Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis

Abstract

Temozolomide (TMZ) represents a standard-of-care chemotherapeutic agent in glioblastoma (GBM). However, the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma. Although specific innovative approaches, such as immunotherapy, have shown favorable clinical outcomes, the inherent invasiveness of most gliomas continues to make them challenging to treat. Consequently, there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development. This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors. A total of 648 proteins exhibiting significant differential expression were identified. Gene Set Enrichment Analysis (GSEA) unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups. Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate (IP3) kinase B (ITPKB) in the recurrence group, correlating with poor survival in glioma patients. In TMZ-resistant cells, the depletion of ITPKB led to an increase in reactive oxygen species (ROS) related to NADPH oxidase (NOX) activity and restored cell sensitivity to TMZ. Mechanistically, the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination. This, in turn, elevated ITPKB stability and impaired ROS production. Furthermore, ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model. These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target for TMZ-resistant GBM.

Fig. 1

Differential expression analysis of the protein ITPKB in primary and recurrent glioma patients. a Six pairs of GBM tissues from glioma patients who received standard TMZ treatment and underwent primary and recurrent surgical treatment, were analyzed by mass spectrometry. A volcano plot represented the differential expression of proteins with a condition of P < 0.05 and an absolute fold change > 1.2. b GSEA pathway enrichment analysis was performed based on the identified differentially expressed proteins between the primary and recurrent groups in the tumors. c Lipid kinases and differentially expressed proteins depicted in a Venn diagram. d–g Expression profiles of differentially expressed lipid kinases (PIP4K2A, ITPKB, ITPK1, and PRKDC) in the proteomic analysis of primary and recurrent GBM tissue from six patients. h The prognostic significance of ITPKB in recurrent glioma patients was analyzed using the CGGA database (http://www.cgga.org.cn/). i, j IHC staining of ITPKB was performed in 19 pairs of primary/recurrent GBM patient specimens who received TMZ chemotherapy at Xiangya Hospital. The represented images are shown and quantification was analyzed by t-test. k, l IHC staining of ITPKB was analyzed in human glioma tissue arrays to assess the ITKPB survival values. The high or low group was reduced by the median total score of 4. The association of ITPKB with progression-free survival and overall survival of glioma patients was assessed using the log-rank test. m, n Survival subgroup analysis was performed using the log-rank test in HGG and LGG patients. Statistical significance is shown as: *p < 0.01, **p < 0.001, ns nonsignificant

Fig. 2

ITPKB-mediates TMZ sensitivity in GBM cells dependent on its kinase activity. a The expression of ITPKB was assessed in TMZ-sensitive (T98G, U118) and resistant (T98G-R, U118-R) cells using Western Blot. b T98G and U118 cells were treated with TMZ for indicated durations, and the cell lysates were blotted with specified antibodies. c After 48 h of TMZ treatment, lysates were collected from both TMZ-sensitive (T98G, U118) and resistant (T98G-R, U118-R) cells. Subsequently, these cell lysates were blotted with the indicated antibodies. d Construction of ITPKB-depleted TMZ-resistant glioma cell lines involved the transduction of T98G-R and U118-R cells with lentivirus encoding control (Ctrl) or ITPKB shRNAs. Cell lysates were then blotted with the specified antibodies. e, f Glioma cells from d were subjected to treatment with indicated doses of TMZ (0, 25, 50, 75, 100 μM) for 10-15 days. Cell survival was determined by colony formation assay. Error bars represent ± SD from three independent experiments. g TMZ-resistant glioma cells stably expressing Ctrl or ITPKB shRNA-1 were transiently transfected with the wild-type (WT) and kinase-dead mutant D897N of ITPKB. Subsequently, the cells were lysed, and Western blot analysis was performed using the specified antibodies. h Glioma cells from g were treated with 100 μM TMZ for 10–15 days, and cell survival was determined by colony formation assay. Error bars represent ± SD from three independent experiments. i TMZ-resistant glioma cells were treated with 20 μM GNF362 and/or 25 μM TMZ, and cell survival was determined by colony formation assay. Error bars represent ± SD from three independent experiments. j The expression of ITPKB in glioma cell lines (U251, U87, U118, U373, U138, LN299, HS683, and T98G) was assessed by western blot, and IC₅₀ of each cell line was calculated after the addition of gradient concentration of TMZ for 96 h. The correlation between ITPKB expression and IC₅₀ was analyzed by Pearson’s test in a panel of glioma cell lines. Statistical significance is indicated as: *p < 0.01, **p < 0.001, ns nonsignificant

Fig. 3

ITPKB participates in TMZ sensitivity through ROS homeostasis. a, b TMZ-sensitive and resistant cells were exposed to 100 μM TMZ for 48 h, and ROS levels were assessed using the DCFDA assay. Data, derived from three independent samples, are presented as mean fold change to control ± SD. c–e T98G-R cells stably expressing Ctrl or ITPKB shRNAs were treated with or without 100 μM TMZ for 48 h. ROS levels were measured using the DCFDA assay, and cell apoptosis was assessed after 500 μM TMZ treatment using the Annexin V-FITC apoptosis kit. Data, from three independent samples, are displayed as mean fold change to control ± SD. f, g TMZ-resistant glioma cells stably expressing Ctrl or ITPKB shRNA-1 were transiently transfected with the wild-type (WT) and kinase-dead mutant D897N of ITPKB. Following 100 μM TMZ treatment, ROS levels were determined by DCFDA assay, and cell apoptosis was assessed after 500 μM TMZ treatment using the Annexin V-FITC apoptosis kit. h, i T98G-R cells stably expressing Ctrl or ITPKB shRNAs were treated with 100 μM TMZ and/or 1000 U/ml antioxidant enzyme Catalase. ROS levels were measured using the DCFDA assay, and relative cell survival was determined by CCK8 assay. Error bars represent ± SD from three independent experiments. j T98G-R cells stably expressing Ctrl or ITPKB shRNAs were treated with or without 100 μM TMZ for 48 h, and NADH Oxidase Activity Assay Kit was used to analyze NOX activity. k, l T98G-R cells stably expressing Ctrl or ITPKB shRNAs were treated with 100 μM TMZ and/or 10 μM NOX1/4 inhibitor GKT137831. ROS was measured using the DCFDA assay. For cell apoptosis, T98G-R cells were treated with 500 μM TMZ and/or 10 μM NOX1/4 inhibitor GKT137831, and assessed by the Annexin V-FITC apoptosis kit. m The level of IP4 was measured in T98G-R cells stably expressing control or ITPKB shRNAs using an ELISA assay. Statistical significance is shown as: *p < 0.01, **p < 0.001

Fig. 4

Trim25 is an ITPKB-binding protein. a Relative ITPKB mRNA levels in six pairs of GBM tissues from Fig. 1a were determined by RT-PCR. b Transcriptional mRNA levels of ITPKB were analyzed in TMZ-sensitive and resistant cells after TMZ treatment by RT-PCR. c A list of ITPKB-binding proteins was identified through mass spectrometric analysis. HEK293T and T98G-R cells expressing Flag-ITPKB were generated, and ITPKB complexes were subjected to mass spectrometric analysis. d Glioma cell lysates from TMZ-sensitive (T98G, U118) and resistant (T98G-R, U118-R) cells were immunoprecipitated with control IgG or anti-Trim25 antibody, followed by blotting with indicated antibodies. e T98G and U118 cells treated with 100 μM TMZ for 48 h had their cell lysates subjected to immunoprecipitation with control IgG or anti-Trim25 antibody. f HEK293T cells expressing wild-type Flag-ITPKB were transfected with V5-tagged Trim25. Cells were pretreated with 100 μM TMZ, then co-treated with 50 μM MG132 for an additional 3 h. Cell lysates were immunoprecipitated with Anti-FLAG® M2 Magnetic Beads, followed by blotting with the indicated antibodies. g, h Representative images of merged Proximity Ligation Assay (PLA) and nuclei (DAPI) channels from PLA experiments. In situ PLA was utilized to assess the interaction between Trim25 and endogenous ITPKB in TMZ-sensitive and resistant glioma cells. Each red dot represents the detection of the Trim25-ITPKB interaction complex, and the graphs representing mean ± SD are shown in (g). The scale bar in the bottom left is 20 μm. i, j Trim25 was found to bind to the kinase domain of ITPKB. HEK293T cells were transiently transfected with V5-Trim25 along with wildtype (WT) and truncated mutants (1–768, 1–800, 768–946, and 800–946 aa) of Flag-ITPKB. The protein interaction was assayed by immunoprecipitation with Anti-FLAG® M2 Magnetic Beads, followed by blotting with the indicated antibodies. Schematic representation of Flag-ITPKB and its deletion mutants is shown in (j)

Fig. 5

ITPKB interaction with Trim25 dependent on K48 ubiquitination at sites K793 and K818. a Depletion of Trim25 induces ITPKB protein levels. T98G-R and U118-R cells were transfected with control (Ctrl) or Trim25 siRNAs, and cell lysates were immunoblotted with the indicated antibodies. b T98G-R and U118-R cells were transiently transfected with Trim25 plasmid, and the cell lysates were immunoblotted with the indicated antibodies. c T98G-R cells transfected with Ctrl or Trim25 plasmid were treated with vehicle or MG132 (50 μM) for 3 h. Cell lysates were then immunoblotted with the indicated antibodies. d T98G-R cells transfected with Ctrl or Trim25 siRNAs were treated with CHX (0.1 mg/mL) and harvested at the indicated times. Cells were lysed, and cell lysates were then immunoblotted with the indicated antibodies. e Quantification of the ITPKB protein levels relative to Actin. f T98G-R cells transfected with Trim25 plasmid were treated with CHX (0.1 mg/mL) and harvested at the indicated times. g Quantification of the ITPKB protein levels relative to Actin. h Cells transfected with Ctrl or Trim25 siRNAs were treated with MG132 for 3 h before harvest. Immunoprecipitation with control IgG and ITPKB was performed, followed by immunoblotting with the indicated antibodies. i, j HEK293T cells expressing Flag-ITPKB were transiently transfected with V5-tagged Trim25 and HA-tagged ubiquitin/K48 ubiquitin. After 48 h, cells were treated with MG132 (50 μM) for 3 h. Cell lysates were immunoprecipitated with Anti-FLAG® M2 Magnetic Beads, and then immunoblotted with the indicated antibodies. k HEK293T cells expressing Flag-ITPKB were transiently transfected with indicated HA-K48 lysine-specific mutant constructs. After 48 h, cells were treated with MG132 for 3 h before collection. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibody. l, m Identification of the ubiquitination sites of ITPKB for its K48-specific polyubiquitination. ITPKB stably knockdown HEK293T cells were transiently transfected with indicated constructs. After 48 h, cells were treated with MG132 for 3 h before collection. Cell lysates were immunoprecipitated and then immunoblotted with the indicated antibodies. n–q Trim25 plasmids were co-transfected with a control vector, ITPKB wildtype, or 2KR mutant in T98G-R cells. ROS levels were measured using the DCFDA assay. Cell apoptosis was assessed by the Annexin V-FITC apoptosis kit. Relative cell survival was determined by CCK8 assay. Statistical significance is shown as: *p < 0.01, **p < 0.001

Fig. 6

Phosphorylation of Trim25 at S100 is required for ITPKB ubiquitination. a Phosphorylation proteomics analysis of Trim25 in primary and recurrence GBM. b The phosphorylation level of Trim25 S100 site in GBM tissue. c Dephosphorylation of the E3 ligase Trim25 resulted in a more pronounced alleviation of ITPKB ubiquitination in a cell-free system compared to mock-treated Trim25. Dephosphorylated or mock-treated Trim25 was incubated with immunopurified Flag-ITPKB, ubiquitin, recombinant E1 (Uba1), and E2 (UbcH5b). ITPKB ubiquitination was then determined using a ubiquitination assay. d The interaction between ITPKB and Trim25 was affected by the Trim25 S100D phosphomimetic mutant and S100A dephosphorylation mutant. e The ubiquitination of ITPKB was altered when the S100 site was mutated to Asp (S100D) or Ala (S100A). HEK293T cells were co-transfected with the respective plasmids and treated with MG132 for 3 h after 48 h of transfection. Cell lysates were immunoprecipitated and blotted with specific antibodies for analysis. Statistical significance is shown as: *p < 0.01

Fig. 7

In vivo regulation of glioma TMZ sensitivity by ITPKB through ROS homeostasis. a Schematic diagram of tumor xenograft experiments using ITPKB knockdown glioma cells. 1 × 10⁷ cells were subcutaneously injected into nude mice. Tumor volumes were measured at indicated days. Mice were sacrificed after 5 weeks. b Tumor images were acquired (n = 5). c Tumor weights were measured and represented as mean tumor weight ± SD. d, e Tumor volume and mice body weight measured on the indicated day, represented as mean ± SD. f Tissues from a were homogenized and centrifuged. The H₂O₂ concentration of each tissue was analyzed by the H₂O₂ assay kit at 560 nm absorbance. g Representative IHC staining image of tissues from a using ITPKB and Ki67 antibodies. h, i Quantifications of ITPKB and Ki67 IHC results. j Schematic diagram of tumor xenograft experiments using combined treatment with GNF362 and TMZ. Tumor images of each group were shown in k, and tumor weight represented as mean ± SD was shown in (l). m, n Tumor volume and mice body weight measured on the indicated day represented as mean ± SD. o Tissues from j were homogenized and centrifuged. The H₂O₂ concertation of each tissue was analyzed by the H₂O₂ assay kit at 560 nm absorbance. p A schematic representation of ITPKB degradation by E3 ligase Trim25, participating in TMZ resistance of glioma through ROS homeostasis. Statistical significance is indicated as: **p < 0.001