Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression

Abstract

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

Fig. 1. Oncogenic function of TRAF6 in AML.

A TRAF6 mRNA expression in healthy BM CD34⁺ cells (n = 12) and AML (n = 451). The data for both healthy BM CD34+ cells and AML patients were retrieved from a published database (BeatAML) [24]. B Immunoblot analysis confirming knockdown of TRAF6 in leukemic cells upon addition of DOX(1 μg/mL). C Viable cell growth of HEL, TF-1, MV4;11, MOLM14 and THP-1 cells transduced with the inducible shTRAF6 was assayed by trypan blue exclusion. The relative cell number was evaluated on 7 days after equal number of the cells were seeded. Data are presented as the means ± SD from biological triplicates. Results are representative of three independent assays. D Representative flow cytometric analysis for the evaluation of cell cycle of HEL cells transduced with inducible shTRAF6. E Percentage of cells in each cell cycle phase, shown as means ± SD from biological replicates (n = 3). These results are representative of two independent assays. F Overview of experimental design to examine the requirement of TRAF6 for the MLL-AF9 leukemic function in vivo. Isolated Lin^- BM cells were transduced with retrovirus encoding MLL-AF9 and GFP. The transduced BM cells were serially replated in methylcellulose medium to select transformed cells. Lethally-irradiated recipient mice (CD45.2) were engrafted with the transformed BM cells along with wild-type BM cells (CD45.1) for radioprotection. From day 14, the recipient mice received intraperitoneal injection of polyinosinic-polycytidylic acid [poly(I:C)] to delete TRAF6, and then were monitored for engraftment and overall survival. G Kaplan-Meier analysis of overall survival of mice engrafted with MLL-AF9;Traf6^+/+ (n = 10) and MLL-AF9;Traf6^−/− (n = 10) AML cells. H Summary of the leukemic cell burden (GFP⁺) in the PB of the mice 10 weeks after transplant with MLL-AF9;Traf6^+/+ (n = 8) and MLL-AF9;Traf6^−/− (n = 10) AML cells. I Representative flowcytometric profiles from EdU assay using MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− AML cells. J Percentage of cells in each cell cycle phase, shown as means ± SD for biological replicates (n = 6). HC, healthy control. *P < 0.05; **P < 0.01, ***P < 0.001.

Fig. 2. TRAF6 expression inversely correlates with mitochondrial function-related gene signatures in AML.

A Normalized enrichment scores (NES) from Gene Set Enrichment Analysis (GSEA) for the top 20 upregulated (red, top) and downregulated (blue, bottom), significantly altered gene sets in TRAF6^low compared to TRAF6^hi AML patient samples from the TCGA AML dataset [3]. Low and high TRAF6 expressions defined by: TRAF6^low, Z score <1; TRAF6^hi, Z score >1. B, C RNA sequencing analysis of two groups: (1) human leukemic cell lines with inducible shTRAF6, treated with or without doxycycline (DOX, 1 μg/mL) for 7 days, and (2) MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells. The mitochondrial states were evaluated using GSEA profiles based on TRAF6^low leukemic cells, with a focus on mitochondria-associated gene signatures organized by their P values (log₁₀). Selected gene set enrichment plots of AML patients stratified based on low/high TRAF6 expression (D), HEL cells transduced with the inducible shTRAF6 (E), and MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells (F). NES normalized enrichment score, DOX doxycyclin.

Fig. 3. Loss of TRAF6 in leukemia cells induces the changes in the mitochondrial function parameters.

A Representative flow cytometry histograms of mitochondrial TMRE levels in HEL cells expressing shTRAF6 or shControl (shCtrl). B Median fluorescent intensity (MFI) of tetramethylrhodamine ethyl ester (TMRE) observed from HEL cells expressing shTRAF6 or shCtrl. Data are presented as the means ± SD from biological replicates (n = 3). Results are representative of two independent assays. C Oxygen consumption rate (OCR) in HEL cells transduced with the inducible shTRAF6. Cells were sequentially treated with oligomycin, fluoro-carbonyl cyanide phenylhydrazone (FCCP), and rotenone/antimycin A at the indicted time points. Data are presented as the means ± SD from technical replicates (n = 4). Results are representative of three independent assays. D Basal respiration, maximal respiration, ATP production and spare respiratory capacities of HEL cells transduced with the inducible shTRAF6 calculated from the data of (C). Data are shown as the means ± SD (n = 4). E Representative flow cytometry histograms of mitochondrial TMRE levels in MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells. F MFI of TMRE observed from MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells. Data are presented as the means ± SD from biological replicates (n = 6). Results are representative of two independent assays. G OCR in MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells. Cells were sequentially treated with oligomycin, FCCP, and rotenone/antimycin A at the indicted time points. Data are presented as the means ± SD from technical replicates (n = 6). Results are representative of two independent assays. H Basal respiration, maximal respiration, ATP production and spare respiratory capacities of MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells calculated from the data of (G). Data are shown as the means ± SD (n = 6). **, P < 0.01; ***, P < 0.001.

Fig. 4. TRAF6 loss in leukemia leads to dynamic alteration of metabolic profile.

A–F Metabolites in HEL cells transduced with the inducible shTRAF6 cultured for 2 days with or without DOX (1 µg/mL) were analyzed using capillary electrophoresis Fourier transform mass spectrometry (CE-FTMS) (n = 3). Hierarchical clustering heatmap analysis (A) and principal component analysis (B) of all metabolites that were detected as a peak by CE-FTMS (n = 483). The concentrations or relative area of selected metabolites in TCA cycle (C), glycolytic pathway (D), pentose phosphate pathway (E) and purine synthesis pathway (F). G6P glucose 6-phosphate, F6P fructose 6-phosphate, FBP fructose 1,6-diphosphate, 6-PG 6-phosphogluconic acid, Ru5P ribulose 5-phosphate, R5P ribose 5-phosphate, PRPP phosphoribosyl pyrophosphate, IMP inosine monophosphate, dATP deoxyadenosine triphosphate, dGTP deoxyguanosine triphosphate. *P < 0.05; **, <0.01; ***P < 0.001.

Fig. 5. Similarity of the leukemic cellular features between loss of OGT and TRAF6.

A Venn diagram of downregulated genes (1.5-fold, P < 0.05) in DOX-treated HEL cells transduced with the inducible shTRAF6 (relative to untreated HEL cells transduced with the inducible shTRAF6) and 130 AML essential genes identified by CRISPR-Cas9 screens [32]. B OGT mRNA expression in AML patients stratified on TRAF6 expression (TRAF6^hi, n = 18; TRAF6^low, n = 21) [3]. C Immunoblot analysis of TRAF6 and OGT in HEL, TF-1, MV4;11 and MOLM14 cells transduced with the inducible shTRAF6, treated with or without DOX (1 μg/mL) for 3 days. D Overall survival of AML patients stratified on OGT expression (OGT^hi, n = 85; OGT^low, n = 87) [3]. High and low OGT expressions defined by: OGT^hi, above median; OGT^low, below median. Survival curves were generated using the BloodSpot database (https://www.fobinf.com/). E Immunoblot analysis of OGT in HEL and TF-1 expressing shOGT or shSCR (Upper panel). Viable cell number of HEL and TF-1 expressing shOGT or shSCR was assayed by trypan blue exclusion. The relative cell number was evaluated 72 h after equal number of the cells were seeded. Data are presented as the means ± SD from technical triplicates. Results are representative of two independent assays. F Viable cell number of HEL and TF-1 exposed to 20 µM of OSMI-1 (OGT-inhibitor) was assayed by trypan blue exclusion. The relative cell number was evaluated 72 h after equal number of the cells were seeded. Data are presented as the means ± SD from technical triplicates. Results are representative of two independent assays. G OCR in HEL cells treated with OSMI-1 (20 μM) for 48 h. Cells were sequentially treated with oligomycin. FCCP, and rotenone/antimycin A at the indicted time points. Data are presented as the means ± SD from technical triplicates. Results are representative of two independent assays. H Basal respiration, maximal respiration, ATP production and spare respiratory capacities of HEL cells treated with OSMI-1 calculated from the data of (G). Data are presented as the means ± SD (n = 3). I Immunoblot analysis of OGT in MLL-AF9;Traf6^+/+ and MLL-AF9;Traf6^−/− leukemic cells. J Viable cell number of MLL-AF9 leukemic cells exposed to 2 µM of OSMI-1 was assayed by trypan blue exclusion. The relative cell number was evaluated 72 h after equal number of the cells were seeded. Data are presented as the means ± SD from technical triplicates. Results are representative of two independent assays. K MLL-AF9 leukemic cells (2μM) for 48h. Cells were sequentially treated with oligomycin. FCCP, and rotenone/antimycin A at the indicted time points. Data are presented as the means ± SD from technical replicates (n = 6). Results are representative of two independent assays. L Basal respiration, maximal respiration, ATP production and spare respiratory capacities of MLL-AF9 leukemic cells treated with OSMI-1 calculated from the data of (K). Data are presented as the means ± SD (n = 6).*P < 0.05; **<0.01; ***P < 0.001.

Fig. 6. O-GlcNAc modification is a potential contributor for TRAF6-mediated metabolic reprogramming of leukemia.

Immunoblot analysis of OGT in HEL (A) and TF-1 (B) transduced with inducible shTRAF6 expressing either control vector or cDNA of OGT, cultured with or without DOX (1 μg/mL) for 3 days (left panel). Viable cell growth of the cells was assayed by trypan blue exclusion (right panel). The normalized cell count, relative to untreated cells, was determined 72 h post-seeding of an equal number of cells. Data are presented as the means ± SD from technical triplicates. Results are representative of two independent assays. C OCR in HEL cells transduced with inducible shTRAF6, expressing control vector or cDNA of OGT, untreated or treated with DOX for 3 days. Cells were sequentially treated with oligomycin. FCCP, and rotenone/antimycin A at the indicted time points. Data are shown as the means ± SD for technical replicate analyses (n = 6). Results are representative of two independent assays. D Basal respiration, maximal respiration, ATP production and spare respiratory capacities of HEL cells calculated from the data of (C). The data are shown as the means ± SD (n = 6). E Schematic of O-GlcNacylation. F Immunoblotting of HEL cells transduced with the inducible shTRAF6, treated with or without DOX (1 μg/mL) for 3 days. G Immunoblotting of HEL cells transduced with the inducible shTRAF6, untreated with DOX, treated with DOX, and treated with DOX and 100 nM of MK8719 (OGA inhibitor). H One hundred thousand HEL cells transduced with inducible shTRAF6 were cultured with 1 μM of MK8719 for 7 days. Viable cell growth of the cells was assayed by trypan blue exclusion. Data are presented as the means ± SD for technical triplicates. Results are representative of two independent assays. I OCR in HEL cells transduced with the inducible shTRAF6 untreated with DOX, treated with DOX (1 μg/mL), and treated with DOX (1 μg/mL) and 200 nM of MK8719 (OGA inhibitor). Cells were sequentially treated with oligomycin. FCCP, and rotenone/antimycin A at the indicted time points. Data are presented as the means ± SD from technical replicate analyses (n = 3–4). Results are representative of two independent assays. J Basal respiration, maximal respiration, ATP production and spare respiratory capacities of HEL cells transduced with the inducible shTRAF6 calculated from the data of (I). Data are shown as the means ± SD (n = 3–4). *P < 0.05; **<0.01; ***P < 0.001.