Loss of TRAF6 impairs leukemic cell function in vitro and in vivo

To examine the role of TRAF6 in AML cells, we first compared the expression of TRAF6 in bone marrow (BM) cells from AML patient samples with that of healthy controls using a publicly available database [24]. RNA-seq analysis revealed that the mRNA levels of TRAF6 in AML were higher compared with that in healthy controls (Fig. 1A), suggesting the potential importance of TRAF6 in leukemogenesis. We previously demonstrated that the loss of TRAF6 in BM cells with a deficiency of Tet2, which is the most frequently observed CHIP-associated mutation, results in transformation to leukemia in mice [20]. However, the role of TRAF6 in leukemia cells, rather than transformation from CHIP-associated mutants, remains unclear. We addressed this by knocking down TRAF6 in various human AML cell lines, including HEL, TF-1, MV4;11, MOLM14, and THP-1, using a doxycycline (DOX)-inducible shRNA system. TRAF6 knockdown notably inhibited proliferation in all tested cell lines except THP-1 (Fig. 1B, C). To identify the basis for the reduced cell number in TRAF6-knockdown leukemia cells, we determined the effect of TRAF6 loss on apoptosis and cell cycle dynamics. Cell cycle analysis revealed that the loss of TRAF6 led to an increased proportion of cells in the G1 phase and a decreased proportion in the S phase among HEL, TF-1, MV4;11, and MOLM14 cells (Fig. 1D-E). Conversely, TRAF6 loss did not significantly impact apoptosis in these cells (data not shown). These observations suggest that the inhibitory effects of TRAF6 loss on leukemic cell proliferation stem from altered cell cycle progression rather than from increased apoptosis. To validate these findings in vivo, we established a myeloid leukemia model induced by the leukemic fusion gene MLL-AF9 [22]. Isolated lineage− (Lin−) BM cells, derived from TRAF6+/+;MxCre and TRAF6flox/flox;MxCre mice [25], were retrovirally transduced with vectors expressing MLL-AF9 (MSCV-IRES-GFP) and then serially replated in methylcellulose medium to select transformed cells. The transformed BM cells (CD45.2+) were transplanted into lethally-irradiated recipient mice along with wild-type BM cells (CD45.1+). At day 14 post-transplant, TRAF6 was deleted by intraperitoneally injecting polyinosinic-polycytidylic acid (pIpC) (Fig. 1F). As expected, the mice engrafted with MLL-AF9;Traf6+/+ cells developed a rapid and fully penetrant AML up to 100 days post-transplantation (Fig. 1G). In contrast, mice engrafted with MLL-AF9;Traf6−/− cells developed a significantly delayed leukemia (Fig. 1G). Consistent with these findings, an immunophenotypic analysis of peripheral blood revealed a reduced leukemic burden in the mice engrafted with MLL-AF9; Traf6−/− cells compared with those engrafted with MLL-AF9;Traf6+/+ cells at the time point (Fig. 1H). Cell cycle assessments in murine MLL-AF9 leukemic cells confirmed that TRAF6 loss impairs cell cycle progression as observed in human leukemic cells (Fig. 1I–J). Together, these findings affirm that TRAF6 loss suppresses leukemic cell function both in vitro and in vivo.

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.

TRAF6 expression in AML is inversely correlated with mitochondria-related gene signatures

To determine the molecular mechanism of the inhibitory effects of TRAF6 loss on leukemia function, we stratified AML patient samples based on TRAF6 expression and compared the gene expression profiles between AML with high (Z score >1.0, hereafter TRAF6hi AML) and low (Z score <1.0, hereafter TRAF6low AML) TRAF6 expression. TRAF6 is not only a central mediator of innate immune signaling, but is also involved in other immune functions, such as T cell receptor (TCR) signaling and immune control mediated by regulatory T cells [26, 27]. As expected, gene set enrichment analysis revealed that immune-related gene sets, such as those associated with FOXP3 targets, MAPK signaling, and TCR signaling, were significantly enriched in TRAF6hi AML cells (Fig. 2A). Interestingly, the most enriched gene sets in TRAF6low AML cells included mitochondrial function-associated processes, such as oxidative phosphorylation (OXPHOS), respiratory electron transport, and the TCA (tricarboxylic acid) cycle (Fig. 2A, B, D). Furthermore, gene sets related to neurodegenerative diseases, such as Huntington’s, Parkinson’s, and Alzheimer’s disease, of which mitochondrial dysfunction plays a central role in pathogenesis, were also overrepresented in TRAF6low AML cells (Fig. 2A, C) [28]. Similarly, knocking down TRAF6 in HEL, MV4;11, MOLM14, and TF-1 cells—but not in THP-1 cells—induced mitochondrial-related gene expression signatures (Fig. 2B, C, E). Notably, the absence of such enrichment in THP-1 cells aligns with our observations that TRAF6 loss did not impact their proliferation, suggesting a possible link between the mitochondrial gene expression profile and the proliferative capacity affected by TRAF6 status (Fig. 2B, C, E). This pattern was also present in murine MLL-AF9;Traf6−/− leukemic cells (Fig. 2B, C, F). These results suggest that TRAF6 loss in AML may be linked to alterations in the expression of genes involved in mitochondrial processes.

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 TRAF6low compared to TRAF6hi AML patient samples from the TCGA AML dataset [3]. Low and high TRAF6 expressions defined by: TRAF6low, Z score <1; TRAF6hi, 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 TRAF6low 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.

Loss of TRAF6 in AML results in the perturbation of mitochondrial function

Mitochondria are cellular organelles that generate energy and metabolites required for cell survival and growth. Energy in the form of adenosine triphosphate (ATP) is primarily generated in mitochondria by the OXPHOS process, in which byproducts of the TCA cycle feed the electron transport chain (ETC) complexes and their electrons pass through the ETC. As the electrons are funneled through the various complexes of the inner mitochondrial membrane, the ETC generates a mitochondrial membrane potential (MMP) that produces ATP [29]. To ascertain the impact of TRAF6 loss on mitochondrial function in AML, we assessed MMP using tetramethylrhodamine-ethyl ester dye in leukemia cells. Post TRAF6 knockdown, HEL and TF-1 cells exhibited a reduction in MMP compared to controls (Fig. 3A, B and Supplemental Fig. 1A). Further evaluation of mitochondrial function with an extracellular flux analyzer indicated a decrease in respiratory capacity in TRAF6-knockdown HEL and TF-1 cells (Fig. 3C, D and Supplemental Fig. 1B). Conversely, MV4;11 and MOLM14 cells did not exhibit notable changes in MMP or respiratory capacity (Supplemental Fig. 1C–F). This lack of change suggests that these cell lines might compensate for mitochondrial function disruption through the upregulation of mitochondrial genes, and that other molecular mechanisms may mitigate the effects of TRAF6 knockdown on their proliferation. Similarly, murine MLL-AF9;Traf6−/− leukemic cells displayed a decreased MMP and reduced mitochondrial respiratory capacity compared to their Traf6+/+ counterparts (Fig. 3E–H). Collectively, these findings indicate that TRAF6 loss can lead to mitochondrial dysfunction in leukemic cells, contributing to the observed phenotypic alterations in a subset of AML cases.

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.

TRAF6 loss in AML gives rise to metabolic alterations

Mitochondria are essential organelles that act as metabolic hubs and signaling platforms within the cell. Therefore, we evaluated the effect of TRAF6 loss on the metabolic profiles of leukemia cells by metabolome analysis. Heatmap and principal component analysis revealed that TRAF6 loss in HEL cells induced dynamic changes in several metabolites (Fig. 4A, B). Since we observed reduced MMP and impaired mitochondrial respiratory capacity in TRAF6 knockdown leukemic cells (Fig. 3A–E), we first noted alterations in intermediates of the TCA cycle (Supplemental Fig. 2A). Consistent with our findings, the level of multiple metabolites in the TCA cycle, including 2-oxoglutaric acid, succinic acid, fumaric acid, and malic acid were reduced in TRAF6 knockdown HEL cells (Fig. 4C). For energy generation through the TCA cycle, glucose in the cytoplasm ultimately breaks down into pyruvic acid, which is transported to the mitochondria and metabolized into acetyl-CoA under aerobic conditions (Supplemental Fig. 2A). The level of most intermediates in the glycolytic pathway, including glucose 6-phosphate (G6P), fructose 6-phosphate, fructose 1,6-diphosphate, and pyruvic acid were reduced in TRAF6 knockdown HEL cells, suggesting a possible cause for the reduction of intermediates in the TCA cycle (Fig. 4D). Furthermore, TRAF6 loss resulted in the reduction of most intermediates in the pentose phosphate pathway (PPP), such as phosphogluconic acid, ribulose 5-phosphate, ribose 5-phosphate, and phosphoribosyl pyrophosphate (Fig. 4E). Because PPP plays important role in purine synthesis (Supplemental Fig. 2A), we examined the metabolites in the purine synthetic pathway. Indeed, the level of inosine monophosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate, which acts as a precursor for nucleic acid synthesis during replication, was decreased upon TRAF6 loss (Fig. 4F). These reductions in a broad spectrum of metabolic intermediates led us to speculate that TRAF6 loss might suppress glucose uptake, thus affecting leukemic cell metabolism. However, assessments of glucose uptake and intracellular glucose levels in TRAF6-knockdown HEL and TF-1 cells revealed minimal changes in these parameters (Supplemental Fig. 3A, B). Furthermore, intracellular glucose level in murine MLL-AF9;Traf6−/− leukemic cells was higher than in control cells (Supplemental Fig. 3A), suggesting that accumulation of unused intracellular glucose due to the impairment of cascade reactions in metabolic pathways. Glucose uptake capacity in murine MLL-AF9;Traf6−/− leukemic cells was suppressed compared with controls (Supplemental Fig. 3B), implying a negative feedback effect for high intracellular glucose levels. These findings indicate that TRAF6 loss leads to a global reduction in metabolic intermediates through mechanisms independent of glucose uptake capacity. Typically, mitochondrial dysfunction prompts enhanced glycolysis to compensate for reduced ATP production by impaired OXPHOS [30]. However, the evaluation of extracellular acidification rate (ECAR) in TRAF6-knockdown HEL and TF-1 cells, as well as murine MLL-AF9;Traf6−/− leukemic cells, revealed the absence of such compensatory action (Supplemental Fig. 3C). This suggests that the metabolic changes induced by TRAF6 loss in leukemic cells are mediated through mechanisms affecting a broad range of cellular processes. Since metabolic reprogramming is a hallmark of malignancy and crucial for supporting the heightened proliferation of tumor cells [31], our results imply that TRAF6 loss in leukemia cells induces metabolic alterations, contributing to their inhibited growth capacity.

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.

OGT is a potential mediator of metabolic reprogramming regulated by TRAF6 in leukemia

To identify the mechanism of metabolic alterations driven by TRAF6 loss in leukemia, we compared the upregulated genes in TRAF6-knockdown HEL cells (1.5-fold, P < 0.05) (Supplemental Table 1) with the 130 previously identified essential genes for AML cell survival in vitro and in vivo [32]. We found that 8 genes overlapped, and O-GlcNAc transferase (OGT) and ETNK1 were associated with mitochondrial function and leukemia cell survival (Fig. 5A) [33, 34]. Stratification of AML patients revealed that TRAF6 gene expression in human AML patient samples was positively correlated with the expression of OGT, but not ETNK1 (Fig. 5B and data not shown). Correspondingly, TRAF6 knockdown in HEL and TF-1 cells also led to reduced OGT protein levels (Fig. 5C). Intriguingly, in MV4;11 and MOLM14 cells, where TRAF6 loss did not significantly affect mitochondrial function (Supplemental Fig. 1C–F), OGT expression was also suppressed (Fig. 5C), suggesting a broader positive correlation between TRAF6 and OGT expression in leukemic cells. Furthermore, stratification of AML patients revealed that OGT-low patients were associated with longer overall survival (P < 0.001) (Fig. 5D). This led us to investigate OGT as a mediator of TRAF6-regulated mitochondrial function in leukemia cells. To evaluate the role of OGT in leukemia progression, we examined the impact of OGT loss or inhibition on leukemic cell growth. Inducing OGT-targeted shRNAs in HEL and TF-1 cells significantly reduced cell numbers (Fig. 5E). Furthermore, treatment with the OGT inhibitor OSMI-1 suppressed cell growth in the leukemic cells, correlating with diminished mitochondrial respiratory function (Fig. 5F–H). Similar trends were observed in murine MLL-AF9 leukemic cells, showing a positive correlation between Traf6 and Ogt levels, and susceptibility to OSMI-1, along with changes in mitochondrial respiratory function (Fig. 5I–L). These findings suggest that OGT loss mimics TRAF6 loss effects on leukemia cell metabolism and growth capacity.

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 (TRAF6hi, n = 18; TRAF6low, 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 (OGThi, n = 85; OGTlow, n = 87) [3]. High and low OGT expressions defined by: OGThi, above median; OGTlow, 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.

O-GlcNAc modification is a potential contributor to TRAF6-mediated metabolic reprogramming and leukemia progression

To investigate OGT’s role in the regulation of mitochondrial function mediated by TRAF6 in leukemia, we utilized a lentiviral system to overexpress OGT in TRAF6 knockdown human leukemic cells, examining its impact on growth capacity. The forced overexpression of OGT successfully counteracted inhibitory effect of TRAF6 loss on the proliferation defect in HEL and TF-1, along with a corresponding recovery in mitochondrial respiratory capacity (Fig. 6A–D). This result suggests a link between OGT and the metabolic dysregulation due to TRAF6 loss, affecting leukemic cell proliferation. However, in murine MLL-AF9;Traf6−/− leukemic cells, OGT overexpression did not rectify the proliferation defect (Supplemental Fig. 4A). This observation aligns with previous research indicating that both excessive and insufficient levels of OGT can negatively impact cell metabolism and proliferation [35].

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.

OGT catalyzes the addition of an O-GlcNAc moiety to serine or threonine residues of protein substrates, while O-GlcNAcase (OGA) removes these modifications (Fig. 6E) [36]. Notably, the level of O-GlcNAc modification was reduced in TRAF6-knockdown HEL and TF-1 cells, as well as in murine MLL-AF9;Traf6−/− leukemic cells (Fig. 6F and Supplemental Fig. 4B). Given that numerous metabolic enzymes are O-GlcNAc targets [36, 37], we hypothesized that the reduction in this modification disrupts metabolic reprogramming caused by TRAF6 loss in leukemia. To test this hypothesis, we examined the effects of the OGA inhibitor MK8719 on the growth capacity of leukemia cells impacted by TRAF6 loss. MK8719 treatment in TRAF6-knockdown HEL cells and murine MLL-AF9;Traf6–/– leukemic cells restored O-GlcNAc levels (Fig. 6G and Supplemental Fig. 4C) and inhibited the effects of TRAF6 loss on growth capacity, correlating with a recovery in mitochondrial respiratory function (Fig. 6H–J and Supplemental Fig. 4D-F). However, MK8719 did not correct the proliferation defect in TRAF6-knockdown TF-1 cells (data not shown). Given that a broad range of metabolic enzymes are targets of O-GlcNAcylation, the metabolic status in leukemia cells is likely finely regulated by the balance between OGT and OGA expression and activity.

Taken together, these results suggest that the fine-tuned and complex regulation of O-GlcNAc modification plays an influential role in TRAF6-mediated metabolic reprogramming of leukemic cells.