The arginine metabolome in acute lymphoblastic leukemia can be targeted by the pegylated-recombinant arginase I BCT-100

Abstract

Arginine is a semi-essential amino acid that plays a key role in cell survival and proliferation in normal and malignant cells. BCT-100, a pegylated (PEG) recombinant human arginase, can deplete arginine and starve malignant cells of the amino acid. Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, yet for patients with high risk or relapsed disease prognosis remains poor. We show that BCT-100 is cytotoxic to ALL blasts from patients in vitro by necrosis, and is synergistic in combination with dexamethasone. Against ALL xenografts, BCT-100 leads to a reduction in ALL engraftment and a prolongation of survival. ALL blasts express the arginine transporter CAT-1, yet the majority of blasts are arginine auxotrophic due to deficiency in either argininosuccinate synthase (ASS) or ornithine transcarbamylase (OTC). Although endogenous upregulation or retroviral transduced increases in ASS or OTC may promote ALL survival under moderately low arginine conditions, expression of these enzymes cannot prevent BCT-100 cytotoxicity at arginine depleting doses. RNA-sequencing of ALL blasts and supporting stromal cells treated with BCT-100 identifies a number of candidate pathways which are altered in the presence of arginine depletion. Therefore, BCT-100 provides a new clinically relevant therapeutic approach to target arginine metabolism in ALL.

Keywords: ALL; arginase; arginine.

Figure 1

BCT‐100 arginine depletion decreases ALL disease burden in vivo. (a) NOG mice were injected with REH‐GFP ALL blasts. BCT‐100 (20 mg/kg) was given by i.v. injection twice a week from D+1. Bone marrow was sampled from the femurs after 2 weeks to assess hCD45+ cells by flow cytometry. BCT‐100 leads to significantly lower ALL engraftment. (b) NOG mice were injected with REH ALL blasts. BCT‐100 (20 mg/kg) was given by i.v. injection twice a week from D+14. Bone marrow was sampled from the femurs after 2 weeks to assess hCD45+ cells by flow cytometry. BCT‐100 leads to significantly lower ALL engraftment. Data are representative of two independent experiments. (c) NOG mice were injected with human ALL blasts, sorted from the blood of a newly diagnosed patient. BCT‐100 (20 mg/kg) was given by i.v. injection twice a week after engraftment was reached. Kaplan‐Meier curves showing a significant prolongation of survival in BCT‐100 treated mice. (d) NOG mice were injected with human ALL blasts, sorted from the blood of a newly diagnosed patient. BCT‐100 (20 mg/kg) was given by i.v. injection twice a week from D+1. Bone marrow was sampled from the femurs after 2 weeks to assess hCD45+ cells by flow cytometry. BCT‐100 leads to significantly lower ALL engraftment. (e) Levels of selected amino acids detected by HPLC‐MS in plasma of BCT‐100 treated or untreated NSG mice normalized to untreated mice. Data for arginine and immediate breakdown products are shown as individual graphs. (f) Plasma from control and BCT‐100 treated NOG mice was collected after 14 days. The concentration of arginine was determined by ELISA. BCT‐100 significantly lowers the plasma arginine concentration in vivo.

Figure 2

BCT‐100 does not affect CNS metabolic profile or ALL disease burden in vivo. (a) NSG mice were injected i.v. with REH cells and BCT‐100 (20 mg/kg) was given by i.v. injection weekly from D+14. Histology of treated and control mice showing no change in ALL engraftment in the CNS at the end of the experiment. Representative images from three of seven mice per group are shown. (b) Quantification of area of ALL CNS infiltration from xenografts showing no significant difference between control and treated mice. (c) Mass‐spectroscopy profiling of CSF from BCT‐100 and control mice revealing no significant change in metabolites. p values are all >0.05 (not shown). (d) Distribution of total radioactivity mice i.v. injected with ¹²⁵I‐PEG‐BCT‐100. The concentration of total radioactivity in tissues of mice at 2, 24, 72 and 168 hrs after intravenous injection of ¹²⁵I‐ PEG‐BCT‐100.The highest total radioactivity level was found in serum, followed by lymph node, spleen, bone marrow and spinal cord (n = 6 mice per time point).

Figure 3

ALL blasts from patients are auxotrophic for arginine. (a) Arginine pathway component expression was determined by qPCR of sorted CD19+ ALL blasts from patients at diagnosis. Patients are identified by unique symbols, which are used consistently throughout the manuscript. AU: arbitrary units. (b) ALL cell lines significantly deplete arginine from the microenvironment. All data are representative of three independent experiments. (c) Staining of bone marrow samples from ALL patients at diagnosis with hematoxylin eosin (upper panel), anti‐ASS (centre panel) and anti‐OTC (lower panel) (Scale = 100 μm). Representative marrows from 2 of 35 patients showing positive antigen staining (right) and negative antigen staining (left). (d) Histoscores of ASS and OTC staining in adult (upper) and pediatric (lower) ALL bone marrow samples. (e) JURKAT, NALM6 and REH cell lines transduced with ASS or OTC genes have increased viability compared with wild‐type cell lines, in cultures with 150 ng/mL BCT‐100 (Histograms). At 600 ng/mL BCT‐100 cell viability of all lines was <15% (red dotted line). Representative of three experiments.

Figure 4

BCT‐100 is cytotoxic against ALL blasts from patients and synergises with dexamethasone. (a) ALL blasts from 14 newly diagnosed patients were cultured with BCT‐100 (0–9,600 ng/mL) for 72 hrs. The percentage of viable blasts relative to untreated was determined by flow cytometry. BCT‐100 leads to a dose‐dependent decrease in ALL blast viability. (b) IC₅₀ values for the activity of BCT‐100 against ALL patient blasts are shown. (c) CAT‐1 expression does not correlate with the percentage of viable cells following 600 ng/mL BCT‐100. (d) ALL blasts from patients were cultured with 600 ng/mL BCT‐100 alone, 600 ng/mL dexamethasone or both for 72 hrs. The percentage of viable cells relative to control after 72 hrs was measured by flow cytometry. BCT‐100 cytotoxicity is synergistic in combination with dexamethasone (BCT vs. combination p = 0.035; dexamethasone vs. combination p = 0.39; two‐way ANOVA: F _(1,24) = 857.2, p < 0.0001). (e) Chou‐Talalay CI for individual patient samples showing synergy between BCT‐100 and dexamethasone. Synergism is defined as CI < 1. (f) The percentage of viable cells following treatment with 600 ng/mL BCT‐100 or 600 ng/mL dexamethasone was correlated. Sensitivity to BCT‐100 does not correlate with sensitivity to dexamethasone.

Figure 5

BCT‐100‐induced cell cycle arrest leads to necrotic cell death. (a) ALL cell lines were cultured with 600 ng/mL BCT‐100. Cell cycle analysis was performed after 72 hrs. BCT‐100 increases the percentage of cells in G0/G1 arrest. (b) Relative expression of cyclins A, B1, D, B2 and E1 in BCT‐100‐treated ALL patients' blasts compared with untreated controls (hashed line) were investigated by qPCR. Representative data of 6 patients are shown. (c) ALL blasts from patients were treated with BCT‐100 (600 ng/mL). Analysis of cell death was performed by transmission electron microscopy. Representative micrographs of three of six patients were shown. Upper panel: untreated cells. Lower panels: post treatment with 600 ng/mL BCT‐100. Features consistent with organelle enlargement, cell membrane permeablisation and cellular fragmentation with 600 ng/mL BCt‐100. Experiments performed on six separate occasions.