Arginine dependence of acute myeloid leukemia blast proliferation: a novel therapeutic target

Abstract

Acute myeloid leukemia (AML) is one of the most common acute leukemias in adults and children, yet significant numbers of patients relapse and die of disease. In this study, we identify the dependence of AML blasts on arginine for proliferation. We show that AML blasts constitutively express the arginine transporters CAT-1 and CAT-2B, and that the majority of newly diagnosed patients' blasts have deficiencies in the arginine-recycling pathway enzymes argininosuccinate synthase and ornithine transcarbamylase, making them arginine auxotrophic. BCT-100, a pegylated human recombinant arginase, leads to a rapid depletion in extracellular and intracellular arginine concentrations, resulting in arrest of AML blast proliferation and a reduction in AML engraftment in vivo. BCT-100 as a single agent causes significant death of AML blasts from adults and children, and acts synergistically in combination with cytarabine. Using RNA sequencing, 20 further candidate genes which correlated with resistance have been identified. Thus, AML blasts are dependent on arginine for survival and proliferation, as well as depletion of arginine with BCT-100 of clinical value in the treatment of AML.

Figure 1

AML blasts are auxotrophic for arginine. (A) AML patients’ blasts and AML cell lines were cultured in complete or arginine-depleted media. The viability of AML blasts from patients and cell lines was assessed by flow cytometry after 72 hours. Arginine depletion leads to a decreased percentage of viable blasts. Representative of 2 independent experiments. (B) Expression of CAT-1, CAT-2A, and CAT-2B in blasts from 10 patients was confirmed by quantitative polymerase chain reaction (qPCR). Patients are identified by unique symbols, which are used consistently throughout the manuscript. (C) Staining of 39 bone marrow samples from AML patients at diagnosis with hematoxylin-eosin (left panel), anti-OTC (center panel), and anti-ASS (right panel). Representative marrows from 2 patients showing positive antigen staining (top) and negative antigen staining (bottom). (D) Histoscores of ASS and OTC staining in adult and pediatric AML bone marrow samples. (E) Plasma from 20 AML patients at diagnosis and 16 healthy donors were analyzed for arginine concentration by enzyme-linked immunosorbent assay (ELISA). Plasma arginine levels are significantly lower in newly diagnosed patients (P < .0001).

Figure 2

BCT-100 arginine depletion reduces the number of viable AML blasts in vitro and in vivo. (A) Plasma from control and BCT-100 treated NOG mice were collected after 14 days. The concentration of arginine was determined by ELISA. BCT-100 significantly lowers the plasma arginine concentration in vivo (P < .0244). (B) NOG mice were injected with HL-60 AML blasts. BCT-100 (5 mg/kg) or cytarabine (25 mg/kg) was given i.p. injections twice a week. Bone marrow was sampled from the femurs after 5 weeks to assess hCD45⁺ cells by flow cytometry. BCT-100 leads to significantly lower AML engraftment (P = .029), equivalent to cytarabine treatment. Data are representative of 2 independent experiments. (C) Untreated and BCT-100 NOG mice engrafted with HL-60 AML showed no significant difference in body weight in response to treatment.

Figure 3

BCT-100 is cytotoxic against primary blasts from patients. (A) AML blasts from 20 newly diagnosed patients were cultured with BCT-100 (0-4000 ng/mL) for 72 hours. The percentage of viable blasts relative to untreated was determined by flow cytometry. BCT-100 leads to a dose-dependent decrease in AML blast viability. (B) IC₅₀ values for the activity of BCT-100 against AML patient blasts are shown. (C) AML blasts from patients were cultured with 600 ng/mL BCT-100 (OBD) alone, 500 ng/mL cytarabine, or both for 72 hours. The percentage of viable cells relative to control after 72 hours was measured by flow cytometry. BCT-100 cytotoxicity is synergistic in combination with cytarabine (BCT vs combination, P = .0054; cytarabine vs combination, P = .0059; 2-way ANOVA: F_(1,57) = 6.405, P < .0001). (D) The percentage of viable cells following treatment with 600 ng/mL BCT-100 and 500 ng/mL cytarabine was correlated. Sensitivity to BCT-100 correlates moderately with sensitivity to cytarabine (r = 0.5128, P = .0208).

Figure 4

BCT-100 depletes arginine intracellularly. (A) Cell lines or patient samples were cultured with BCT-100 (600 ng/mL) for 72 hours. Intracellular arginine concentrations were measure by ELISA. BCT-100 causes a depletion of intracellular arginine. Data are representative of 2 independent experiments. (B) Internalization of BCT-100-AF647. AML blasts (top panel) and HL60 cells (bottom panel) were incubated with fluorescently labeled BCT-100 for 8 hours. Unbound drug was removed with stripping buffer and extensive washing. Nucleus was stained with 4,6 diamidino-2-phenylindole (DAPI). Images of representative cells were collected by LSM510 system (Zeiss). Arrows indicate intracellular localization of labeled BCT-100; arrowheads indicate surface-bound drug. Scale, 10 μm. Representative patient sample of 3 different patient samples.

Figure 5

BCT-100 halts proliferation and cell cycle arrest. (A) BCT-100 halts AML cell division. CFSE-labeled cell lines were cultured in the presence of 600 ng/mL BCT-100. Representative histogram plots shown. Independent experiments were performed on 2 separate occasions. (B) Cell lines were cultured with BCT-100 (0-2000 ng/mL) for 72 hours. AML proliferation was measured by ³H-thymidine incorporation after 72 hours. Data are representative of 2 independent experiments. BCT-100 causes a dose-dependent decrease in AML proliferation. (C) AML cell lines were cultured with 600 ng/mL BCT-100. Cell cycle analysis was performed after 72 hours. BCT-100 increases the percentage of cells in G0/G1 arrest. Representative histogram plots for untreated and treated HL-60 shown. Independent experiments were performed on 4 separate occasions. (D) Table showing the relative percentages of cells in G0/G1, S, G2/M based on flow cytometry cell cycle analysis.

Figure 6

BCT-100 induced cell cycle arrest leads to necrotic cell death. (A) Relative expression of cyclins A, B, E in BCT-100–treated AML patient blasts compared with untreated controls (hashed line) were investigated by qPCR. Representative data of 4 patients shown. (B) AML blasts from patients were treated with BCT-100 (600 ng/mL) or cytarabine (500 ng/mL) for 72 hours. Analysis of cell death was performed by transmission electron microscopy. Representative micrographs of 2 of 5 patients shown. Left panel, Untreated cells. Middle panels, Posttreatment with 600 ng/mL BCT-100. Features consistent with organelle enlargement and cell membrane permeabilization. Right panels, Posttreatment with 500 ng/mL cytarabine. Features consistent with nuclear fragmentation bodies and preserved membrane integrity. Experiments performed on 3 separate occasions. (C) Sensitivity to BCT-100 does not correlate with CAT1 expression (D) and only mildly with CAT-2B expression (r = −0.31, P = .41).