) after 24 h. The inhibition of phosphor-Akt (Ser473) expression was also confirmed by the immunofluorescence assay (S2 Fig.). These results indicated that FD268 did effectively inhibit PI3K signaling cascades in HL-60 and MOLM-16. We further determined the effects of FD268 on mTOR activity downstream the PI3K/Akt signaling. Western blot assay showed that FD268 dose-dependently inhibited the phosphorylation of mTOR at Ser2448 site, showing a 50% and 80% decrease of the initial level in HL-60 and MOLM-16 induced by 1 μM FD268 after 24 h respectively. Furthermore, FD268 almost fully inhibited the expression of mTOR downstream factors p-4E-BP1 (Ser65) and p-p70S6K (Thr389) at 1 μM concentration. These results revealed that FD268 exerted the antiproliferation and the induction of apoptosis by inhibition of PI3K/Akt/mTOR signaling pathway in HL-60 and MOLM-16.

Fig 6. FD268 inhibits the PI3K/Akt/mTOR signaling in HL-60 and MOLM-16 cells.

Fig 6

Open in a new tab

HL-60 and MOLM-16 cells were treated with FD268 at increasing concentrations for 24h and harvested to measure the levels of PI3K pathway proteins by western blot. (A) AKT, p-AKT (Ser473), p-AKT (Thr308), mTOR, and p-mTOR (Ser2448) was detected. (B) The level of phosphorylated mTOR target proteins: p-4E-BP1 and p70 ribosomal protein S6 kinase (p-p70S6K) was detected by western blot. FD268 concentration-dependently inhibited the phosphorylation of AKT, mTOR and downstream targets of PI3K pathway. Intensity of the bands was measured, and the fold change of the intensity was compared with that of the control and indicated below the bands. β-actin was used as a loading control.

3.4 FD268 regulates cell survival related genes and pathways in AML cells

To further provide a detailed and unbiased view of the complex and dynamic nature of the transcriptome, RNA-seq analyses were performed on FD268-treated HL-60 cells (0.125 and 0.5 μM respectively for 24 h). All data were analyzed after quality screening, and DESeq2 software was used for screening differentially expressed genes between control and FD268-treated groups (adjusted p-value < 0.05; fold change > 1). There were 194 upregulated and 148 downregulated genes observed in 0.125 μM FD268 treated group. With the increase of concentration to 0.5 μM, the numbers of upregulated and downregulated genes also increased to 655 and 240, respectively. A heat map (Fig 7A) and volcano plots (S3 Fig) of the differentially expressed genes (DEGs) showed a clear distinction between control group and FD268-treated groups. Among the FD268-treated groups, BCL2L2, JUN, TARF11 and TNFRSF10C were related to apoptosis; CDKN2A, CDKN2B and TGFB2 were related to cell cycle, ABCA3 and ABCC11 were related to ATP binding. It is worth to mention that a few genes with a role in acute myeloid leukemia: GRB2, CSFIR, MARCKS and PDGFB were upregulated; FLT1, IL6, PIK3C2B, BALAP3 and MAPK10 were downregulated (Fig 7B–7D).

Fig 7. FD268 induces differential regulation of gene expression in HL-60 cells.

Fig 7

Open in a new tab

(A) HL-60 cells were treated with 0.125 and 0.5 μM FD268 for 24 h at the indicated concentrations and collected for RNAseq analysis. Hierarchical clustering shows deferentially expressed mRNA patterns between the control and FD268-treated groups. (B) Volcano plot of deferentially expressed mRNAs of 0.5 μM FD268-treated group. A total of 895 deferentially expressed mRNAs were identified, including 655 upregulated and 240 downregulated mRNAs. (C) (D) Crucial regulators of cell proliferation screened from each FD268-treated group in HL-60 cells. DESeq2 was used to screen differentially expressed genes among different groups (|log2FC| ≥1.5 and P value≥0.05).

GO analysis was used to identify the functional categories of these deferentially expressed genes. A total of 1669 GO terms in the category of biological processes were enriched at an FDR threshold of < 0.05. The top eight significantly enriched biological processes were inflammatory response, defense response, response to wounding, regulation of response to stimulus, secretion, secretion by cell, cytokine production and immune response (S4 Fig and S2 Table). Pathways were annotated and categorized in KEGG database for the identified differential target genes (S3 Table). Top 6 pathways were therefore isolated and shown in the statistical diagram (Fig 8A). In the 0.125 μM FD268 treated group, downregulated genes were mainly enriched in Toll-like receptor signaling pathway, PI3K-Akt signaling pathway and cytosolic DNA-sensing pathway. PI3K-Akt signaling pathway was significantly enriched with extremely low p‑value, indicating that FD268 significantly altered PI3K-Akt pathway genes in human leukemia HL-60 cells which was consistent with the above western blot results.

Fig 8. FD268 regulates cell survival related genes and pathways in HL-60 cells.

Fig 8

Open in a new tab

(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of RNA-seq data in 0.125 μM FD268-treated group. The top six enriched pathways of common deferentially expressed genes are shown. The DEGs are involved in several metabolic pathways related to cell survival including PI3K-Akt signaling pathway and a set of main metabolism and signal transduction pathways was involved. (B) Overlapped deferentially expressed target genes of 0.125 μM FD268-treated and 0.5 μM FD268-treated group. (C) Expression profiles of overlapped common of target genes. The color in heatmap represents the normalized expression value.

Furthermore, a set of genes were identified by overlapping the deferentially expressed target genes of 0.125 μM FD268-treated and 0.5 μM FD268-treated group (S4 Table). ABCA3, IL6, RHOB, NOXA1, C1RL, LRRC25, RASAL1 and SULF2 were significantly regulated by FD268 dose-dependently (Fig 8B and 8C). Among these targets, ATP transmembrane transporter subfamily protein ABCA3 and NADPH oxidase activator NOXA1 are related to apoptosis; LRRC25 promotes autophagy and induces the inhibition of NF-κB signaling pathway [38]; cytokine interleukin 6 is also closely related to PI3K pathway and cell differentiation [39, 40]. The alternation of these target genes was verified by RT-qPCR (S5 Fig). Therefore, it is reasonable to conclude that FD268 interrupt cell proliferation via regulating a bunch of crucial PI3K/Akt related genes and pathways.

4 Discussion

Inhibition of PI3K is believed to induce apoptosis in cancer cells [41]. Developed in parallel to those of PI-103 and CAL-101 analogous [42, 43], pyridinesulfonamide derivatives are another class of PI3K inhibitors generally characterized by occupying the affinity pocket of PI3K with pyridinesulfonamide motif leaving the hinge region and hydrophobic region of PI3K to the conjugated groups. FD268 is the one conjugated with 7-azaindole motif showing the potent activities against class I PI3K [32]. Owing the verified hyperactivation of PI3K/Akt/mTOR in AML, targeting PI3K is therefore of great therapeutic interest for the treatment of AML. Although the pyridinesulfonamide derivatives represented a large number of PI3K inhibitors developed recently, their efficacies as well as plausible mechanistic paths in AML are seldom explored. Thus, whether and how FD268 suppresses the proliferation and promotes apoptosis induction through inhibition of the PI3K network activity in AML cell lines received our great interest.

We examined for the first time the in vitro activity of FD268 on a panel of human AML cell lines including HL-60, MOLM-16, Mv-4-11 and KG-1, which are characterized by different PI3K/Akt/mTOR pathway activation patterns. Our studies demonstrated that FD268 significantly suppresses cell proliferation in a dose-dependent manner for the tested cell lines with the potent activities superior to that of PI-103 and CAL-101. Given its high anti-proliferation activity on HL-60 and MOLM-16, FD268-induced cell cycle arrest and apoptosis were examined and confirmed on these two cell lines respectively. Meanwhile, FD268 exerted the anti-proliferation through induction of cell cycle arrest at G0/G1 phase on HL-60 while at G2/M phase on MOLM-16. Furthermore, dose-dependent expression of apoptosis-related gene Bcl-2, Bad and Mcl-1 was confirmed on HL-60 while not on MOLM-16. Nevertheless, western-blot analyses revealed similar dose-dependent expression of pro- and antiapoptotic proteins on both HL-60 and MOLM-16. Although chemotherapy-induced differential transcriptional and protein expression as well as the cell-line-dependent regulation of cell cycle arrest are common, it raises the question whether they are strongly associated with the inhibition of PI3K/Akt/mTOR signaling pathway by FD268. Previous studies have revealed that Akt is a primary mediator in this pathway [44, 45]. Its activation promotes cell cycle progression by downregulation of cyclin D1, maintains cell survival through inactivation of Bad meanwhile increase of the expression of Bcl-2 protein, and promotes cell growth by phosphorylation of the downstream mTOR. Treated with FD268, our studies documented the reduction of PI3K/Akt process and downregulation in the level of phosphorylated mTOR as well as its downstream targets p70S6K at Thr389 and 4EBP1 in both HL-60 and MOLM-16 cells, which is in agreement with those well-known PI3K inhibitors in treatment of AML [26, 46, 47]. Although it is hard to reveal the cell-line-dependent regulation of cell cycle arrest and completely map the mechanisms of actions in AML, our studies, together with KEGG pathway analysis, demonstrated the capacity of FD268 in suppressing cell proliferation and inducing apoptosis on AML through inhibition of PI3K/Akt/mTOR signaling pathway (Fig 9).

Fig 9. Schematic for the plausible mechanisms by which PI3K pathway inhibition mediated the anti-leukemic activity of FD268 AML cells.

Fig 9

Open in a new tab

Via inhibition of the PI3K/Akt/mTOR signaling pathway, compound FD268 diminished the survival and proliferative capacity of the AML cells. The suppression of PI3K induced apoptotic cell death through upregulation of proapoptotic protein Bad and downregulation of antiapoptotic Mcl-1, and induced mitochondrial dependent cell apoptosis by activation of the caspase-3.

5 Conclusion

Our studies have documented for the first time that FD268, a pyridinesulfonamide derivative characterized by conjugation of 7-azaindole motif, possesses significant anti-leukemic activity towards AML cell lines by inhibition of PI3K/Akt/mTOR signaling pathway, thus supporting further development of pyridinesulfonamide derivatives as PI3K inhibitors for pre-clinical evaluation in treatment of AML.

Supporting information

S1 Fig. Morphological changes HL-60 and MOLM-16 treated with FD268 for 48h.

Cellular morphology of HL-60 and MOLM-16 treated with various concentrations of FD268 at 48 h. Phase contrast microscopy images and fluorescence microscopy images after staining with DAPI were observed. Cells incubated with DMSO (0.01%) were used control. Scale bar: 20 μm.

(TIF)

Click here for additional data file. (6.1MB, tif)
S2 Fig. FD268 concentration-dependently inhibited the phosphorylation of AKT (Ser473) in HL-60 cells.

HL-60 cells were treated with indicated concentrations of FD268 for 24 h. The p-AKT(Ser473) expression and location was detected by immunofluorescence assay. The fluorescence microscopy images after staining with DAPI were observed. Scale bar: 20 μm.

(TIF)

Click here for additional data file. (6.5MB, tif)
S3 Fig. Differentially expressed gene (DEGs) in HL-60 cells treated with FD268.

DESeq2 was used to screen differentially expressed genes among different groups (|log2FC| ≥1and P value≥0.05). Volcano plot of deferentially expressed genes. (A) 0.125 μM FD268-treated group. (b) 0.5 μM FD268-treated group. 194 up-regulated and 184 down-regulated mRNAs were screened by comparison in group FD268-0.125μM, 655 up-regulated and 240 down-regulated mRNAs were screened by comparison in group FD268-0.5μM.

(TIF)

Click here for additional data file. (1.2MB, tif)
S4 Fig. Gene Ontology (GO) analysis of RNA-seq data.

The top ten significantly enriched biological processes (BP), cell component (CC), molecule function (MF) are shown.

(TIF)

Click here for additional data file. (1.7MB, tif)
S5 Fig. Expression level of overlapped common of target genes dose-dependently regulated by FD268.

HL-60 cells were treated by FD268 (0.125μM, 0.5 μM) for 24 h. The expression of target genes ABCA3, C1RL, LRRC25, NOXA1, RASAL1, AULF2 and IL-6 in HL-60 cells were determined using qRT-PCR analysis.

(TIF)

Click here for additional data file. (469.2KB, tif)
S1 Table. The sequence of primers used for q-PCR.

(XLSX)

Click here for additional data file. (10.1KB, xlsx)
S2 Table. A total of terms annotated and categorized for the identified differential target genes.

(XLSX)

Click here for additional data file. (163.6KB, xlsx)
S3 Table. KEGG pathways annotated and categorized for the identified differential target genes.

(XLSX)

Click here for additional data file. (18.4KB, xlsx)
S4 Table. Target genes identified by overlapping the deferentially expressed of 0.125 μM FD268-treated and 0.5 μM FD268-treated group.

(XLSX)

Click here for additional data file. (61.4KB, xlsx)
S1 File. Raw images of Western blot from Figs 3–6.

(DOCX)

Click here for additional data file. (2.2MB, docx)
S1 Raw images

(PDF)

Click here for additional data file. (3.1MB, pdf)

Data Availability

All relevant data are within the article and its Supporting information files.

Funding Statement

The author Zhou.YM received the financial support from Zhuhai-Fudan Innovation Institute, the National Key Technologies R&D Program of China (2017YFA0205103), and Fudan-SIMM Joint Research Program (Grant No. FUSIMM20172005); author Lu.L received funding from the Commission for Science and Technology of Shanghai Municipality (20ZR1404800). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Choi KD, Vodyanik M, Slukvin II. Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells. Nat Protoc. 2011;6(3):296–313. Epub 2011/03/05. doi: 10.1038/nprot.2010.184 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Deschler B, Lubbert M. Acute myeloid leukemia: epidemiology and etiology. Cancer. 2006;107(9):2099–107. Epub 2006/10/05. doi: 10.1002/cncr.22233 . [DOI] [PubMed] [Google Scholar]
  • 3.Short NJ, Rytting ME, Cortes JE. Acute myeloid leukaemia. Lancet. 2018;392(10147):593–606. Epub 2018/08/07. doi: 10.1016/S0140-6736(18)31041-9 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Rodriguez-Ariza A, Lopez-Pedrera C, Aranda E, Barbarroja N. VEGF targeted therapy in acute myeloid leukemia. Crit Rev Oncol Hematol. 2011;80(2):241–56. Epub 2010/11/03. doi: 10.1016/j.critrevonc.2010.09.009 . [DOI] [PubMed] [Google Scholar]
  • 5.Medeiros BC, Fathi AT, DiNardo CD, Pollyea DA, Chan SM, Swords R. Isocitrate dehydrogenase mutations in myeloid malignancies. Leukemia. 2017;31(2):272–81. Epub 2016/10/11. doi: 10.1038/leu.2016.275 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kindler T, Lipka DB, Fischer T. FLT3 as a therapeutic target in AML: still challenging after all these years. Blood. 2010;116(24):5089–102. doi: 10.1182/blood-2010-04-261867 [DOI] [PubMed] [Google Scholar]
  • 7.Sadarangani A, Pineda G, Lennon KM, Chun HJ, Shih A, Schairer AE, et al. GLI2 inhibition abrogates human leukemia stem cell dormancy. J Transl Med. 2015;13:98. Epub 2015/04/19. doi: 10.1186/s12967-015-0453-9 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fu JR, Liu WL, Zhou JF, Sun HY, Xu HZ, Luo L, et al. Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via Pl3-kinase/Akt signaling pathways. Acta Pharmacol Sin. 2006;27(6):685–93. doi: 10.1111/j.1745-7254.2006.00335.x [DOI] [PubMed] [Google Scholar]
  • 9.Boudny M, Trbusek M. ATR-CHK1 pathway as a therapeutic target for acute and chronic leukemias. Cancer Treat Rev. 2020;88. ARTN 102026, doi: 10.1016/j.ctrv.2020.102026 [DOI] [PubMed] [Google Scholar]
  • 10.Yu J, Jiang PYZ, Sun H, Zhang X, Jiang Z, Li Y, et al. Advances in targeted therapy for acute myeloid leukemia. Biomark Res. 2020;8:17. Epub 2020/06/02. doi: 10.1186/s40364-020-00196-2 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kayser S, Levis MJ. Advances in targeted therapy for acute myeloid leukaemia. Br J Haematol. 2018;180(4):484–500. Epub 2017/12/02. doi: 10.1111/bjh.15032 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Stanchina M, Soong D, Zheng-Lin B, Watts JM, Taylor J. Advances in Acute Myeloid Leukemia: Recently Approved Therapies and Drugs in Development. Cancers (Basel). 2020;12(11). Epub 2020/11/04. doi: 10.3390/cancers12113225 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Yang J, Nie J, Ma X, Wei Y, Peng Y, Wei X. Targeting PI3K in cancer: mechanisms and advances in clinical trials. Mol Cancer. 2019;18(1):26. Epub 2019/02/21. doi: 10.1186/s12943-019-0954-x . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Manning BD, Toker A. AKT/PKB Signaling: Navigating the Network. Cell. 2017;169(3):381–405. Epub 2017/04/22. doi: 10.1016/j.cell.2017.04.001 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Nepstad I, Hatfield KJ, Gronningsaeter IS, Reikvam H. The PI3K-Akt-mTOR Signaling Pathway in Human Acute Myeloid Leukemia (AML) Cells. Int J Mol Sci. 2020;21(8). Epub 2020/04/25. doi: 10.3390/ijms21082907 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Herschbein L, Liesveld JL. Dueling for dual inhibition: Means to enhance effectiveness of PI3K/Akt/mTOR inhibitors in AML. Blood Rev. 2018;32(3):235–48. doi: 10.1016/j.blre.2017.11.006 [DOI] [PubMed] [Google Scholar]
  • 17.Martelli AM, Evangelisti C, Chiarini F, McCubrey JA. The phosphatidylinositol 3-kinase/Akt/mTOR signaling network as a therapeutic target in acute myelogenous leukemia patients. Oncotarget. 2010;1(2):89–103. doi: 10.18632/oncotarget.114 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Brenner AK, Tvedt THA, Bruserud O. The Complexity of Targeting PI3K-Akt-mTOR Signalling in Human Acute Myeloid Leukaemia: The Importance of Leukemic Cell Heterogeneity, Neighbouring Mesenchymal Stem Cells and Immunocompetent Cells. Molecules. 2016;21(11). ARTN 1512, doi: 10.3390/molecules21111512 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Darici S, Alkhaldi H, Horne G, Jorgensen HG, Marmiroli S, Huang X. Targeting PI3K/Akt/mTOR in AML: Rationale and Clinical Evidence. J Clin Med. 2020;9(9). Epub 2020/09/17. doi: 10.3390/jcm9092934 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Horne GA, Kinstrie R, Copland M. Novel drug therapies in myeloid leukemia. Pharm Pat Anal. 2015;4(3):187–205. Epub 2015/06/02. doi: 10.4155/ppa.15.3 . [DOI] [PubMed] [Google Scholar]
  • 21.Pal I, Mandal M. PI3K and Akt as molecular targets for cancer therapy: current clinical outcomes. Acta Pharmacol Sin. 2012;33(12):1441–58. doi: 10.1038/aps.2012.72 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Allegretti M, Ricciardi MR, Licchetta R, Mirabilii S, Orecchioni S, Reggiani F, et al. The pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia Sci Rep. 2016;6. ARTN 20743. doi: 10.1038/srep20743 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sadeghi S, Esmaeili S, Pourbagheri-Sigaroodi A, Safaroghli-Azar A, Bashash D. PI3K Abrogation Using Pan-PI3K Inhibitor BKM120 Gives Rise to a Significant Anticancer Effect on AML-Derived KG-1 Cells by Inducing Apoptosis and G2/M Arrest. Turk J Hematol. 2020;37(3):167–76. doi: 10.4274/tjh.galenos.2020.2019.0440 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Deng L, Jiang L, Lin XH, Tseng KF, Liu Y, Zhang X, et al. The PI3K/mTOR dual inhibitor BEZ235 suppresses proliferation and migration and reverses multidrug resistance in acute myeloid leukemia. Acta Pharmacol Sin. 2017;38(3):382–91. Epub 2017/01/04. doi: 10.1038/aps.2016.121 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Nguyen LX, Sesay A, Mitchell BS. Effect of CAL-101, a PI3Kdelta inhibitor, on ribosomal rna synthesis and cell proliferation in acute myeloid leukemia cells. Blood Cancer J. 2014;4:e228. Epub 2014/07/12. doi: 10.1038/bcj.2014.49 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Park S, Chapuis N, Bardet V, Tamburini J, Gallay N, Willems L, et al. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML. Leukemia. 2008;22(9):1698–706. Epub 2008/06/13. doi: 10.1038/leu.2008.144 . [DOI] [PubMed] [Google Scholar]
  • 27.Miller MS, Thompson PE, Gabelli SB. Structural Determinants of Isoform Selectivity in PI3K Inhibitors. Biomolecules. 2019;9(3). Epub 2019/03/01. doi: 10.3390/biom9030082 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yang C, Xu C, Li Z, Chen Y, Wu T, Hong H, et al. Bioisosteric replacements of the indole moiety for the development of a potent and selective PI3Kdelta inhibitor: Design, synthesis and biological evaluation. Eur J Med Chem. 2021;223:113661. Epub 2021/07/09. doi: 10.1016/j.ejmech.2021.113661 . [DOI] [PubMed] [Google Scholar]
  • 29.Knight SD, Adams ND, Burgess JL, Chaudhari AM, Darcy MG, Donatelli CA, et al. Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin. ACS Med Chem Lett. 2010;1(1):39–43. Epub 2010/04/08. doi: 10.1021/ml900028r . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Barton N, Convery M, Cooper AWJ, Down K, Hamblin JN, Inglis G, et al. Discovery of Potent, Efficient, and Selective Inhibitors of Phosphoinositide 3-Kinase delta through a Deconstruction and Regrowth Approach. J Med Chem. 2018;61(24):11061–73. Epub 2018/12/12. doi: 10.1021/acs.jmedchem.8b01556 . [DOI] [PubMed] [Google Scholar]
  • 31.Tian C, Yang C, Wu T, Lu M, Chen Y, Yang Y, et al. Discovery of cinnoline derivatives as potent PI3K inhibitors with antiproliferative activity. Bioorg Med Chem Lett. 2021;48:128271. Epub 2021/07/21. doi: 10.1016/j.bmcl.2021.128271 . [DOI] [PubMed] [Google Scholar]
  • 32.Yang CB, Zhang X, Wang Y, Yang YT, Liu XF, Deng ML, et al. Discovery of a Novel Series of 7-Azaindole Scaffold Derivatives as PI3K Inhibitors with Potent Activity. Acs Med Chem Lett. 2017;8(8):875–80. doi: 10.1021/acsmedchemlett.7b00222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Matsuo Y, Drexler HG, Kaneda K, Kojima K, Ohtsuki Y, Hara M, et al. Megakaryoblastic leukemia cell line MOLM-16 derived from minimally differentiated acute leukemia with myeloid/NK precursor phenotype. Leuk Res. 2003;27(2):165–71. Epub 2003/01/16. doi: 10.1016/s0145-2126(02)00081-4 . [DOI] [PubMed] [Google Scholar]
  • 34.Lange B, Valtieri M, Santoli D, Caracciolo D, Mavilio F, Gemperlein I, et al. Growth factor requirements of childhood acute leukemia: establishment of GM-CSF-dependent cell lines. Blood. 1987;70(1):192–9. Epub 1987/07/01. . [PubMed] [Google Scholar]
  • 35.Saito H, Bourinbaiar A, Ginsburg M, Minato K, Ceresi E, Yamada K, et al. Establishment and characterization of a new human eosinophilic leukemia cell line. Blood. 1985;66(6):1233–40. Epub 1985/12/01. . [PubMed] [Google Scholar]
  • 36.Koeffler HP, Golde DW. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science. 1978;200(4346):1153–4. Epub 1978/06/09. doi: 10.1126/science.306682 . [DOI] [PubMed] [Google Scholar]
  • 37.Gallagher R, Collins S, Trujillo J, McCredie K, Ahearn M, Tsai S, et al. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood. 1979;54(3):713–33. Epub 1979/09/01. . [PubMed] [Google Scholar]
  • 38.Feng Y, Duan T, Du Y, Jin S, Wang M, Cui J, et al. LRRC25 Functions as an Inhibitor of NF-kappaB Signaling Pathway by Promoting p65/RelA for Autophagic Degradation. Sci Rep. 2017;7(1):13448. Epub 2017/10/19. doi: 10.1038/s41598-017-12573-3 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Zhou J, Sun X, Zhang J, Yang Y, Chen D, Cao J. IL-34 regulates IL-6 and IL-8 production in human lung fibroblasts via MAPK, PI3K-Akt, JAK and NF-kappaB signaling pathways. Int Immunopharmacol. 2018;61:119–25. Epub 2018/06/02. doi: 10.1016/j.intimp.2018.05.023 . [DOI] [PubMed] [Google Scholar]
  • 40.Duluc D, Delneste Y, Tan F, Moles MP, Grimaud L, Lenoir J, et al. Tumor-associated leukemia inhibitory factor and IL-6 skew monocyte differentiation into tumor-associated macrophage-like cells. Blood. 2007;110(13):4319–30. Epub 2007/09/13. doi: 10.1182/blood-2007-02-072587 . [DOI] [PubMed] [Google Scholar]
  • 41.Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev. 2004;30(2):193–204. Epub 2004/03/17. doi: 10.1016/j.ctrv.2003.07.007 . [DOI] [PubMed] [Google Scholar]
  • 42.Knight ZA, Chiang GG, Alaimo PJ, Kenski DM, Ho CB, Coan K, et al. Isoform-specific phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold. Bioorg Med Chem. 2004;12(17):4749–59. Epub 2004/09/11. doi: 10.1016/j.bmc.2004.06.022 . [DOI] [PubMed] [Google Scholar]
  • 43.Lannutti BJ, Meadows SA, Herman SE, Kashishian A, Steiner B, Johnson AJ, et al. CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability. Blood. 2011;117(2):591–4. Epub 2010/10/21. doi: 10.1182/blood-2010-03-275305 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995;376(6541):599–602. Epub 1995/08/17. doi: 10.1038/376599a0 . [DOI] [PubMed] [Google Scholar]
  • 45.Revathidevi S, Munirajan AK. Akt in cancer: Mediator and more. Semin Cancer Biol. 2019;59:80–91. Epub 2019/06/08. doi: 10.1016/j.semcancer.2019.06.002 . [DOI] [PubMed] [Google Scholar]
  • 46.Chapuis N, Tamburini J, Green AS, Vignon C, Bardet V, Neyret A, et al. Dual inhibition of PI3K and mTORC1/2 signaling by NVP-BEZ235 as a new therapeutic strategy for acute myeloid leukemia. Clin Cancer Res. 2010;16(22):5424–35. Epub 2010/10/05. doi: 10.1158/1078-0432.CCR-10-1102 . [DOI] [PubMed] [Google Scholar]
  • 47.Li J, Wang X, Ma C, Xu S, Xu M, Yang J, et al. Dual PI3K/mTOR inhibitor NVPBEZ235 decreases the proliferation of doxorubicinresistant K562 cells. Mol Med Rep. 2021;23(4). Epub 2021/07/06. doi: 10.3892/mmr.2021.11940 . [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Francesco Bertolini

8 Aug 2022

PONE-D-22-00370

A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid Leukemia

PLOS ONE

Dear Dr. Zhou,

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 by both Reviewers.

Please submit your revised manuscript by Sep 19 2022 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: https://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,

Francesco Bertolini, MD, PhD

Academic Editor

PLOS ONE

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

2. Thank you for including your ethics statement:  "N/A".   

For studies reporting research involving human participants, PLOS ONE requires authors to confirm that this specific study was reviewed and approved by an institutional review board (ethics committee) before the study began. Please provide the specific name of the ethics committee/IRB that approved your study, or explain why you did not seek approval in this case.

Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

4. 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. 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: Congratulations on the excellent article. Some considerations:

- in keywords: you do not need to include those that are already mentioned in the title (pyridinesulfonamide; acute myeloid leukemia; Apoptosis)

- objectives are not well highlighted

- in some tests, in the text of materials and methods, it is not clear whether triplicates and 3 independent experiments were carried out, or only 3 independent experiments. They are only explained in the subtitles.

- in figure 4c the name of the cell lines was missing

- the work has been done with two cell types, but in RNAseq only one cell line was chosen, but there was no explanation for this.

Reviewer #2: The manuscript “A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid Leukemia” by Chen et al. presents the results on therapeutic activity of novel and highly active FD268 inhibitor of PI3K. The authors found that FD268 inhibits the viabilities of AML cells with the efficacies superior to other two PI3K inhibitors, PI-103 and CAL-101. They showed that FD268 suppresses cell proliferation, induces cell cycle arrest and apoptosis of AML cells through the inhibition of PI3K activity.

In my humble opinion, I think that this work will be of interest to a broad audience.

Overall, the manuscript is well written and experiments were properly designed and executed. However, some minor corrections and improvements are necessary prior to its acceptance.

Comments and Suggestions:

Line 90: repeating words “and and”.

Line 108: Should read “…and treated with FD268…”

Line 178: Should read “…plates and treated with FD268 (0.125, 0.5 µM) or DMSO for 24 h. Cells were harvested…”

Line 225: Should read “The values were shown as the…”

Lines 249-250: Fig. 3C: there is no dose dependent inhibition of cleavage of caspase 3 and PARP, but increase instead. The sentence should be corrected as “…proteins were consequently examined by western blot assay, which showed a dose dependent increase of both caspase-3 and PARP cleavage (Fig 3C).”.

Line 258: Should read “Statistical significance is represented with asterisks *P < 0.05 and ***P < 0.001.”, as there is no **P < 0.01. It might be also better to use white background for S phase data, as labels/asterisks are barely visible in Figure 2B.

Figure 4C: Indicate which blots represent HL-60 and MOLM-16 cells. Label for cell lines is missing.

Line 288: The authors should mention here that the protein levels of other markers of apoptosis, e.g. Bcl-2, Bax and Bcl-XL, did not change significantly in FD268-treated cells.

Figure 5C: Label of MOLM-16 has to be corrected (swapped letters). The same applies to Figure 6A and 6B.

Figure 9: Shouldn´t the cleaved caspase-3 and PARP lead to cell death/apoptosis, instead of cell survival (check the bottom right corner)?

********** 

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: No

**********

[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.

PLoS One. 2022 Nov 22;17(11):e0277893. doi: 10.1371/journal.pone.0277893.r002

Author response to Decision Letter 0

21 Sep 2022

Dear Editor and reviewers，

Thank you for your decision and constructive comments on our manuscript entitled “A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid Leukemia”. Those comments are all valuable and helpful for revising and improving our paper. We have studied all comments carefully and have made conscientious correction in the manuscript. Revised portion are marked in red and highlighted in this paper.

Revision notes, point-to-point, are given as follows:

Reviewer #1:

Comment 1: in keywords: you do not need to include those that are already mentioned in the title (pyridinesulfonamide; acute myeloid leukemia; Apoptosis).

Response 1: Thank you for the suggestion about the key words. The key words that mentioned in the title has been replaced with the the following words: PI3K inhibitor, Haematological cancer and HL-60.

Comment 2: objectives are not well highlighted.

Response 2: Thank you for this valuable feedback. Our research have documented for the first time that a novel pyridinesulfonamide scaffold possesses significant anti-leukemic activity towards AML cells, the objective of our paper is about the early exploration of the mechanism of novel drug development. In the text of this paper, the objectives was not well highlighted due to the ways of language expression. We have further clarified this in the end of the Introduction section (lines 69-71), and the Conclusion section ( lines 457-461).

Comment 3: in some tests, in the text of materials and methods, it is not clear whether triplicates and 3 independent experiments were carried out, or only 3 independent experiments. They are only explained in the subtitles.

Response 3: Thank you for your questions. All the cells in each cell-based assay were seeded and harvested in triplicates, and at least three independent experiments were carried out in the CCK-8 cell proliferation assay and cell apoptosis assay. We have in added examinations in the text of materials and methods (lines 104-105, lines 120-121 and lines 145-146).

Comment 4: in figure 4c the name of the cell lines was missing.

Response 4: Thanks for pointing out about the missing details, the name of cell lines has been added in Figure 4C.

Comment 5: the work has been done with two cell types, but in RNA-seq only one cell line was chosen, but there was no explanation for this.

Response 5: We appreciate for this question. Based on the previous studies of compound FD268, we found that FD268 has potential AML cell proliferation inhibitory activity, and further screening assay showed that the HL-60 cell line was more sensitive to compound FD268, so in the subsequent mechanistic explorations we chose HL-60 for the RNA-seq analysis.

Reviewer #2:

Comment 1: Line 90: repeating words “and and”.

Comment 2: Line 108: Should read “…and treated with FD268…”

Comment 3: Line 178: Should read “…plates and treated with FD268 (0.125, 0.5 µM) or DMSO for 24 h. Cells were harvested…”

Comment 4: Line 225: Should read “The values were shown as the…”

Response 1-4: Thanks for the kind comments. The tense and repetition mistakes have been corrected in the manuscript.

Comment 5: Lines 249-250: Fig. 3C: there is no dose dependent inhibition of cleavage of caspase 3 and PARP, but increase instead. The sentence should be corrected as “…proteins were consequently examined by western blot assay, which showed a dose dependent increase of both caspase-3 and PARP cleavage (Fig 3C).”

Response 5: Thanks for the comments. In Figure. 3C, the western blot was applied to detect cleaved PARP, cleaved caspase-3 and caspase-3 as indicators of apoptotic cell death. The cleavage of caspase-3 and PARP increased dose dependently. We have changed the sentences as suggested. (Lines 249-250)

Comment 6: Line 258: Should read “Statistical significance is represented with asterisks *P < 0.05 and ***P < 0.001.”, as there is no **P < 0.01. It might be also better to use white background for S phase data, as labels/asterisks are barely visible in Figure 2B.

Response 6: Thanks for the valuable suggestion and we have removed the “**P < 0.01” in statistical significance annotation of Figure 2. The background for S phase data bar have been re-edited to highlight labels/asterisks.

Comment 7: Figure 4C: Indicate which blots represent HL-60 and MOLM-16 cells. Label for cell lines is missing.

Response 7: Thanks for pointing out about the missing, the name of cell lines has been added in Figure 4C.

Comment 8: Line 288: The authors should mention here that the protein levels of other markers of apoptosis, e.g. Bcl-2, Bax and Bcl-XL, did not change significantly in FD268-treated cells.

Response 8: Thanks for this kind suggestion. At protein level, FD268 increased the expression of Bad, meanwhile inhibited Mcl-1 in both HL-60 and MOLM-16 dose-dependently. While, the protein levels of other markers of apoptosis, Bcl-2, Bax and Bcl-XL, did not change significantly in FD268-treated cells. We have added a sentence to to clarify this. (Lines 291-292)

Comment 9: Figure 5C: Label of MOLM-16 has to be corrected (swapped letters). The same applies to Figure 6A and 6B.

Response 9: Thanks for pointing out the mistakes, the label of MOLM-16 have been corrected in Figure 5C, 6A and 6B.

Comment 10: Figure 9: Shouldn´t the cleaved caspase-3 and PARP lead to cell death/apoptosis, instead of cell survival (check the bottom right corner)?

Response 10: Thank you for this valuable comment. In this diagram, we meant to show that the compound FD268 induced apoptosis through the activation of caspase-3 and PARP cleavage, so the label “cell Survival” have been changed into “Cell Apoptosis”.

After careful revision, we worked on the manuscript for a long time and fixed all of these format or grammar issues on the manuscript. Thanks again to the reviewers and editors for you hard work!

Sincerely yours，

Prof. Yaming Zhou

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (15.2KB, docx)

Decision Letter 1

Francesco Bertolini

5 Oct 2022

PONE-D-22-00370R1A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid LeukemiaPLOS ONE

Dear Dr. Zhou,

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.

==============================

ACADEMIC EDITOR: Please insert comments here and delete this placeholder text when finished. Be sure to:

  • Indicate which changes you require for acceptance versus which changes you recommend

  • Address any conflicts between the reviews so that it's clear which advice the authors should follow

  • Provide specific feedback from your evaluation of the manuscript

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data. 

==============================

Please submit your revised manuscript by Nov 19 2022 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: https://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,

Jianhong Zhou

Support Staff - Editorial

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.

Additional Editor Comments (if provided):

[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: All comments have been addressed

**********

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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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: Yes

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: Yes

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: Now, The manuscript is much better! All notes have been answered.

The reading is better understood after all mistakes are solved.

Reviewer #2: The authors have revised the manuscript according to my suggestions and recommendations. I have no further comments.

**********

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: No

**********

[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.

PLoS One. 2022 Nov 22;17(11):e0277893. doi: 10.1371/journal.pone.0277893.r004

Author response to Decision Letter 1

3 Nov 2022

Dear reviewers,

Thank you for your constructive comments on our manuscript (ID: PONE-D-22-00370R2). Those comments are all valuable and helpful for revising and improving our paper. We have considered all comments carefully and made conscientious correction. Attached please find the revised manuscript entitled “A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid Leukemia” for your consideration. Point-to-point responses for the original manuscript were given in the file ‘Response to reviewers.docx’ and the correction were highlighted in the revised manuscript with track changes. There is no correction according to the recent comments for the R1 version.

Thank you very much for your time! We would be glad to respond to any further questions and comments that you may have.

Sincerely yours

Prof. Yaming Zhou

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (15.7KB, docx)

Decision Letter 2

Jianhong Zhou

6 Nov 2022

A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid Leukemia

PONE-D-22-00370R2

Dear Dr. Zhou,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jianhong Zhou

Staff Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Jianhong Zhou

10 Nov 2022

PONE-D-22-00370R2

A Pyridinesulfonamide Derivative FD268 Suppresses Cell Proliferation and Induces Apoptosis via Inhibiting PI3K Pathway in Acute Myeloid Leukemia

Dear Dr. Zhou:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Francesco Bertolini

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Morphological changes HL-60 and MOLM-16 treated with FD268 for 48h.

    Cellular morphology of HL-60 and MOLM-16 treated with various concentrations of FD268 at 48 h. Phase contrast microscopy images and fluorescence microscopy images after staining with DAPI were observed. Cells incubated with DMSO (0.01%) were used control. Scale bar: 20 μm.

    (TIF)

    Click here for additional data file. (6.1MB, tif)
    S2 Fig. FD268 concentration-dependently inhibited the phosphorylation of AKT (Ser473) in HL-60 cells.

    HL-60 cells were treated with indicated concentrations of FD268 for 24 h. The p-AKT(Ser473) expression and location was detected by immunofluorescence assay. The fluorescence microscopy images after staining with DAPI were observed. Scale bar: 20 μm.

    (TIF)

    Click here for additional data file. (6.5MB, tif)
    S3 Fig. Differentially expressed gene (DEGs) in HL-60 cells treated with FD268.

    DESeq2 was used to screen differentially expressed genes among different groups (|log2FC| ≥1and P value≥0.05). Volcano plot of deferentially expressed genes. (A) 0.125 μM FD268-treated group. (b) 0.5 μM FD268-treated group. 194 up-regulated and 184 down-regulated mRNAs were screened by comparison in group FD268-0.125μM, 655 up-regulated and 240 down-regulated mRNAs were screened by comparison in group FD268-0.5μM.

    (TIF)

    Click here for additional data file. (1.2MB, tif)
    S4 Fig. Gene Ontology (GO) analysis of RNA-seq data.

    The top ten significantly enriched biological processes (BP), cell component (CC), molecule function (MF) are shown.

    (TIF)

    Click here for additional data file. (1.7MB, tif)
    S5 Fig. Expression level of overlapped common of target genes dose-dependently regulated by FD268.

    HL-60 cells were treated by FD268 (0.125μM, 0.5 μM) for 24 h. The expression of target genes ABCA3, C1RL, LRRC25, NOXA1, RASAL1, AULF2 and IL-6 in HL-60 cells were determined using qRT-PCR analysis.

    (TIF)

    Click here for additional data file. (469.2KB, tif)
    S1 Table. The sequence of primers used for q-PCR.

    (XLSX)

    Click here for additional data file. (10.1KB, xlsx)
    S2 Table. A total of terms annotated and categorized for the identified differential target genes.

    (XLSX)

    Click here for additional data file. (163.6KB, xlsx)
    S3 Table. KEGG pathways annotated and categorized for the identified differential target genes.

    (XLSX)

    Click here for additional data file. (18.4KB, xlsx)
    S4 Table. Target genes identified by overlapping the deferentially expressed of 0.125 μM FD268-treated and 0.5 μM FD268-treated group.

    (XLSX)

    Click here for additional data file. (61.4KB, xlsx)
    S1 File. Raw images of Western blot from Figs 3–6.

    (DOCX)

    Click here for additional data file. (2.2MB, docx)
    S1 Raw images

    (PDF)

    Click here for additional data file. (3.1MB, pdf)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (15.2KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (15.7KB, docx)

    Data Availability Statement

    All relevant data are within the article and its Supporting information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

    RESOURCES