Recent studies have revealed that targeting amino acid metabolic enzymes is a promising strategy in cancer therapy. Acute myeloid leukemia (AML) downregulates the expression of argininosuccinate synthase (ASS1), a recognized rate-limiting enzyme for arginine synthesis, and yet displays a critical dependence on extracellular arginine for survival and proliferation. This dependence on extracellular arginine, also known as arginine auxotrophy, suggests that arginine deprivation would be a treatment strategy for AML. NEI-01, a novel arginine-depleting enzyme, is capable of binding to serum albumin to extend its circulating half-life, leading to a potent anticancer activity. Here we reported the preclinical activity of NEI-01 in arginine auxotrophic AMLs. NEI-01 efficiently depleted arginine both in vitro and in vivo. NEI-01-induced arginine deprivation was cytotoxic to arginine auxotrophic AML cells through induction of cell-cycle arrest and apoptosis. Furthermore, the potent anti-leukemia activities of NEI-01 were observed in three different types of mouse models including human cell line-derived xenograft, mouse cell line-derived homografts in syngeneic mice and patient-derived xenograft. This preclinical data provide strong evidence to support the potential use of NEI-01 as a therapeutic approach in AML treatment.

Acute myeloid leukemia (AML) is one of the most common acute leukemias that progress quickly. From 1990 to 2017, the incidence of AML gradually increased (1). In 2015, AML affected about one million people and resulted in 147,000 deaths globally (2, 3). The general therapeutic strategy in patients with AML has not changed substantially in more than 30 years. The traditional therapy consists of two chemotherapy phases: remission induction and consolidation. The cure rate is about 35% in people under 60 years old and 10% over 60 years old (4). A large amount of patients with AML are not able to receive intensive chemotherapy during the induction phase due to age, comorbidities, and toxicity profile. The outcome in these patients remains dismal, with a median survival of only 5 to 10 months (5). Thus, new therapeutic approaches are urgently needed.

Drug-induced-specific nutrient deprivation is one of the promising strategies as tumor cells have a much higher nutrient demand compared with normal cells (6). Arginine is a multifunctional amino acid and is indispensable for synthesis of proteins, nitric oxide, urea, polyamines, proline, glutamate, creatine, and agmatine (7). Arginine and its downstream products have been implicated in tumor development (8). Certain tumors require arginine for survival and proliferation, making them either arginine auxotrophic or partially auxotrophic (9). The expression of arginine biosynthetic enzymes, such as argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL), are considered key determinants of the auxotrophic status of tumor cells (10). As noted downregulation of ASS1 by either promoter methylation or by HIF1α is associated with increased metastasis, intrinsic chemoresistance, and poor prognosis (11–13). Arginine deprivation is becoming a recognized treatment strategy for therapy-refractory malignancies (14).

UALCAN database (http://ualcan.path.uab.edu/index.html) analysis has shown that AML has the lowest expression of ASS1 over the various types of cancer (Supplementary Fig. S1). Lower expression of ASS1 is associated with poorer survival in AML patients (Supplementary Fig. S2A). Most AML patients except AML M3 show low or absent ASS1 expression (Supplementary Fig. S2B), leading to arginine auxotrophy, which renders AML vulnerable to arginine deprivation therapy. Pegylated arginine depleting enzymes such as recombinant human arginase (rh-Arg1-PEG) and arginine deiminase (ADI-PEG) have been demonstrated to be cytotoxic to arginine auxotrophic AMLs (15, 16). PEGylation can change the physical and chemical properties of the biomedical molecule, resulting in an improvement in the pharmacokinetic behavior of the therapeutic proteins (17). However, PEGylation can also lead to a loss in binding affinity due to steric interference with the drug-target binding interaction (18). Moreover, another disadvantage of which cannot be neglected, is the presence of antibodies against PEG (anti-PEG Abs) produced by the immune system. It has also been reported that the existence of anti-PEG Abs is correlated with loss of therapeutic efficacy and increase in adverse effects in clinics (19). Fusion of therapeutic proteins to albumin-binding domain is one of alternative strategies (20). Here we tested the activities of NEI-01 (21), a novel arginine-depleting enzyme fused to an albumin binding domain, in ASS1-deficient AMLs both in vitro and in vivo. This preclinical data provide a rationale for a clinical evaluation of NEI-01 in arginine auxotrophic AMLs.

Cell lines

Human AML cell line KG-1 (CVCL_0374), Kasumi-1 (CVCL_0589), HL-60 (CVCL_0002), U-937 (CVCL_0007), MV4–11 (CVCL_0064), AML-193 (CVCL_1071), THP-1 (CVCL_0006), mouse AML cell line C1498 (CVCL_3494), and human normal fibroblast cell line HFF-1 (CVCL_3285) were purchased from ATCC during July 2017 to February 2019. The culture condition was listed in Supplementary Materials and Methods. All human cell lines have been authenticated using short tandem repeat analysis by Pangenia lifesciences Ltd. and mouse AML cell line C1498 was authenticated using short tandem repeat analysis by ATCC during January to May 2021. Cell lines were tested mycoplasma free and the latest date for testing was April to May 2021. Cells used for experiments were kept in continuous culture for no more than 15 passages.

Western blot analysis

Total protein was extracted from cells following lysis in the RIPA lysis buffer (Thermo Fisher Scientific) supplemented with the protease inhibitor cocktail (Calbiochem) at 4°C for 30 minutes. The resulting debris was removed by centrifugation and the supernatant containing the protein lysate was collected. The cellular protein content was determined using BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of protein was electrophoresed by 12% SDS-PAGE and separated proteins were transferred to low fluorescence PVDF membranes (Bio-Rad Laboratories). After being blocked with 5% nonfat milk in TBST, the membranes were incubated with the primary antibodies at 4°C overnight, followed by 1:20,000 HRP-conjugated secondary antibodies (anti-rabbit, anti-mouse; Abcam Scientific) at room temperature for 1 hour. The primary antibodies were: anti-ASS1 antibody (1:5,000, Cell Signaling Technology), anti-ASL antibody (1: 5,000, Abcam Scientific), and anti-GAPDH (1:20,000, Cell Signaling Technology). Immunoreactive bands were visualized using an Enhanced Chemiluminescence Kit (Bio-Rad Laboratories) and photographed by ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

qPCR

Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. One hundred nanograms of RNA was used to synthesize cDNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad Laboratories) following the manufacturer's instructions. qPCR was carried out by StepOnePlus Real-Time PCR Sytem (Thermo Fisher Scientific), with reagent GoTaq qPCR Master Mix (Promega Corporation). Two microliters of cDNA was used per 20 μL reaction with the following conditions: denaturation at 95°C for 2 minutes followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 minute. Product specificity was evaluated by melting curves. Primer sequences are as follows:

  • human ASS1, forward 5′-AGCTCAGCTGCTACTCACTGG-3′ and reverse 5′-TTGAACCGGTTGTAGAATTCAG-3′;

  • human ASL, forward 5′-GGAAGCTGTGTTTGAAGTGTCA-3′ and reverse 5′- CATGTTCTCTTGGTGAATCTGC-3′;

  • human GAPDH, forward 5′-AGCCACATCGCTCAGACAC-3′ and reverse 5′-GCCCAATACGACCAAATCC-3′. Human GAPDH were used as endogenous control.

Methylation-specific PCR (MS-PCR)

Genomic DNA was extracted from cells using the Zymo Quick-DNA Microprep Plus Kit (Cambridge Bioscience Ltd.) following the manufacturer's instructions. Bisulphite conversion of genomic DNA was performed using the Zymo EZ DNA Methylation Kit (Cambridge Bioscience Ltd.) following the manufacturer's instructions. MS-PCR was carried out to determine the methylation status of ASS1. In all, 100 ng of bisulphite-modified DNA was used as templates for PCR reactions with primers specific for methylated or unmethylated sequences. Zymo Human HCT116 DKO Methylated and nonmethylated DNA (Cambridge Bioscience Ltd.) were used as positive and negative controls, respectively. Primer sequences are methylated forward 5′-GTAGGAGGGAAGGGGTTTTC-3′; methylated reverse 5′-GCAAAAAACAAATAACCCGAA-3′; unmethylated forward 5′-GTAGGAGGGAAGGGGTTTTT-3′ and unmethylated reverse 5′-ACAAAAAACAAATAACCCAAA-3′. PCR conditions were as follows: preheating at 95°C for 10 minutes, 8 cycles of 95°C for 2 minutes, 52°C for 30 seconds, and 72°C for 30 seconds were followed by 32 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 30 seconds, then a final extension at 72°C for 7 minutes. PCR products were electrophoresed through 2% agarose gels, stained with SYBR Safe DNA Gel Stain and visualized using ChemiDoc Touch Imaging System.

Cell viability assay

AML cells (1 × 105/mL) were treated with NEI-01 at the indicated concentrations. Seventy-two hours after treatment, cells were harvested and counted using the Trypan Blue Exclusion Method with Countess II FL Automated Cell Counter (Thermo Fisher Scientific).

Extracellular arginine/citrulline detection

AML cells (1 × 105/mL) were treated with NEI-01 at the indicated concentrations (range from 0.064 to 10,000 ng/mL). Seventy-two hours after treatment, the medium was collected by centrifugation at 2,000 rpm for 5 minutes, and mixed with 50% sulfosalicylic acid (w/v). The mixture was incubated at 4°C overnight and centrifuged at 15,000 rpm at 4°C for 10 minutes to remove the protein precipitate. The samples were mixed with a lithium loading buffer, filtered with a 0.45 μm filter, and analyzed by 30+ Amino Acid Analyzer according to the manufacturer's instructions (Biochrom).

Cell cycle and apoptosis analysis

A total of 1 × 106 AML cells were treated with NEI-01 (100 ng/mL) and harvested after the indicated incubation period. For cell-cycle analysis, AML cells were washed with PBS and fixed with cold 70% ethanol at 4°C for 30 minutes. After washing with PBS, cells were treated with RNase A (100 μg/mL; Abcam Scientific) and stained with propidium iodide (PI) solution (50 μg/mL; Abcam Scientific) at 4°C for 30 minutes. Samples were analyzed by a FACSVia Flow Cytometer (BD Biosciences). For apoptosis analysis, Annexin-V-FITC/PI Kit (BD Biosciences) was used following the manufacturer's instructions. In brief, AML cells were washed with PBS, resuspended in an annexin binding buffer, and labeled with Annexin V and PI in the dark. Samples were analyzed by a FACSVia Flow Cytometer (BD Biosciences).

Animal studies

Efficacy studies in cell line-derived xenograft (CDX) and syngeneic mouse models were performed according to protocols approved by the Animal Experimentation Ethics Committee of The Hong Kong Polytechnic University.

Efficacy studies in patient-derived xenograft (PDX) were conducted by Crown Bioscience in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Crown Bioscience. AM5512 was obtained from the samples of AML patient and all procedures were approved by the institutional review boards (IRB) of the hospital, following the principles of the Helsinki declaration.

Human HL-60 and mouse C1498 cells were transducted with RediFect Red-FLuc-GFP lentiviral particles (PerkinElmer). A stable clone production (HL-60-gfp-Luc+ or C1498-gfp-Luc+ cells) that coexpress firefly luciferase (Luc) and GFP was obtained by sorting of GFPhi cell population using flow cytometry (BD FACSAria III cell sorter; BD Biosciences).

For orthtopic CDX model, 5 × 106 well-established HL-60-gfp-Luc+ cells per mouse in 100 μL PBS were intravenously transplanted into the NOD/SCID mice (female, 6–7-week-old). For subcutaneous CDX model, 5 × 106 HL-60 cells with 20% Corning Matrigel Matrix (Corning Incorporated) were subcutaneously inoculated into the right flank of each BALB/c nude mouse (female, 6–7-week-old). For the syngeneic model, 5 × 106 well-established C1498-gfp-Luc+ cells per mouse in 100 μL PBS were intravenously transplanted into the C57BL/6 mice (female, 6–7-week-old). For PDX model, 1 × 106 AM5512 patient-derived AML cells per mouse were intravenously transplanted into the NOD/SCID mice (female, 3–4-week-old). All of the mice were treated with NEI-01 or vehicle for 4 weeks.

Amino acid detection in mouse plasma

Mouse blood was collected weekly from the saphenous vein before treatment. Plasma was obtained by centrifugation at 6,000 rpm for 10 minutes at 4°C. Amino acids arginine and citrulline in mouse plasma were quantified by 30+ Amino Acid Analyzer (Biochrom) as described previously (21).

In vivo bioluminescent imaging (BLI)

Following intravenous transplantation with HL-60-gfp-Luc+ or C1498-gfp-Luc+ cells, the tumor burden was monitored twice a week by in vivo BLI. The mice were anesthetized using 2% isoflurane and intraperitoneally injected with 150 mg/kg D-luciferin 15 minutes before acquiring an image. The photons emitted from tumor burden were detected with In Vivo Animal Imaging System (Perkin-Elmer). The whole-body bioluminescent signal was quantified by Living Image in vivo software (Perkin-Elmer).

Flow cytometry analysis

For HL-60-gfp-Luc+ orthotopic model, the bone, liver, and spleen were dissected at the termination of the experiment. Mononuclear cells collected from bone marrow (BM), spleen, and liver were analyzed by BD Accuri C6 Plus flow cytometer (BD Biosciences). The presence of GFP+ population was considered as AML tumor burden.

For PDX model, peripheral blood (PB) was weekly collected via retro-orbital bleeding. Bone, liver, and spleen were dissected at the termination of the experiment. Mononuclear cells collected from PB, BM, spleen, and liver were stained with FITC conjugated mouse anti-human CD45 (BioLegend). Stained cells were analyzed on the BD Accuri C6 Plus flow cytometer (BD Biosciences). The presence of the human CD45+ population was considered as AML tumor burden.

Subcutaneous xenograft measurement

Following subcutaneous inoculation with HL-60 cells, the length and width of the tumors were measured twice a week with a caliper. Tumor volume was calculated using the formula: Tumor volume = (width2 × length)/2. The tumors were dissected and weighted at the termination of the experiment.

Statistical analysis

Data are expressed as mean ± SD. P values were calculated with two-tail unpaired Student t test. Results with P value < 0.05 were considered statistically significant.

ASS1 and ASL status affect the response of AML cell lines to NEI-01 treatment

ASS1 and ASL were found to be differentially expressed in various AML cell lines. Western blot analysis results showed that ASS1 expression was high in Kasumi-1, U937, MV-4–11, and THP-1 cells, but low or absent in KG-1, HL-60, and AML-193 cells. However, ASL expression was high in most of AML cells, except HL-60 and MV-4–11 cells (Fig. 1A). Similar results were obtained using qPCR (Fig. 1B). Flow cytometry analysis was used to confirm the ASS1 expressions (Supplementary Fig. S3).

Figure 1.

ASS1 and/or ASL status affect the response of AML cell lines to NEI-01 treatment. A, ASS1 and ASL protein expressions were detected in seven AML cell lines and one normal fibroblast cell line (HFF-1) using Western blot analysis. B, ASS1 and ASL gene expression in seven AML cell lines was quantified by qPCR and normalized to that in HFF-1 cells. Experiments were performed in triplicate and repeated three times. Data are expressed as mean ± SD. C, Treatment with NEI-01 significantly reduced the cell viability of AML cells. AML cells were treated with NEI-01 at the various concentrations for 72 hours. The cells were harvested and counted using the Trypan Blue Exclusion method. Experiment was performed in triplicate and repeated three times. Data are expressed as mean ± SD. P value was calculated using Student t test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). D, Methylation status of ASS1 gene was assessed in AML cell lines by MS-PCR. U, unmethylated DNA; M, methylated DNA; MC, methylation control; UC, unmethylation control; NC, negative control.

Figure 1.

ASS1 and/or ASL status affect the response of AML cell lines to NEI-01 treatment. A, ASS1 and ASL protein expressions were detected in seven AML cell lines and one normal fibroblast cell line (HFF-1) using Western blot analysis. B, ASS1 and ASL gene expression in seven AML cell lines was quantified by qPCR and normalized to that in HFF-1 cells. Experiments were performed in triplicate and repeated three times. Data are expressed as mean ± SD. C, Treatment with NEI-01 significantly reduced the cell viability of AML cells. AML cells were treated with NEI-01 at the various concentrations for 72 hours. The cells were harvested and counted using the Trypan Blue Exclusion method. Experiment was performed in triplicate and repeated three times. Data are expressed as mean ± SD. P value was calculated using Student t test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). D, Methylation status of ASS1 gene was assessed in AML cell lines by MS-PCR. U, unmethylated DNA; M, methylated DNA; MC, methylation control; UC, unmethylation control; NC, negative control.

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KG-1 and HL-60 cells with absent ASS1 expression had a good in vitro response to NEI-01. More than 70% killing was seen in KG-1 and HL-60 cells after NEI-01 treatment. However, AML-193 (with low ASS1 but high ASL expressions) and MV-4–11 (with high ASS1 but low ASL expressions) cells had a partial response to NEI-01. 50% to 70% killing was seen in AML-193 and MV-4–11 cells after NEI-01 treatment. In contrast, Kasumi-1, U937, and THP-1 cells with both high ASS1 and ASL expressions were relatively resistant to NEI-01. Less than 20% killing was seen in Kasumi-1, U937, and THP-1 cells after NEI-01 treatment (Fig. 1C). These results indicated that ASS1 and ASL expressions affected the sensitivity of AML cells to NEI-01 treatment.

To further investigate the potential epigenetic regulation of ASS1, MS-PCR was carried out. Five of seven AML cell lines had evidence of methylation of the ASS1 promoter. Two AML cell lines (MV-4–11 and THP-1) with high expression of ASS1 mRNA and protein, had not shown detectable methylation in ASS1 promoter (Fig. 1D).

NEI-01-induced arginine deprivation caused cell-cycle arrest and induced apoptosis in HL-60 cells in vitro

To further investigate the cytotoxic effect of NEI-01 on AMLs, we compared ASS1-deficient HL-60 cells and ASS1-proficient THP-1 cells. It was found that NEI-01 (≥8 ng/mL) effectively depleted the extracellular arginine both of HL-60 and THP-1 cells. However, this NEI-01-induced arginine deprivation was only cytotoxic to HL-60 cells, but not to THP-1 cells (Fig. 2A).

Figure 2.

NEI-01-induced arginine deprivation had a cytotoxic effect on HL-60 through induction of cell cycle arrest and apoptosis. A, Treatment with NEI-01 dose-dependently depleted extracellular arginine both in AML cell lines HL-60 and THP-1, but only reduced the cell viability of HL-60. HL-60, and THP-1 cells were treated with NEI-01 at various concentrations for 72 hours. Arginine concentration quantified by amino acid analyzer is shown as columns, whereas cell viability tested by trypan blue exclusion method is shown as lines. Experiment was performed in triplicate and repeated three times. Data are expressed as mean ± SD. B and C, Treatment with NEI-01 induced cell cycle arrest in sub-G1 stage and apoptosis in HL-60 cells, but not in THP-1 cells. HL-60 and THP-1 cells were treated with 100 ng/mL NEI-01 for different durations. Cell-cycle analysis was performed using PI staining and flow cytometry. Apoptotic status of cells was examined by flow cytometry using PI and annexin V double-staining. Viable cells are Annexin V negative and PI negative; early apoptotic cells are Annexin V positive and PI negative; and late apoptotic cells are Annexin V positive and PI positive. Experiment was repeated three times. Data are expressed as mean ± SD. P value was calculated using Student t test (*, P < 0.05; **, P < 0.01).

Figure 2.

NEI-01-induced arginine deprivation had a cytotoxic effect on HL-60 through induction of cell cycle arrest and apoptosis. A, Treatment with NEI-01 dose-dependently depleted extracellular arginine both in AML cell lines HL-60 and THP-1, but only reduced the cell viability of HL-60. HL-60, and THP-1 cells were treated with NEI-01 at various concentrations for 72 hours. Arginine concentration quantified by amino acid analyzer is shown as columns, whereas cell viability tested by trypan blue exclusion method is shown as lines. Experiment was performed in triplicate and repeated three times. Data are expressed as mean ± SD. B and C, Treatment with NEI-01 induced cell cycle arrest in sub-G1 stage and apoptosis in HL-60 cells, but not in THP-1 cells. HL-60 and THP-1 cells were treated with 100 ng/mL NEI-01 for different durations. Cell-cycle analysis was performed using PI staining and flow cytometry. Apoptotic status of cells was examined by flow cytometry using PI and annexin V double-staining. Viable cells are Annexin V negative and PI negative; early apoptotic cells are Annexin V positive and PI negative; and late apoptotic cells are Annexin V positive and PI positive. Experiment was repeated three times. Data are expressed as mean ± SD. P value was calculated using Student t test (*, P < 0.05; **, P < 0.01).

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Cell-cycle analysis showed that NEI-01 caused a significant increase of HL-60 cells in the sub-G1 phase (P < 0.01) and a corresponding decrease in the S and G2/M phase. The percentage of HL-60 cells in sub-G1 phase was increased from 5.45 ± 2.21% (nontreated control) to 7.94 ± 0.80% (6 hours NEI-01 treatment), 23.10 ± 1.68% (24 hours NEI-01 treatment), 27.23 ± 4.53% (48 hours NEI-01 treatment), and 32.37 ± 2.80% (72 hours NEI-01 treatment; Fig. 2B). These results indicated that NEI-01 caused HL-60 cell-cycle arrest at sub-G1 phase, resulting in the inhibition of its cell growth. Similar results were found in mouse ASS1-deficient AML C1498 cells (Supplementary Fig. S4).

Apoptosis analysis, in addition, showed that NEI-01 caused a significant decrease in viable cells (Annexin-V negative, PI negative) of HL-60, but increases in its both early apoptotic (Annexin-V positive, PI negative) and late apoptotic (Annexin-V positive, PI positive) cells (Fig. 2C). This NEI-01 induced apoptosis on HL-60 cells was most pronounced after 48 hours. In nontreated HL-60 cells, there were a high percentage of viable cells (91.80 ± 1.56%), but a low percentage of early apoptotic (5.90 ± 1.79%) and late apoptotic cells (2.21 ± 0.31%). After 48 hours NEI-01 treatment on HL-60 cells, the percentages of the viable cells were significantly decreased to 59.63 ± 9.38% (P < 0.01); the early and late apoptotic cells were significantly increased to 33.73 ± 10.19% (P < 0.05) and 6.44 ± 1.02% (P < 0.05), respectively. Similar results were found in mouse ASS1-deficient AML C1498 cells (Supplementary Fig. S4).

There was no significant difference in cell-cycle distribution between NEI-01-treated and nontreated THP-1 cells (Fig. 2B). Also no apoptosis induction was found in NEI-01-treated THP-1 cells (Fig. 2C).

NEI-01 has shown an antileukemia activity in human AML CDX

CDX model is the most commonly used in oncology drug development for more than 40 years. Here, we tested the antileukemia activities of NEI-01 in ASS1-deficient AML CDX model.

Human ASS1-deficient HL-60 cells stably expressing luciferase and GFP (HL-60-gfp-Luc+) were intravenously transplanted into NOD/SCID mice to establish an AML orthotopic CDX model. Seven days later, the mice (n = 10) were intravenously treated with 280 U/kg NEI-01 or PBS twice a week for 4 weeks. NEI-01, as a potent arginine-depleting enzyme (21), effectively depleted the circulating arginine and it was accompanied by increases in the product of arginine breakout, citrulline in AML mice (Fig. 3A). Treatment with NEI-01 significantly reduced the bioluminescence signals, indicating that slowed down the leukemia progression of AML mice bearing HL-60-gfp-Luc+ xenografts (Fig. 3B and C). At the termination of the experiment (1 week after the fourth dosing), it was found that there was a significant decrease of leukemia cells in BM and spleens, but not in livers (Fig. 3D).

Figure 3.

NEI-01 showed antileukemia activities in AML cell line HL-60-derived xenografts. A, NEI-01 efficiently depleted circulating arginine (left) and increased citrulline (right) in AML mice bearing HL-60-gfp-Luc+ xenografts. Human HL-60-gfp-Luc+ cells obtained by lentiviral transduction were intravenously transplanted into NOD/SCID mice. 7 days later, the mice were randomly assigned to NEI-01 280 twice a week group (n = 10) and intravenously received NEI-01 at 280 U/kg twice a week. The mice in control group (n = 10) intravenously received PBS only. Blood was collected weekly before each treatment. The plasma obtained after centrifuge was subject to quantify the concentrations of arginine and citrulline in mouse plasma by amino acid analyzer. B and C, Treatment with NEI-01 significantly slowed down the leukemia growth in HL-60-gfp-Luc+ bearing mice. The leukemia growth, shown as the increase in bioluminescent signal was monitored twice a week by IVIS and the signal intensity was quantified by Living Image in vivo software. D, Treatment with NEI-01 significantly reduced the HL-60-gfp-Luc+ leukemia cells in BM and spleen, but not in liver at the termination. BM, spleens and livers were dissected at the termination (28 days after the initial treatment). Mononuclear cells from BM, spleens and livers were analyzed by flow cytometry. The percentage of GFP+ population was considered as leukemia cells. E and F, Treatment with NEI-01 significantly inhibited the growth of HL-60 xenografts and caused a reduction in the tumor weight. Human HL-60 cells were subcutaneously inoculated into the nude mice. Twelve days later, the mice were randomly assigned to NEI-01 280 twice a week group (n = 9) and intraperitoneally received NEI-01 at 280 U/kg twice a week. Control group (n = 9) intraperitoneally received PBS only. The tumor volume was monitored twice a week and calculated using: tumor volume = (width2 × length)/2. The tumors were harvested and weighted at the termination (28 days after the initial treatment). Data are expressed as mean ± SD. P value was calculated using Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

NEI-01 showed antileukemia activities in AML cell line HL-60-derived xenografts. A, NEI-01 efficiently depleted circulating arginine (left) and increased citrulline (right) in AML mice bearing HL-60-gfp-Luc+ xenografts. Human HL-60-gfp-Luc+ cells obtained by lentiviral transduction were intravenously transplanted into NOD/SCID mice. 7 days later, the mice were randomly assigned to NEI-01 280 twice a week group (n = 10) and intravenously received NEI-01 at 280 U/kg twice a week. The mice in control group (n = 10) intravenously received PBS only. Blood was collected weekly before each treatment. The plasma obtained after centrifuge was subject to quantify the concentrations of arginine and citrulline in mouse plasma by amino acid analyzer. B and C, Treatment with NEI-01 significantly slowed down the leukemia growth in HL-60-gfp-Luc+ bearing mice. The leukemia growth, shown as the increase in bioluminescent signal was monitored twice a week by IVIS and the signal intensity was quantified by Living Image in vivo software. D, Treatment with NEI-01 significantly reduced the HL-60-gfp-Luc+ leukemia cells in BM and spleen, but not in liver at the termination. BM, spleens and livers were dissected at the termination (28 days after the initial treatment). Mononuclear cells from BM, spleens and livers were analyzed by flow cytometry. The percentage of GFP+ population was considered as leukemia cells. E and F, Treatment with NEI-01 significantly inhibited the growth of HL-60 xenografts and caused a reduction in the tumor weight. Human HL-60 cells were subcutaneously inoculated into the nude mice. Twelve days later, the mice were randomly assigned to NEI-01 280 twice a week group (n = 9) and intraperitoneally received NEI-01 at 280 U/kg twice a week. Control group (n = 9) intraperitoneally received PBS only. The tumor volume was monitored twice a week and calculated using: tumor volume = (width2 × length)/2. The tumors were harvested and weighted at the termination (28 days after the initial treatment). Data are expressed as mean ± SD. P value was calculated using Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

Human ASS1-deficient HL-60 cells were subcutaneously inoculated into the athymic nude mice to establish an AML subcutaneous CDX model. Twelve days later, the xenografts reached 100 mm3 and the mice (n = 9) were intraperitoneally treated with 280 U/kg NEI-01 or PBS twice a week for 4 weeks. Treatment with NEI-01 significantly inhibited the tumor growth (Fig. 3E) and caused a significant reduction in the tumor weights (P < 0.001, 0.27 ± 0.21 g in NEI-01 treatment group vs. 1.04 ± 0.39 g in control group, Fig. 3F).

NEI-01 has shown an antileukemia activity in mouse AML cell line-derived homografts in syngeneic mice

It is well known that arginine has an important role in the immune system. Impact of arginine metabolism was reported to affect the immune response, helping the cancer cells escape immune recognition and elimination, and resulting in the growth of many cancers (22). Here we tested the antileukemia activity of NEI-01-induced arginine deprivation in immunocompetent mouse models with AML.

Mouse ASS1-deficient C1498 cells stably expressing luciferase and GFP (C1498-gfp-Luc+ cells) were intravenously transplanted into immunocompetent C57BL/6 mice to establish an AML syngeneic mouse model. Three days later, the mice were intravenously treated with 140 U/kg NEI-01 once a week (n = 9), 280 U/kg NEI-01 once a week (n = 9), 280 U/kg NEI-01 twice a week (n = 8) or vehicle (n = 8) for 4 weeks. NEI-01 effectively depleted the circulating arginine in these syngeneic AML mice (Fig. 4A), which is the similar result found in the immunocompromised mice bearing HL-60 xenografts (Fig. 3A). An aggressive disease progression observed in PBS-treated control mice, resulted in all deaths within 28 days after the leukemia cell transplantation. Treatment with NEI-01 significantly prolonged the overall survival by at least 5 days compared with controls, with median overall survival of ≥29 days in NEI-01 treated mice and 24 days in control mice, respectively (Fig. 4B). The in vivo BLI results showed that treatment with NEI-01 significantly reduced the bioluminescence signals, indicating that slowed down the leukemia progression in a dose-dependent manner (Fig. 4C and D).

Figure 4.

NEI-01 showed antileukemia activities in AML syngeneic mouse model. A, NEI-01 efficiently depleted circulating arginine (left) and increased citrulline (right) in syngeneic AML mice. Mouse C1498-gfp-Luc+ cells obtained by lentiviral transduction were intravenously transplanted into C57BL/6 mice. Three days later, the mice were randomly assigned to NEI-01 140 once a week (n = 8), NEI-01 280 once a week (n = 9), and NEI-01 280 twice a week (n = 9) groups. The mice intravenously received NEI-01 at 140 U/kg once a week, 280 U/kg once a week, or 280 U/kg twice a week, respectively. The mice in control group (n = 8) intravenously received PBS only. Blood from the mice (n = 5) was collected weekly before each treatment. The plasma obtained after centrifuge was subject to quantify the concentrations of arginine and citrulline in mouse plasma by amino acid analyzer. B, Kaplan–Meier survival curves showed that treatment with NEI-01 significantly prolonged the survival of C1498-gfp-Luc+ bearing mice as compared with vehicle control by a log-rank test. C and D, Treatment with NEI-01 significantly decreased the leukemia burden in C1498-gfp-Luc+ bearing mice. Leukemia burden, shown as bioluminescent signal, was detected twice a week by IVIS and quantified by Living Image in vivo software. Data are expressed as mean ± SD.

Figure 4.

NEI-01 showed antileukemia activities in AML syngeneic mouse model. A, NEI-01 efficiently depleted circulating arginine (left) and increased citrulline (right) in syngeneic AML mice. Mouse C1498-gfp-Luc+ cells obtained by lentiviral transduction were intravenously transplanted into C57BL/6 mice. Three days later, the mice were randomly assigned to NEI-01 140 once a week (n = 8), NEI-01 280 once a week (n = 9), and NEI-01 280 twice a week (n = 9) groups. The mice intravenously received NEI-01 at 140 U/kg once a week, 280 U/kg once a week, or 280 U/kg twice a week, respectively. The mice in control group (n = 8) intravenously received PBS only. Blood from the mice (n = 5) was collected weekly before each treatment. The plasma obtained after centrifuge was subject to quantify the concentrations of arginine and citrulline in mouse plasma by amino acid analyzer. B, Kaplan–Meier survival curves showed that treatment with NEI-01 significantly prolonged the survival of C1498-gfp-Luc+ bearing mice as compared with vehicle control by a log-rank test. C and D, Treatment with NEI-01 significantly decreased the leukemia burden in C1498-gfp-Luc+ bearing mice. Leukemia burden, shown as bioluminescent signal, was detected twice a week by IVIS and quantified by Living Image in vivo software. Data are expressed as mean ± SD.

Close modal

NEI-01 has shown an antileukemia activity in AML PDX

PDX model has increasingly become a preferred model for drug discovery and translation in the past decade. Here, we tested the antileukemia activities of NEI-01 in ASS1-deficient AML PDX model.

Human ASS1-deficient AM5512 cells were intravenously transplanted into NOD/SCID mice to establish an AML PDX model. Twenty-two days later, the PB engraftment of hCD45+ AML cells reached approximately 1.33% and the mice (n = 10) were intravenously treated with 140 U/kg NEI-01, 280 U/kg NEI-01, or vehicle once a week for 4 weeks. Treatment with NEI-01 at either 140 or 280 U/kg, significantly inhibited the leukemia cell growth in PB (Fig. 5A). In addition, the leukemia burden in BM, liver, and spleen was also effectively decreased (Fig. 5B). Excitingly, treatment with NEI-01 eliminated nearly all AML burden in BM (5.38 ± 3.19% and 3.13 ± 3.44% respectively in NEI-01 140 and 280 U/kg once a week) and spleens (2.40 ± 1.50% and 2.38 ± 1.95% respectively in NEI-01 140 and 280 U/kg once a week), whereas an antileukemia activity of NEI-01 was present in liver in a dose-dependent manner.

Figure 5.

NEI-01 showed antileukemia activities in AML PDXs. A, Treatment with NEI-01 significantly inhibited leukemia cell growth in PB. B, Treatment with NEI-01 significantly reduced the leukemia burden in BM, spleen and liver at the termination. AML patient-derived AM5512 cells were intravenously transplanted into the NOD/SCID mice. Twenty-two days later, the mice were randomly assigned to NEI-01 140 once a week (n = 10) and NEI-01 280 once a week (n = 10) groups. The mice intravenously received NEI-01 at 140 and 280 U/kg once a week, respectively. The mice in control group (n = 10) intravenously received vehicle only. PB was collected weekly. BM, spleens, and livers were dissected at the termination (28 days after the initial treatment). Mononuclear cells from PB, BM, livers, and spleens were stained with FITC conjugated mouse antihuman CD45 and analyzed by flow cytometry. The percentage of hCD45+ population was considered as leukemia burden. Data are expressed as mean ± SD. P values were calculated using Student t test (**, P < 0.01; ***, P < 0.001).

Figure 5.

NEI-01 showed antileukemia activities in AML PDXs. A, Treatment with NEI-01 significantly inhibited leukemia cell growth in PB. B, Treatment with NEI-01 significantly reduced the leukemia burden in BM, spleen and liver at the termination. AML patient-derived AM5512 cells were intravenously transplanted into the NOD/SCID mice. Twenty-two days later, the mice were randomly assigned to NEI-01 140 once a week (n = 10) and NEI-01 280 once a week (n = 10) groups. The mice intravenously received NEI-01 at 140 and 280 U/kg once a week, respectively. The mice in control group (n = 10) intravenously received vehicle only. PB was collected weekly. BM, spleens, and livers were dissected at the termination (28 days after the initial treatment). Mononuclear cells from PB, BM, livers, and spleens were stained with FITC conjugated mouse antihuman CD45 and analyzed by flow cytometry. The percentage of hCD45+ population was considered as leukemia burden. Data are expressed as mean ± SD. P values were calculated using Student t test (**, P < 0.01; ***, P < 0.001).

Close modal

Histologic staining showed that a large number of leukemic cells were visible in the BM cavity of mice from the control group. Treatment with NEI-01 at either 140 or 280 U/kg, significantly reduced the leukemia cells, as well as recovered the hematopoietic system. Like most cases in patients with AML, the leukemic cells infiltrated the liver and spleen in AML mice. However, treatment with NEI-01 significantly decreased the leukemic infiltration and recovered their normal structures (Supplementary Fig. S5). This result also supported the recently reported idea about the effects of arginine deprivation on cell migration and invasion (9).

AML blast cells have a much higher nutrient demand to support their rapid growth, and eventually affect the production and function of normal blood cells. Drug-induced nutrient deprivation directly depletes the nutrient in circulating system and is considered as more effective for treatment of hematologic malignancies. One of the successful examples is asparaginase which is used as a first-line treatment for acute lymphoblastic leukemia. Here, we have tested the preclinical activities of NEI-01-induced arginine deprivation in AMLs.

Both ASS1 and ASL control the endogenous arginine synthesis. ASS1 expression has been highly recommended as a predictive biomarker for sensitivity to arginine deprivation therapy (23, 24). In our present study, significant correlation was observed among low ASS1 mRNA levels, low ASS1 protein expression, and ASS1 promoter methylation in AMLs. The ASS1-deficient AML cells were found to be sensitive to NEI-01-induced arginine deprivation both in vitro and in vivo. However, no ASL-negative tumor has been described in the literature to date (10). Interestingly, both previous and our studies have indicated that ASL is highly expressed in some ASS1-deficient tumors (25–27), and supported the secondary role of ASL in modulating tumoral arginine auxotrophy and sensitivity to arginine depleting enzymes (28).

The mechanism of arginine deprivation for cancer therapy has been studied by several groups. It has been indicated that arginine deprivation can potentially modulate numerous cellular and signaling pathways rendering their cytotoxic and cytostatic pathways (29). In our present study, NEI-01-induced arginine deprivation prompted ASS1-deficient AML cell death through induction of sub-G1 cell-cycle arrest and apoptosis. Comparable effects were found in pancreatic cancer, cholangiocarcinoma, small cell lung cancer, and leukemia when treated with ADI-PEG or rh-Arg1-PEG (15, 30–33). Besides the role in apoptosis induction, arginine deprivation has been reported to have anti-angiogenic effects as well as inhibition of de novo protein synthesis. It is worth noting that the antitumor potential of arginine deprivation is not only simply accredited to its action as arginine depleting enzyme but also to several other mechanisms important in the cellular functioning of tumor cells (29). Indeed, much attention has been devoted to the combination of arginine deprivation with either chemo- or immune-therapy. The encouraging results were observed when combining ADI-PEG20 and cytarabine for AML treatment both in preclinical and clinical studies (16, 34). Further studies will be conducted to investigate the combination effects between NEI-01 and other chemo- or immune-therapy agents.

Preclinical study is essential to predict the clinical outcome during the drug development. Here we used three different types of mouse models to synthetically evaluate the NEI-01 therapeutic efficacy in ASS1-deficient AMLs. The results showed that NEI-01 has the potent antileukemia activities not only in immunocompromised (CDX and PDX) mouse models, but also in immunocompetent (syngeneic) mouse models. Comparable effects were found in ADI-PEG treated PDX (16) and rh-Arg1-PEG treated CDX (15). Previous study has indicated that arginine deprivation impedes proliferation and cell cycle progression of activated T cells, leading to impaired antitumor immunity (35, 36). Therefore, it is important to evaluate the therapeutic efficacy of arginine deprivation in immunocompetent mouse models. Here we report for the first time that NEI-01-induced arginine deprivation significantly inhibited the leukemia growth as well as prolonged the overall survival in immunocompetent (syngeneic) AML mice. It is still unclear how the immunity works with the leukemia cells under NEI-01 induced arginine deprivation. Previous studies have shown that T-cell responses could be impaired by depleting arginine, however this impairment could be revised by addition of citrulline (37). Fortunately, NEI-01 is designed to hydrolyze arginine into citrulline and ammonia. T cells increase citrulline uptake and upregulate ASS1 expression to make sufficient arginine for their survival, proliferation, and functions (38). In addition, NEI-01-induced arginine deprivation could result in cell-cycle arrest and apoptosis in arginine auxotrophic AMLs. The apoptotic cell death has been indicated to be associated with enhanced infiltration of lymphocytes, yielding boosted antitumor immune surveillance (39).

In conclusion, NEI-01, as a modified arginine depleting enzyme, shows a promising preclinical performance in antileukemia activities that may pave the way for clinical trials.

Y-c. Leung reports other support from new epsilon innovation limited during the conduct of the study; other support from other outside the submitted work; also has a patent for Patent Nos. 16/946,590, PCT/CN2020/098681, and 110105746 pending. Y. Cai reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. J.P.H. Chow reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. Y-O. Leung reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. X. Lu reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. C-H. Yuen reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. W.L. Lee reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. K-C. Chau reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. L.L. Yang reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. R.M.H. Wong reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. J.Y.T. Lam reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. D.T.L. Chow reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. S.H.K. Chung reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work. S-Y. Kwok reports other support from New Epsilon Innovation Limited during the conduct of the study; other support from New Epsilon Innovation Limited outside the submitted work; also has a patent for 16/946,590, PCT/CN2020/098681, and 110105746 pending.

Y. Cai: Conceptualization, formal analysis, investigation, writing–original draft. J.P.H. Chow: Data curation, validation, investigation. Y.-O. Leung: Data curation, formal analysis, methodology. X. Lu: Data curation, investigation, methodology. C.-H. Yuen: Data curation, investigation, methodology. W.L. Lee: Data curation, investigation, methodology. K.-C. Chau: Data curation, investigation, methodology. L.L. Yang: Data curation, investigation, methodology. R.M.H. Wong: Data curation, investigation, methodology. J.Y.T. Lam: Data curation, investigation, methodology. D.T.L. Chow: Data curation, investigation, methodology. S.H.K. Chung: Data curation, investigation, methodology. S-Y. Kwok: Conceptualization, formal analysis, supervision, project administration, writing–review and editing. Y.-c. Leung: Conceptualization, formal analysis, supervision, writing–review and editing.

We are grateful to New Epsilon Innovation Limited for providing NEI-01. We thank Crown Bioscience, Inc., for providing AML patient derived cells and helping with NEI-01 in vivo efficacy test on AM5512 PDX model. We thank University Research Facility in Life Sciences (The Hong Kong Polytechnic University) for equipment support. We also acknowledge support from the Hong Kong Polytechnic University PTEC Grant P19-0121 (to Y-c. Leung). This work was supported by New Epsilon Innovation Limited.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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