Inhibiting glutamine uptake represents an attractive new strategy for treating acute myeloid leukemia

Abstract

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.

Figure 1

Glutamine removal inhibits mTORC1 activity and induces apoptosis in AML cells. (A) The MOLM-14 and OCI-AML3 cell lines, and primary AML cells from a representative patient, were cultured for 6 or 24 hours with or without 4 mM glutamine (Gln) and/or 0.4 mM leucine (Leu). Cells were lysed in Laemmli buffer and western blotting was performed to analyze the p70S6K T389 and 4E-BP1 S65 phosphorylation status. (B) MOLM-14, OCI-AML3, MV4-11, and HL-60 cell lines were cultured with or without Gln for 24 hours and apoptosis was quantified by flow cytometry analysis of Annexin-V binding. (C) The indicated cell lines were cultured for 24 hours with or without glutamine and then assessed for caspase-3 and PARP cleavage by western blotting. (D) Normal CD34⁺ cell were obtained from 3 healthy allogeneic transplant donors after positive sorting and primary AML cells from 8 AML patients were also tested. Cells were cultured 48 hours with or without glutamine (4 mM), and the apoptotic responses were quantified by flow cytometry analysis of Annexin-V binding.

Figure 2

Targeting the SLC1A5 glutamine transporter has proapoptotic effects and in vivo antileukemic activity. (A) SLC1A5 expression was studied by western blotting in the 4 indicated AML cell lines. (B-F) MOLM-14 cells were infected with a lentiviral vector expressing either an inducible SLC1A5 or a control (CTR) shRNA. Stably infected cell lines were established after selection in puromycin. (B) MOLM-14 cells infected with SLC1A5 or CTR shRNA were cultured with or without doxycycline for the indicated times. The inhibition of SLC1A5 expression with doxycycline was assessed by western blotting. (C) MOLM-14/SLC1A5 shRNA cells and MOLM-14/CTR shRNA cells were starved or not for 6 hours of glutamine and leucine to inhibit mTORC1, as detected by the inhibition of p70S6K T389 phosphorylation. Previously starved cells were then stimulated for 15 or 30 minutes with leucine + glutamine in the presence or absence of doxycycline. The reactivation of mTORC1 was then determined. (D) Apoptosis was evaluated in MOLM-14/SLC1A5 shRNA cells and MOLM-14/CTR shRNA cells in the presence of doxycycline by flow cytometry analysis of Annexin-V fixation or by western blotting analysis of PARP and caspase-3 cleaved fragments. Analysis was performed every day from day 2 to day 6. (E) MOLM-14/SLC1A5shRNA cells (red) or MOLM-14/CTR shRNA cells (black) were injected into nude mice. The mice were then treated with doxycycline by oral gavage, and the tumor volumes were determined at the indicated times. The means of the individual tumor sizes were then plotted (n = 8 in each group). (F) Kaplan-Meier survival curves of nude mice treated with doxycycline. (G) SLC1A5 expression was detected by western blotting analysis of tumor extracts from MOLM-14/SLC1A5 shRNA or MOLM-14/CTR shRNA mice treated with doxycycline (day 22).

Figure 3

The glutaminase activity of l-ase inhibits mTORC1 and protein translation in AML cells. (A) OCI-AML3 cells were cultured for 24 hours with the indicated doses of E coli l-ase, and the levels of glutamine and asparagine in the intracellular compartment were quantified by ion exchange chromatography. (B) The OCI-AML3 cell line was cultured for 6 or 24 hours with or without E coli or E chrysanthemi l-ases at an equal level of asparaginase activity (0.1-10 UI/mL) and then lysed in Laemmli buffer. Western blotting was performed with anti-phospho-p70S6K T389, anti-phospho-4E-BP1 S65, anti-p70S6K, anti-4E-BP1, and anti-actin antibodies. (C) Cells were treated as indicated in B, and the level of Gln in the extracellular medium was quantified by ion exchange chromatography. (D) Primary bone marrow leukemic cells from AML patients were treated as in A, and the same analysis was assessed by western blotting. The intensities of phospho-p70S6K T389, phospho-4E-BP1 S65, and actin signals were quantified in different AML samples. Ratios of phospho-p70S6K T389 or phospho-4E-BP1 S65 to actin were calculated and assigned a reference value of 1 in the control culture. (E) HEK-293T cells were transfected with a Flag-Raptor-Rheb15 expression vector or a control vector and then cultured for 24 hours with or without glutamine at 4 mM or E coli l-ase (10 UI/mL). Western blotting was then performed with anti-flag, anti-actin, or anti-phospho-p70S6K T389 antibodies. (F) [S³⁵] methionine pulses were performed in MOLM-14, OCI-AML3, MV4-11, and HL-60 cells to evaluate the global protein synthesis profile with or without E coli l-ase at 10 UI/mL during a 6-hour period. (G) OCI-AML3, MOLM-14, MV4-11, and HL-60 cell lines and a representative AML sample were cultured for 24 hours with or without E coli l-ase. The expression of Mcl-1 and c-Myc was then analyzed by western blotting.

Figure 4

l-ase induces apoptosis in leukemic cells. OCI-AML3 cells and primary AML samples were cultured for 24 or 48 hours in 10% FBS MEM with or without 0.1, 1, or 10 UI/mL of E coli or E chrysanthemi l-ase. Apoptosis was determined by Annexin V ^+/− 7AAD binding using flow cytometry (A-D) or by western blotting using an anti-caspase 3 antibody (E).

Figure 5

l-ase increases GS expression in AML, which may represent a mechanism of resistance to l-ase. (A) MOLM-14, OCI-AML3, and primary AML blast cells from 3 patients were cultured over a 24-hour period with or without 0.1, 1, or 10 UI/mL E coli l-ase. GS expression was determined by western blotting. (B) OCI-AML3 and MV4-11 cells were stably infected with a lentiviral vector expressing either a CTR or a GS shRNA and then cultured with or without 10 UI/mL E coli l-ase and with or without glutamine at 4 mM for 24 hours. Western blotting was then performed to assess the GS expression level. (C) The same cell lines were tested by flow cytometry analysis of Annexin V fixation for apoptosis induction after 24 hours in the presence or absence of E coli l-ase at 10 UI/mL and with or without glutamine starvation.

Figure 6

l-ase induces strong protective autophagy in AML cells. (A-B) OCI-AML3 cells were cultured for 6 or 24 hours in 10% FCS MEM with or without 0.1, 1, and 10% FCS MEM with or without 0.1, 1, or 10 UI/mL of E coli l-ase and with or without Gln as indicated. Autophagy was detected by western blotting using anti-LC3b and p62/SQSTM1 antibodies. (C) Representative electron micrographs of OCI-AML3 cells collected after 24 hours of culture in MEM with or without E coli l-ase (1 UI/mL) and without glutamine. Arrows indicate autophagic vesicles. Upper and lower panels show 2 different scales. (D) OCI-AML3 cells were transfected with anti-ATG5 or anti-beclin siRNAs using the Amaxa nucleofector kit. Two days after transfection, cells were cultured for 24 hours with or without E coli l-ase at 10 UI/mL and western blotting was performed to verify ATG5 and Beclin knockdowns, respectively. (E) Viability was measured in OCI-AML3 cells (transfected or not with anti-ATG5 or anti-Beclin siRNA and treated or not with E coli l-ase) by flow cytometry analysis of Annexin V fixation. The histograms shown represent the mean values of 3 independent experiments.