Based on the combined transcriptomic and metabolomic analysis of the regulatory role of GLUL in drug resistance in chronic myeloid leukemia

doi:10.11958/20252219

Abstract

Abstract:

Objective To investigate the differences of gene expression and metabolomics between K562 cells and their resistant counterpart, K562-G01 cells, and analyze the potential role of glutamate-ammonia ligase (GLUL) in regulating the drug resistance of K562-G01 cells. Methods K562 and resistant K562-G01 cells were cultured in vitro. Differentially expressed genes, metabolites and related pathways in the resistant cells were identified through transcriptomics and metabolomics. The association between transcriptomic and metabolomic pathways was analyzed to identify common signaling pathways. Differential gene expression was detected by qPCR and Western blot assay. Flow cytometry was employed to evaluate the effect of GLUL gene interference on cell apoptosis. Results Transcriptomic analysis showed that 2 423 genes were upregulated, and 3 321 genes were downregulated in K562-G01 cells. Metabolomic analysis revealed that 570 metabolites were upregulated and 779 were downregulated in K562-G01 cells. Transcriptomic and metabolomic association analysis indicated that alanine, aspartate and glutamate metabolism pathway were involved in the development of drug resistance in K562 cells. qPCR and Western blot results demonstrated that GLUL expression was upregulated in K562-G01 cells (P<0.05). Further investigation showed that interfering with GLUL expression promoted apoptosis in both K562 and K562-G01 cells and enhanced the drug sensitivity to imatinib (P<0.05). Conclusion Activation of alanine, aspartate and glutamate metabolism pathways may contribute to the drug resistance of K562-G01 cells. Interfering with GLUL expression significantly promotes apoptosis in drug resistant cells and enhances the sensitivity to chemotherapy drugs.

Key words: leukemia, myelogenous, chronic, BCR-ABL positive, drug resistance, neoplasm, computational biology, glutamate-ammonia ligase

CLC Number:

R733.722

Figures/Tables 9

References 21

[1]	MOJIDRA R, HOLE A, IWASAKI K, et al. DNA fingerprint analysis of raman spectra captures global genomic alterations in imatinib-resistant chronic myeloid leukemia: a potential single assay for screening imatinib resistance[J]. Cells, 2021, 10(10):2506. doi:10.3390/cells10102506.
[2]	BADRALEXI I, BORDEI A, HALANAY A, et al. Dynamics of chronic myeloid leukemia under imatinib treatment:a study of resistance development[J]. Mathematics, 2025, 12(24):3937. doi:10.3390/math12243937.
[3]	HE Y, DING J, LIU L, et al. Investigation of TSRP reverses imatinib resistance through the PI3K / Akt pathway in chronic myeloid leukemia[J]. Ann Hematol, 2024, 103(12):5285-5296. doi:10.1007/s00277-024-06099-8.
[4]	MOJIDRA R, GARDI N, BAGAL B, et al. Genomic analysis identifies an incipient signature to forecast imatinib resistance before start of treatment in patients with chronic myeloid leukemia[J]. Adv Biomark Sci Technol, 2025, 7:59-64. doi:10.1016/j.abst.
[5]	ZHANG R, HAO J, YU H, et al. circ_SIRT1 upregulates ATG12 to facilitate Imatinib resistance in CML through interacting with EIF4A3[J]. Gene, 2024,893:147917. doi:10.1016/j.gene.2023.147917.
[6]	LI C, WEN L, DONG J, et al. Alterations in cellular metabolisms after TKI therapy for Philadelphia chromosome-positive leukemia in children: A review[J]. Front Oncol, 2022,12:1072806. doi:10.3389/fonc.2022.1072806.
[7]	KO B W, HAN J, HEO J Y, et al. Metabolic characterization of imatinib-resistant BCR-ABL T315I chronic myeloid leukemia cells indicates down-regulation of glycolytic pathway and low ROS production[J]. Leuk Lymphoma, 2016, 57(9):2180-2188. doi:10.3109/10428194.2016.1142086.
[8]	BHINGARKAR A, VANGAPANDU H V, RATHOD S, et al. Amino acid metabolic vulnerabilities in acute and chronic myeloid leukemias[J]. Front Oncol, 2021,11:694526. doi:10.3389/fonc.2021.694526.
[9]	吴珺, 陆爱东, 张乐萍, 等. 儿童核心结合因子相关性急性髓系白血病疗效及预后因素分析[J]. 中华血液学杂志, 2019, 40(1):52-57.
	WU J, LU A D, ZHANG L P, et al. Study of clinical outcome and prognosis in pediatric core binding factor-acute myeloid leukemia[J]. Chin J Hematol, 2019, 40(1):52-57. doi:10.3760/cma.j.issn.0253-2727.2019.01.010.
[10]	LIANG X, ZHOU J, LI C, et al. The roles and mechanisms of TGFB1 in acute myeloid leukemia chemoresistance[J]. Cell Signal, 2024,116:111027. doi:10.1016/j.cellsig.2023.111027.
[11]	ZHONG L, XIAO J L, LUO J, et al. Adsl,as a hub for amino acid metabolism and glucose metabolism,promotes the development of multiple myeloma[J]. Blood, 2024, 144(Supplement1):6824. doi:10.1182/blood-2024-204474.
[12]	POTETI M, MENEGAZZI G, PEPPICELLI S, et al. Glutamine availability controls bcr/abl protein expression and functional phenotype of chronic myeloid leukemia cells endowed with stem/progenitor cell potential[J]. Cancers (Basel), 2021, 13(17):4372. doi:10.3390/cancers13174372.
[13]	IJARE O, BASKIN D, PICHUMANI K. CBMT-01. alanine fuels energy metabolism of glioblastoma cells[J]. Neuro-Oncology, 2019, 21(Supplement6):32-33. doi:10.1093/neuonc/noz175.123.
[14]	HELENIUS I T, MADALA H R, YEH J J. An asp to strike out cancer? therapeutic possibilities arising from aspartate's emerging roles in cell proliferation and survival[J]. Biomolecules, 2021, 11(11):1666. doi:10.3390/biom11111666.
[15]	XUE W, WU K, GUO X, et al. The pan-cancer landscape of glutamate and glutamine metabolism: a comprehensive bioinformatic analysis across 32 solid cancer types[J]. Biochim Biophys Acta Mol Basis Dis, 2024, 1870(2): 166982. doi:10.1016/j.bbadis.2023.166982.
[16]	LAI Y C, LIN G, HO K C, et al. Aspartate and acetate fuel gastrointestinal stromal tumors beyond the warburg effect[J]. Ann Surg Open, 2022, 3(4):e224. doi:10.1097/AS9.0000000000000224.
[17]	MUTHU M, KUMAR R, SYED KHAJA A S, et al. GLUL ablation can confer drug resistance to cancer cells via a malate-aspartate shuttle-mediated mechanism[J]. Cancers (Basel), 2019, 11(12):1945. doi:10.3390/cancers11121945.
[18]	WU J, DENG P, ZOU L, et al. A phase Ⅱclinical study on apatinib plus vinorelbine in refractory her2-negative breast cancer and its metabolic implications of drug resistance[J]. Curr Cancer Drug Targets, 2025, 25(10):e15680096303785.doi:10.2174/0115680096303785240822155217.
[19]	PENG C J, FAN Z, LUO J S, et al. The potential transcriptomic and metabolomic mechanisms of ATO and ATRA in treatment of FLT3-ITD acute myeloid leukemia[J]. Technol Cancer Res Treat, 2024,23:15330338231223080. doi:10.1177/15330338231223080.
[20]	SINGH P, YADAV R, VERMA M, et al. Antileukemic activity of hsa-miR-203a-5p by limiting glutathione metabolism in imatinib-resistant K562 cells[J]. Curr Issues Mol Biol, 2022, 44(12):6428-6438. doi:10.3390/cimb44120438.
[21]	NAKA K. New routes to eradicating chronic myelogenous leukemia stem cells by targeting metabolism[J]. Int J Hematol, 2021, 113(5):648-655. doi:10.1007/s12185-021-03112-y.

基因名称	引物/干扰序列（5′→3′）	产物大小/bp
Homo β-actin	上游：CCCTGGAGAAGAGCTACGAG	180
Homo β-actin	下游：CGTACAGGTCTTTGCGGATG	180
Homo GLUL	上游：ATTACTGTGGTGTGGGAGCA	200
Homo GLUL	下游：ACGATGCAAGATGAAACGGG	200
NC	UUCUCCGAACGUGUCACGUTT-
NC	ACGUGACACGUUCGGAGAATT
si-GLUL-1	ATTCCATGAAACCTCCAACATT-
si-GLUL-1	TGTTGGAGGTTTCATGGAATTT
si-GLUL-2	GCTGCCATGTTTCGGGACCCCTTTT-
si-GLUL-2	TGTTGGAGGTTTCATGGAATTT
si-GLUL-3	CTGCGACCCCTTTTCGGTGACTT-
si-GLUL-3	GTCACCGAAAAGGGGTCGCAGTT

基因名称	引物/干扰序列（5′→3′）	产物大小/bp
Homo β-actin	上游：CCCTGGAGAAGAGCTACGAG	180
Homo β-actin	下游：CGTACAGGTCTTTGCGGATG	180
Homo GLUL	上游：ATTACTGTGGTGTGGGAGCA	200
Homo GLUL	下游：ACGATGCAAGATGAAACGGG	200
NC	UUCUCCGAACGUGUCACGUTT-
NC	ACGUGACACGUUCGGAGAATT
si-GLUL-1	ATTCCATGAAACCTCCAACATT-
si-GLUL-1	TGTTGGAGGTTTCATGGAATTT
si-GLUL-2	GCTGCCATGTTTCGGGACCCCTTTT-
si-GLUL-2	TGTTGGAGGTTTCATGGAATTT
si-GLUL-3	CTGCGACCCCTTTTCGGTGACTT-
si-GLUL-3	GTCACCGAAAAGGGGTCGCAGTT

细胞	n	基因FPKM
细胞	n	ALDH4A1	GLUL	ADSS2	GFPT2
K562	3	15.71±4.08	74.28±4.56	7.96±2.16	0.04±0.02
K562-G01	3	24.68±0.24	110.71±2.72	19.45±3.32	0.19±0.09
t		4.509^＊	3.699^＊	2.884^＊	2.823^＊
细胞	n	代谢物丰度
细胞	n	琥珀酸半醛	N-乙酰天冬氨酰谷氨酸	γ-氨基丁酸	5-磷酸核糖胺
K562	6	6 455.00±1 081.87	68 900.00±141.42	514.00±1.41	6 495.00±742.46
K562-G01	6	32 000.00±21 071.78	22 400.00±27 152.90	8 950.00±3 747.67	46 500.00±24 041.63
t		3.976^＊＊	3.987^＊＊	3.204^＊＊	4.188^＊＊

细胞	n	基因FPKM
细胞	n	ALDH4A1	GLUL	ADSS2	GFPT2
K562	3	15.71±4.08	74.28±4.56	7.96±2.16	0.04±0.02
K562-G01	3	24.68±0.24	110.71±2.72	19.45±3.32	0.19±0.09
t		4.509^＊	3.699^＊	2.884^＊	2.823^＊
细胞	n	代谢物丰度
细胞	n	琥珀酸半醛	N-乙酰天冬氨酰谷氨酸	γ-氨基丁酸	5-磷酸核糖胺
K562	6	6 455.00±1 081.87	68 900.00±141.42	514.00±1.41	6 495.00±742.46
K562-G01	6	32 000.00±21 071.78	22 400.00±27 152.90	8 950.00±3 747.67	46 500.00±24 041.63
t		3.976^＊＊	3.987^＊＊	3.204^＊＊	4.188^＊＊

组别	GLUL基因		GLUL蛋白
组别	K562	K562-G01	K562	K562-G01
对照组	1.00±0.00	1.00±0.00	0.66±0.07	0.88±0.24
si-GLUL-1组	0.81±0.19	0.83±0.21	0.71±0.03	0.86±0.01
si-GLUL-2组	0.44±0.12^ab	0.39±0.13^ab	0.34±0.07^ab	0.49±0.05^ab
si-GLUL-3组	0.64±0.03^ac	0.62±0.08^ac	0.52±0.20^ac	0.73±0.03^ac
F	24.112^＊＊	18.443^＊＊	19.695^＊＊	87.524^＊＊