Synergistic Effects of Glutamine Deprivation and Metformin in Acute Myeloid Leukemia

Abstract

Objective

The metabolic reprogramming of acute myeloid leukemia (AML) cells is a compensatory adaptation to meet energy requirements for rapid proliferation. This study aimed to examine the synergistic effects of glutamine deprivation and metformin exposure on AML cells.

Methods

SKM-1 cells (an AML cell line) were subjected to glutamine deprivation and/or treatment with metformin or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES, a glutaminase inhibitor) or cytarabine. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis and reactive oxygen species (ROS) by flow cytometry. Western blotting was conducted to examine the levels of apoptotic proteins, including cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, the human long noncoding RNA (lncRNA) microarray was used to analyze gene expression after glutamine deprivation, and results were confirmed with quantitative RT-PCR (qRT-PCR). The expression of metallothionein 2A (MT2A) was suppressed using siRNA. Cell growth and apoptosis were further detected by CCK-8 assay and flow cytometry, respectively, in cells with MT2A knockdown.

Results

Glutamine deprivation or treatment with BPTES inhibited cell growth and induced apoptosis in SKM-1 cells. The lncRNA microarray result showed that the expression of MT family genes was significantly upregulated after glutamine deprivation. MT2A knockdown increased apoptosis, while proliferation was not affected in SKM-1 cells. In addition, metformin inhibited cell growth and induced apoptosis in SKM-1 cells. Both glutamine deprivation and metformin enhanced the sensitivity of SKM-1 cells to cytarabine. Furthermore, the combination of glutamine deprivation with metformin exhibited synergistic antileukemia effects on SKM-1 cells.

Conclusion

Targeting glutamine metabolism in combination with metformin is a promising new therapeutic strategy for AML.

References

[1] Short NJ, Rytting ME, Cortes JE. Acute myeloid leukaemia. Lancet, 2018, 392(10147): 593-606

[2] Döhner H, Estey EH, Amadori S, et al.. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European Leukemia Net. Blood, 2010, 115: 453-474

[3] Stahl M, Lu BY, Kim TK, et al.. Novel Therapies for Acute Myeloid Leukemia: Are We Finally Breaking the Deadlock?. Target Oncol, 2017, 12(4): 413-447

[4] Li T, Le A. Glutamine Metabolism in Cancer. Adv Exp Med Biol, 2018, 1063: 13-32

[5] Choi YK, Park KG. Targeting Glutamine Metabolism for Cancer Treatment. Biomol Ther, 2018, 26(1): 19-28

[6] Matre P, Velez J, Jacamo R, et al.. Inhibiting glutaminase in acute myeloid leukemia: metabolic dependency of selected AML subtype. Oncotarget, 2016, 7(48): 79722-79735

[7] Willems L, Jacque N, Jacquel A, et al.. Inhibiting glutamine uptake represents an attractive new strategy for treating acute myeloid leukemia. Blood, 2013, 1229(20): 3521-3532

[8] Sahra IB, Laurent K, Loubat A, et al.. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene, 2008, 27(25): 3576-3586

[9] Sabnis HS, Bradley HL, Tripathi S, et al.. Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine. Leuk Res, 2016, 50: 132-140

[10] Yi Y, Gao LF, Wu M, et al.. Metformin Sensitizes Leukemia Cells to Vincristine via Activation of AMP-activated Protein Kinase. J Cancer, 2017, 8(13): 2636-2642

[11] Lin SC, Hardie DG. AMPK: Sensing Glucose as well as Cellular Energy Status. Cell Metab, 2018, 27(2): 299-313

[12] Tanaka K, Sasayama T, Irino Y, et al.. Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment. J Clin Invest, 2015, 125(4): 1591-1602

[13] Moarii M, Papaemmanuil E. Classification and risk assessment in AML: integrating cytogenetics and molecular profiling. Hematology Am Soc Hematol Educ Program, 2017, 2017(1): 37-44

[14] Döhner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med, 2015, 373: 1136-1152

[15] Stein EM, Tallman MS. Emerging therapeutic drugs for AML. Blood, 2016, 127(1): 71-78

[16] Yang X, Wang JX. Precision therapy for acute myeloid Leukemia. J Hematol Oncol, 2018, 11(1): 3

[17] Wise DR, Thompson CB. Glutamine Addiction: A New Therapeutic Target in Cancer. Trends Biochem Sci, 2010, 35(8): 427-433

[18] Katt WP, Cerione RA. Glutaminase regulation in cancer cells: a druggable chain of events. Drug Discov Today, 2014, 19(4): 450-457

[19] Freisinger E, Vašák M. Cadmium in metallothioneins. Met Ions Life Sci, 2013, 11: 339-371

[20] Higashimoto M, Isoyama N, Ishibashi S, et al.. Tissue-dependent preventive effect of metallothionein against DNA damage in dyslipidemic mice under repeated stresses of fasting or restraint. Life Sci, 2009, 84(17–18): 569-575

[21] Shimoda R, Achanzar WE, Qu W, et al.. Metallothionein is a potential negative regulator of apoptosis. Toxicol Sci, 2003, 73(2): 294-300

[22] Safe S, Nair V, Karki K. Metformin-induced anticancer activities: recent insights. Biol Chem, 2018, 399(4): 321-335

[23] Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types. Cancer Res, 2011, 71(9): 3196-3201

[24] Asik A, Kayabasi C, Yelken BO, et al.. Antileukemic effect of paclitaxel in combination with metformin in HL-60 cell line. Gene, 2018, 647: 213-220

[25] Wang FF, Liu ZF, Zeng JS, et al.. Metformin synergistically sensitizes FLT3-ITD-positive acute myeloid leukemia to sorafenib by promoting mTOR-mediated apoptosis and autophagy. Leuk Res, 2015, 39(12): 1421-1427

[26] Tamburini J, Chapuis N, Bardet V, et al.. Mammalian target of rapamycin (mTOR) inhibition activates phosphatidylinositol 3-kinase/Akt by up-regulating insulin-like growth factor-1 receptor signaling in acute myeloid leukemia: rationale for therapeutic inhibition of both pathways. Blood, 2008, 111(1): 379-382

[27] Santos CD, Demur C, Bardet V, et al.. A critical role for Lyn in acute myeloidleukemia. Blood, 2008, 111(4): 2269-2279

[28] Bhaskar PT, Hay N. The two TORCs and Akt. Dev Cell, 2007, 12(4): 487-502

[29] Geldermalsen MV, Wang Q, Nagarajah R, et al.. ASCT2/SLC1A5 controls glutamine uptake and tumor growth in triple-negative basal-like breast cancer. Oncogene, 2016, 35(24): 3201-3208

[30] Andrzejewski S, Gravel SP, Pollak M, et al.. Metformin directly acts on mitochondria to alter cellular bioenergetics. Cancer Metab, 2016, 2: 12

[31] Elgogary A, Xu QG, Poore B, et al.. Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer. Proc Natl Acad Sci USA, 2016, 113(36): E5328-E5336

[32] Quijano C, Trujillo M, Castro L, et al.. Interplay between oxidant species and energy metabolism. Redox Biol, 2016, 8: 28-42

[33] Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol, 2014, 24(10): R453-R462

[34] Dunn JD, Alvarez LA, Zhang XZ, et al.. Reactive oxygen species and mitochondria: A nexus of cellular homeostasis. Redox Biol, 2015, 6: 472-485

[35] Jiang L, Shestov AA, Swain P, et al.. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature, 2016, 532(7598): 255-258