USP10 promotes glioma stem cell maintenance and glioblastoma growth by antagonizing DTX3L-mediated SATB2 ubiquitination

Domain: biology
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
Special AT-rich sequence-binding protein 2 (SATB2) is a nuclear matrix-associated protein with a pivotal role in glioblastoma (GBM) progression. However, the mechanisms underpinning aberrant SATB2 expression remain elusive. Here, we identify the ubiquitin specific peptidase 10 (USP10) as a deubiquitinase and the deltex E3 ubiquitin ligase 3L (DTX3L) as a ubiquitin ligase of SATB2 in glioma stem cells (GSCs). USP10 and DTX3L regulate SATB2 ubiquitination at the K266 residue through mutually exclusive interactions and opposing activities. USP10, enriched in GSCs, is induced by transcription factor YY2. Knockdown of USP10 or overexpression of DTX3L markedly downregulates SATB2, resulting in the inhibition of GSC self-renewal and GBM growth, which can be rescued by the overexpression of SATB2. Importantly, pharmacological inhibition of USP10 by Wu-5 effectively suppresses tumor growth. These findings highlight the antagonistic roles of USP10 and DTX3L in the regulation of GBM malignancy and propose USP10 as a potential therapeutic target.

Introduction
Glioblastoma (GBM) is the most prevalent and devastating brain tumor in adults. The standard therapy for GBM is surgery followed by radiotherapy and chemotherapy. Despite remarkable progress in the treatment of other solid tumors, the prognosis for GBM patients remains bleak, with the median survival of GBM patients remains less than 16 months1,2. The malignant features of GBM are thought to be attributable to glioma stem cells (GSCs), a fraction of stem cell-like cancer cells that possess extraordinary capabilities for rapid proliferation, self-renewal, and tumorigenesis3–5. These GSCs have been shown to play a significant role in the initiation, progression, recurrence and resistance to therapy of GBM6–9. Therefore, the development of novel therapeutic strategies targeting GSCs could provide significant insights that may enhance the efficacy of GBM treatments.

Special AT-rich sequence-binding protein 2 (SATB2) is a nuclear matrix-associated transcriptional factor that interacts with the nuclear matrix attachment regions of DNA, thereby regulating chromatin remodeling and gene transcription10–12. SATB2 recruits chromatin-remodeling proteins to the matrix attachment regions, either to activate or repress gene transcription11,13,14. This protein has been shown to play a crucial role in the progression of various cancer types, including hepatocellular carcinoma, renal cell carcinoma, prostate cancer, triple-negative breast cancer, and others15–18. In GBM, SATB2 is overexpressed in GBM tissues when compared to low-grade gliomas, and it is predominantly enriched in GSCs19. Disruption of SATB2 significantly suppresses GSC proliferation and self-renewal19. Moreover, SATB2 plays a key role in the progression of GBM and significantly contributes to the development of resistance to temozolomide and radiation therapy19,20. However, the precise mechanisms underlying SATB2 dysregulation in GSCs remain unclear.

The ubiquitin-proteasome system constitutes the primary pathway for non-lysosomal proteolysis of intracellular proteins and is accountable for the degradation of more than 80% of cellular proteins21,22. Ubiquitination, which is a process that marks proteins for degradation, plays a crucial supportive role in this regulatory mechanism23. This process is reversible and can be mediated by a diverse group of enzymes known as deubiquitylating enzymes, or termed deubiquitinases24,25. USP10, a member of ubiquitin-specific protease (USP) family, is one such deubiquitinase26. It has been implicated in the development of various cancer types, including triple-negative breast cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, and others27–29. Recent studies have indicated that disruption of USP10 can induce apoptosis in GBM cells and suppress proneural-to-mesenchymal transition, a critical process in GBM progression30,31. Deltex E3 ubiquitin ligase 3L (DTX3L) is an E3 ubiquitin ligase belonging to the deltex family32. This protein is associated with the occurrence and development of various cancer types, such as pancreatic cancer, cervical carcinoma, prostate cancer, and others33–35. However, the functional roles of USP10 and DTX3L in GSC biology remain largely enigmatic.

In this study, we identify that deubiquitinase USP10 and E3 ubiquitin ligase DTX3L function in concert to regulate the ubiquitination and stability of SATB2. Our data reveal that USP10 antagonizes DTX3L-mediated ubiquitination at the K266 residue, thereby stabilizing the SATB2 protein, which in turn contributes to the malignant phenotype of GSCs and the growth of GBM tumors. Notably, pharmacological inhibition of USP10 by a small molecule inhibitor, Wu-5, promotes SATB2 ubiquitination and thereby suppresses GSC proliferation, self-renewal, and tumor growth. These findings indicate that USP10 could be a promising target for patients with GBM.

Results
USP10 and DTX3L interact with SATB2 in GSCs
To determine whether the SATB2 protein is degraded through the ubiquitin-proteasome pathway or the lysosomal degradation pathway, we first checked the SATB2 protein levels in two patient-derived GSCs after blocked de novo protein synthesis using cycloheximide (CHX). GSCs were functionally validated as described in Methods. We found the SATB2 protein levels were decreased in a time-dependent manner (Supplementary Fig. 1a). On the contrary, the protein levels of SATB2 were gradually increased after treatment with the proteasome inhibitor MG-132 (Supplementary Fig. 1b). However, the protein levels of SATB2 remained unchanged after treatment with the lysosome inhibitor Bafilomycin A1 (Supplementary Fig. 1c). These results indicate that the degradation of SATB2 is mediated through the ubiquitin-proteasome system. To identify the potential deubiquitinases and ubiquitin ligases that regulate the ubiquitination and stability of SATB2, we performed mass spectrometry to analyze the SATB2-interacting proteins pulled down by the anti-Myc antibody from GSCs overexpressing Myc-tagged SATB2. Notably, among the top candidates of potential SATB2 binding partners, we identified the deubiquitinase USP10 and the E3 ubiquitin ligase DTX3L (Fig. 1a, b and Supplementary Table. 1).

To validate the interactions between SATB2 and either USP10 or DTX3L, we performed co-immunoprecipitation (CoIP) experiments in HEK293T cells that were co-expressing SATB2 with a Myc tag and either USP10 or DTX3L with a Flag tag. CoIP assay revealed that exogenous USP10 or DTX3L was readily detected in exogenous SATB2 pull-down complexes and vice versa (Fig. 1c–f). These interactions were confirmed by showing that endogenous USP10 or DTX3L could be detected in immunoprecipitates of endogenous SATB2 in GSCs (Fig. 1g). To determine the specific region required for SATB2-USP10 or SATB2-DTX3L interaction, we generated various truncated mutants of SATB2-Myc, USP10-Flag, and DTX3L-Flag (Fig. 1h–j). CoIP assays showed that the central domain of SATB2 (amino acids 232–437) and the C-terminal USP domain of USP10 (amino acids 401–798) are required for interaction with each other (Fig. 1k, l). In addition, our results also showed that the central domain of SATB2 (amino acids 232–437) and the central domain of DTX3L (amino acids 201–554) are necessary for their interaction (Fig. 1m, n). Taken together, these data suggest that deubiquitinase USP10 and E3 ubiquitin ligase DTX3L interact with SATB2 in GSCs, suggesting that USP10 and DTX3L may regulate SATB2 stability.

USP10 and DTX3L are preferentially expressed by GSCs
Considering that SATB2 is reported to be preferentially expressed by GSCs19,20, we subsequently aimed to verify if USP10 and DTX3L are also predominantly expressed by GSCs within GBM. We initially examined the protein expression of USP10 and DTX3L in five pairs of matched GSCs and non-stem tumor cells (NSTCs). Immunoblot (IB) analysis showed that USP10 and DTX3L were preferentially expressed in GSCs compared with matched NSTCs (Fig. 2a). In addition, the enrichment of SATB2 in GSCs, as shown in Fig. 2a, aligns with the results of our previous study19. Moreover, qPCR analysis also revealed that the mRNA levels of USP10 and DTX3L were expressed at much higher levels in GSCs than in NSTCs (Fig. 2b, c). We next compared the expression between USP10 and DTX3L and the results showed that the expression of USP10 was significantly higher than DTX3L in GSCs (Fig. 2d). As deubiquitinase and E3 ubiquitin ligase exert antagonistic effects on protein stability, the higher expression of USP10 than DTX3L may account for the abundant expression of SATB2 in GSCs. Immunofluorescent staining revealed that USP10 and DTX3L were predominantly located in the cytoplasm, while SATB2 was primarily found in the nuclei in GSCs (Fig. 2e, f). This observation suggests that the cytoplasmic SATB2 may be translocated to the nucleus after deubiquitination. To verify this hypothesis, we first performed immunofluorescent staining of SATB2 in GSCs after treatment with MG-132 or DMSO. The results showed that MG-132 treatment induced nuclear export of SATB2 in GSCs (Supplementary Fig. 1d). Furthermore, the ubiquitination assay detected ubiquitination of SATB2 in the cytoplasm but not in the nucleus (Supplementary Fig. 1e). These results confirm that the cytoplasmic SATB2 is translocated to the nucleus following deubiquitination. Subsequently, to confirm the preferentially expression of USP10 and DTX3L by GSCs in GBM, we examined the expression pattern of USP10 and DTX3L in several human GBM tumors. Our data confirmed that USP10 and DTX3L were preferentially expressed in glioma cells expressing the GSC marker SOX2 (Fig. 2g, h and Supplementary Fig. 1f, g). Furthermore, USP10 and DTX3L were both co-localized with SATB2 in human primary GBMs (Fig. 2i, j and Supplementary Fig. 1h, i). Collectively, these data demonstrate that both USP10 and DTX3L are preferentially expressed by GSCs in human GBM tumors.

USP10 is required for GSC self-renewal and GSC-driven tumor growth
As USP10 is enriched in GSCs, we therefore explored the role of USP10 in GSC maintenance and GSC-driven tumor growth by using two independent shRNAs targeting USP10. As expected, lentiviral-mediated expression of two independent USP10 shRNAs significantly reduced USP10 protein levels in GSCs, which also induced a notable decrease of SATB2 protein levels (Fig. 3a). However, the mRNA levels of SATB2 remained unchanged after USP10 disruption (Fig. 3b). In contrast, SATB2 disruption did not alter USP10 mRNA or protein levels (Supplementary Fig. 2a, b). Immunofluorescent staining also revealed that disruption of USP10 induced nuclear export of endogenous SATB2, which is consistent with our prediction (Supplementary Fig. 2c). FOXM1 is an important downstream target that mediates SATB2-promoted GSC proliferation and self-renewal19. We therefore investigated the impact of USP10 disruption on FOXM1 expression in GSCs, our results demonstrated that disrupting USP10 also reduced the mRNA and protein expression of FOXM1 (Supplementary Fig. 2d, e). However, this intervention had no effect on the expression of DTX3L (Supplementary Fig. 2d, e). As SATB2 is crucial for GSC maintenance, reduced expression of SATB2 protein by USP10 disruption may impact GSC functions. Our data demonstrated that silencing USP10 markedly inhibited GSC growth as detected by cell titer assay and reduced DNA replication as assayed by EdU incorporation assay (Fig. 3c and Supplementary Fig. 2f, g). In contrast, silencing USP10 had little effect on the growth of NSTCs (Supplementary Fig. 2h, i). Silencing USP10 also impaired GSC self-renewal as assessed by tumorsphere formation and in vitro limiting dilution assays (Fig. 3d–g). Consistently, silencing USP10 decreased the expression of the GSC marker SOX2 (Supplementary Fig. 2j). To verify whether the deubiquitinase activity of USP10 is required for GSC functions, we examined whether ectopic expression of wild-type (WT) or catalytically inactive mutant (C424A, CA) of USP10 could rescue the effects caused by USP10 disruption. We employed an additional USP10 shRNA that specifically targets the 3’ UTR of the endogenous USP10 mRNA (designated as shUSP10-3’) to achieve the knockdown of USP10 expression; thus, we were able to simultaneously silence endogenous USP10 and overexpress exogenous USP10 in GSCs. IB analysis showed that WT USP10, but not CA mutant, could rescue the decreased SATB2 protein levels caused by knockdown of endogenous USP10 (Supplementary Fig. 3a). In addition, ectopic expression of WT USP10, but not CA mutant, rescued the impaired GSC proliferation and self-renewal caused by endogenous USP10 knockdown (Supplementary Fig. 3b-f). These results demonstrate that USP10 regulates SATB2 protein levels and GSC functions through its deubiquitinase activity.

To investigate the influence of USP10 on GSC-driven tumor growth in vivo, GSCs expressing shNT or shUSP10 were intracranially injected into the brains of immunodeficient mice. Animals bearing xenografts derived from GSCs expressing shUSP10 displayed extended survival and reduced tumor volumes than the control group (Fig. 3h, i). Immunofluorescent staining of Ki67 and SOX2 also demonstrated that disruption of USP10 markedly inhibited cell proliferation and reduced GSC population in GBM xenografts (Fig. 3j–m). Additionally, immunofluorescent staining of neuronal markers (MAP2 and TUBB3) and the astrocyte marker (GFAP) revealed that disruption of USP10 significantly increased the number of MAP2-, TUBB3-, and GFAP-positive cells (Supplementary Fig. 4a, b). This finding demonstrates that USP10 disruption promotes the differentiation of GSCs in GBM xenografts. Furthermore, immunofluorescent staining confirmed a marked reduction in USP10 and SATB2 expression in xenografts expressing shUSP10 (Supplementary Fig. 4a, b). Collectively, our data demonstrate that USP10 plays a critical role in promoting GSC self-renewal and GSC-driven tumor growth.

DTX3L impairs GSC self-renewal and GBM tumor growth
As the expression of DTX3L is much lower than USP10 in GSCs, we examined the role of DTX3L in GSCs by overexpressing of DTX3L. We found that overexpression of DTX3L reduced SATB2 protein levels, but this overexpression had no effect on SATB2 mRNA levels (Fig. 4a, b). In addition, the overexpression of DTX3L led to a reduction in FOXM1 expression, whereas it had no significant impact on USP10 expression and ubiquitination (Supplementary Fig. 5a–c). Based on these findings, we hypothesized that DTX3L may also exert an influence on GSC functions. Our results showed that overexpression of DTX3L significantly suppressed GSC growth and reduced DNA replication (Fig. 4c–e). Tumorsphere formation and in vitro limiting dilution assays demonstrated that overexpression of DTX3L impaired GSC self-renewal (Fig. 4f–i). Consistently, DTX3L overexpression also decreased the expression of SOX2 (Supplementary Fig. 5d). We also examined whether the DTX3L mutant (C561S and C564S, 2CS), which lacks E3 ubiquitin ligase activity, could regulate SATB2 protein levels and GSC functions. IB analysis indicated that overexpression of DTX3L 2CS did not result in the inhibition of SATB2 protein levels (Supplementary Fig. 5e). Moreover, DTX3L 2CS overexpression had only a weak promoting effect on ubiquitination of SATB2 (Supplementary Fig. 5f, g). Consistently, it showed no significant impact on GSC proliferation and self-renewal (Supplementary Fig. 5h–l). We subsequently investigated whether the overexpression of DTX3L influenced GSC-driven tumor growth in mouse brains. The results showed that mice bearing tumors derived from DTX3L overexpressing GSCs showed extended survival and reduced tumor masses (Fig. 4j, k). Additionally, tumors derived from DTX3L-overexpressing GSCs contained much fewer proliferative cells and GSC populations than those of the control tumors (Fig. 4l–o). Furthermore, DTX3L overexpression markedly promoted the differentiation of GSCs in GBM xenografts, as indicated by immunofluorescence of MAP2, TUBB3, and GFAP (Supplementary Fig. 6a, b). Immunofluorescent analysis further confirmed that DTX3L expression was elevated and SATB2 levels were reduced in xenografts derived from DTX3L-overexpressing GSCs (Supplementary Fig. 6a, b). Taken together, we conclude that DTX3L exerts inhibitory effects on GSC self-renewal and GBM growth.

DTX3L ubiquitinates SATB2, whereas USP10 mediates SATB2 deubiquitination
Our results have demonstrated that the E3 ubiquitin ligase DTX3L reduced the protein levels of SATB2, while the deubiquitinase USP10 was responsible for preserving SATB2 protein levels. To investigate the impact of DTX3L or USP10 on SATB2 protein stability, DTX3L-overexpressing or USP10 silencing GSCs were subjected to CHX treatment. IB analysis demonstrated that overexpression of DTX3L or disruption of USP10 led to destabilization of the SATB2 protein (Fig. 5a-d and Supplementary Fig. 7a–d). Given that SATB2 is degraded through the ubiquitin-proteasome pathway, we sought to verify that DTX3L and USP10 regulate the turnover of SATB2 via the ubiquitin-proteasome pathway. The results showed that MG-132 treatments inhibited the decreased protein levels of SATB2 induced by DTX3L overexpression or USP10 disruption (Fig. 5e, f). These data indicate that DTX3L and USP10 regulate SATB2 stability in a proteasome-dependent manner. To determine if DTX3L indeed catalyzes the polyubiquitination of SATB2, we co-expressed SATB2-Myc and DTX3L-Flag along with His-ubiquitin in HEK293T cells. As shown in Fig. 5g, overexpression of DTX3L promoted the ubiquitination of SATB2 in HEK293T cells. Consistently, overexpression of DTX3L promoted the ubiquitination of SATB2 in GSCs (Fig. 5h). Subsequently, we co-expressed SATB2-Myc and USP10-Flag along with His-ubiquitin in HEK293T cells. The results demonstrated that overexpression of USP10 reduced the ubiquitination of SATB2 in HEK293T cells (Fig. 5i). In addition, disruption of USP10 enhanced the ubiquitination of SATB2 in GSCs (Fig. 5j). Polyubiquitination can occur through seven different Lys residues on ubiquitin36. Accordingly, we generated ubiquitin vectors with intact K6, K11, K27, K29, K33, K48, or K63 linkages to examine which type of linkage on the SATB2 protein is modulated by DTX3L or USP10. As depicted in Fig. 5k, l, DTX3L robustly stimulated the assembly of K48-linked polyubiquitination, while showing no effect on other ubiquitin linkage types. Further analysis revealed that USP10 specifically disassembled K48-linked polyubiquitination without affecting other polyubiquitin chain types (Fig. 5m, n). These results indicate that DTX3L promotes K48-linked polyubiquitination of SATB2, whereas USP10 prevents this polyubiquitination type of SATB2.

As SATB2 interacts with both DTX3L and USP10 through its central domain, we speculated that DTX3L and USP10 may act as competitors for binding to SATB2. We therefore co-expressed DTX3L-HA and USP10-Flag with SATB2-Myc in HEK293T cells and the result showed that overexpression of USP10 suppressed the interaction between SATB2 and DTX3L (Supplementary Fig. 7e). Consistently, disruption of USP10 increased the endogenous interaction between SATB2 and DTX3L in GSCs (Supplementary Fig. 7f). In addition, overexpression of USP10 prevented the reduced protein levels of SATB2 induced by DTX3L overexpression in GSCs (Supplementary Fig. 7g). To confirm whether DTX3L and USP10 indeed function as an opposing pair of E3 ubiquitin ligase and deubiquitinase to regulate SATB2 ubiquitination and stability, we first co-expressed SATB2-Myc, DTX3L-HA, USP10-Flag with His-ubiquitin in HEK293T cells. CoIP assay revealed that overexpression of USP10 significantly reduced the ubiquitination of SATB2 induced by DTX3L overexpression (Supplementary Fig. 7h). Overexpression of USP10 also reduced the ubiquitination of endogenous SATB2 induced by DTX3L overexpression in GSCs (Supplementary Fig. 7i, j). Furthermore, silencing DTX3L decreased the ubiquitination of endogenous SATB2 induced by USP10 disruption in GSCs (Supplementary Fig. 7k, l). In vitro ubiquitination assays demonstrated that DTX3L directly promoted the ubiquitination of SATB2 (Supplementary Fig. 7m), whereas USP10 antagonized DTX3L-mediated SATB2 ubiquitination (Supplementary Fig. 7n). Functionally, overexpression of both DTX3L and USP10 prevented the inhibitory effect on GSC growth that was induced by DTX3L overexpression alone (Supplementary Fig. 7o). Conversely, simultaneous disruption of both DTX3L and USP10 attenuated the inhibitory effect on GSC growth that was triggered by USP10 disruption alone (Supplementary Fig. 7p). Collectively, we conclude that deubiquitinase USP10 maintains the protein levels of SATB2 by antagonizing DTX3L-mediated ubiquitination.

Overexpression of SATB2 rescues the effects caused by USP10 disruption or DTX3L overexpression
Given that disruption of USP10 or overexpression of DTX3L reduced SATB2 protein levels, we next explored whether exogenous overexpression of SATB2 could rescue the effects induced by USP10 disruption or DTX3L overexpression. The reduction in SATB2 levels caused by USP10 disruption or DTX3L overexpression was corrected to match the levels of endogenous SATB2 in the control group by introducing additional SATB2 through exogenous overexpression (Fig. 6a, b and Supplementary Fig. 8a, b). Exogenous overexpression of SATB2 rescued the impaired GSC proliferation and self-renewal caused by USP10 disruption or DTX3L overexpression (Fig. 6c-h and Supplementary Fig. 8c–j). Importantly, overexpression of SATB2 largely attenuated the increased survival of mice bearing xenografts derived from USP10 silencing or DTX3L overexpressing GSCs (Fig. 6i, j and Supplementary Fig. 9a, b). Consistently, overexpression of SATB2 restored the GSC-driven tumor growth impaired by USP10 disruption or DTX3L overexpression (Fig. 6k, l and Supplementary Fig. 9c, d). Furthermore, immunofluorescent staining confirmed that overexpression of SATB2 restored in vivo cell proliferation impaired by USP10 disruption or DTX3L overexpression, and rescued the reduced GSC population caused by USP10 disruption or DTX3L overexpression (Fig. 6m–p and Supplementary Fig. 9e–h). Taken together, these data demonstrate that overexpression of SATB2 is able to rescue the effects caused by USP10 disruption or DTX3L overexpression, indicating that USP10 and DTX3L regulate GSC self-renewal and GSC-driven tumor growth by controlling the protein levels of SATB2.

USP10 and DTX3L co-regulate the ubiquitination of SATB2 at K266
To pinpoint the lysine residues within human SATB2 protein that are regulated by USP10 or DTX3L, we generated a series of truncated SATB2 mutants and introduced them into HEK293T cells. Notably, among the various mutants, only the expression of the SATB2 mutant encompassing amino acids 232 to 437 exhibited a significant increase in response to USP10 overexpression (Fig. 7a). In addition, overexpression of DTX3L also inhibited the expression of this SATB2 mutant (amino acids 232–437), without affecting the expression of other mutants (Fig. 7b). Subsequently, we individually mutated all lysine residues within the SATB2 mutant (amino acids 232–437) to arginine. The results revealed that the SATB2-K266R mutant was uniquely unaffected by the overexpression of USP10 or DTX3L (Fig. 7c, d). Protein sequence alignment analysis revealed that the domain containing K266 within SATB2 protein is highly conserved across seven distinct species (Fig. 7e). To confirm the ubiquitination site of SATB2, we performed mass spectrometry to analyze the immunoprecipitated SATB2 proteins from HEK293T cells co-expressing SATB2-Myc and His-ubiquitin. The results showed that ubiquitin moieties were exclusively detected on residue K266 (Fig. 7f), confirming that the K266 site is the ubiquitination site of SATB2. To ascertain whether K266 of SATB2 is regulated by USP10 or DTX3L in GSCs, we mutated K266 within the full-length SATB2 and introduced this mutant into GSCs. The results confirmed that the expression of the SATB2-K266R mutant was unaffected by USP10 disruption or DTX3L overexpression in GSCs (Fig. 7g–j). Additionally, to verify whether DTX3L directly catalyzes the ubiquitination of SATB2 at the K266 residue, we co-expressed Myc-tagged WT SATB2 or K266R (KR) mutant SATB2 with DTX3L-Flag and His-ubiquitin in HEK293T cells. CoIP experiment demonstrated that overexpression of DTX3L promoted SATB2 ubiquitination, while it had no obvious effect on the ubiquitination of SATB2-K266R mutant (Fig. 7k). Furthermore, overexpression of USP10 reduced SATB2 ubiquitination and showed no effect on the ubiquitination of the SATB2-K266R mutant (Fig. 7l). Collectively, these data reveal that the K266 within SATB2 is crucial for the deubiquitination and ubiquitination by USP10 and DTX3L.

YY2 activates USP10 transcription in GSCs
Given the abundant expression of USP10 mRNA by GSCs, we explored the mechanism of increased USP10 transcription in GSCs. We analyzed the human USP10 promoter region (−2,000 to +100) using JASPAR database to predict transcription factors that regulate USP10 transcription. PBX homeobox 2 (PBX2), YY2 transcription factor (YY2), and nuclear factor, interleukin 3 regulated (NFIL3) were the top three transcription factors with the highest scores (Supplementary Table 2). Additionally, we have enumerated the potential binding motifs for each of these transcription factors (Supplementary Table 2). We next silenced each of these transcription factors and found that only the disruption of YY2 inhibited the mRNA expression of USP10 (Supplementary Fig. 10a–c). IB analysis revealed that disruption of YY2 reduced USP10 and SATB2 protein levels (Supplementary Fig. 10d). Moreover, IB analysis demonstrated that YY2 and USP10 were preferentially expressed in GSCs compared with matched NSTCs (Supplementary Fig. 10e). To examine whether YY2 and USP10 are colocalized in GBM, we examined the expression pattern of YY2 and USP10 in human GBM tissues. Immunofluorescent staining demonstrated that YY2 was co-localized with USP10 in human GBM specimens (Supplementary Fig. 10f, g). Further analysis confirmed that YY2 was indeed enriched in nuclei of glioma cells expressing the GSC marker SOX2 (Supplementary Fig. 10h, i). To test whether YY2 directly stimulates the transcription of USP10, we performed a luciferase reporter assay. The results revealed that overexpression of YY2 increased USP10 promoter activity (Supplementary Fig. 10j). However, the activating effect by YY2 was completely eliminated upon mutation of the YY2-binding site within the USP10 promoter (Supplementary Fig. 10j). Chromatin immunoprecipitation (ChIP) assay confirmed that YY2 was present on its own binding site on the proximal USP10 promoter in GSCs (Supplementary Fig. 10k). Taken together, these results suggest that YY2 activates USP10 transcription by binding to USP10 promoter in GSCs.

USP10 inhibitor Wu-5 suppresses GSC self-renewal and GSC-driven tumor growth
To evaluate the therapeutic potential of USP10 in GBM, we investigated whether pharmacologic inhibition of USP10 by a small molecule inhibitor named Wu-5 could impact GSCs and GSC-driven tumor growth. Cell viability assay revealed that 8 and 16μM Wu-5 dose-dependently inhibited GSC growth, whereas lower concentrations (2 and 4μM) showed no significant effect (Fig. 8a). Wu-5 treatment also impaired GSC self-renewal and downregulated SOX2 expression in a dose-dependent manner (8 and 16μM) (Fig. 8b–e and Supplementary Fig. 11a). Furthermore, Wu-5 (8 and 16μM) dose-dependently induced GSC apoptosis, as demonstrated by the elevated levels of apoptotic markers Cleaved Caspase-7 and Cleaved PARP (Supplementary Fig. 11b). As expected, Wu-5 treatment reduced SATB2 protein levels in a dose- and time-dependent manner (Fig. 8f and Supplementary Fig. 11c). We also observed that Wu-5 treatment reduced USP10 protein levels in a dose- and time-dependent manner (Fig. 8f and Supplementary Fig. 11c), which is in line with the findings from previous research37,38. Nonetheless, treatment with 8 and 16μM Wu-5 had no impact on SATB2 and USP10 mRNA levels (Fig. 8g and Supplementary Fig. 11d). Consistently, Wu-5 treatment at 8 and 16μM dose-dependently increased the ubiquitination of SATB2 in GSCs (Fig. 8h). To determine whether SATB2 overexpression can rescue the effects of Wu-5 treatment on GSC maintenance, we transduced GSCs with SATB2-Myc and treated them with Wu-5 (16μM). IB analysis showed that ectopic SATB2 expression restored SATB2 to endogenous levels, which had been reduced by Wu-5 treatment (Supplementary Fig. 11e). Importantly, restoring SATB2 to endogenous levels significantly rescued the Wu-5-induced impairment of GSC proliferation and self-renewal (Supplementary Fig. 11f–j). These data indicate that Wu-5 treatment inhibits GSC maintenance via the USP10-SATB2 axis. We then assessed the therapeutic effects of Wu-5 on mice bearing GSC-derived xenografts. Consistent with our in vitro findings, Wu-5 treatment effectively prolonged the survival of mice bearing GSC-derived tumors and reduced tumor volumes in mouse brains (Fig. 8i–k). Notably, no significant alterations in body weight were observed throughout the treatment period (Supplementary Fig. 12a, b). Wu-5 administration also significantly reduced Ki67-positive proliferative cells and SOX2-positive GSC populations in tumors (Fig. 8l–o). Moreover, immunofluorescent staining for MAP2, TUBB3, and GFAP revealed that Wu-5 robustly promoted GSC differentiation in GBM xenografts (Supplementary Fig. 12c–f). Additionally, Wu-5 treatment reduced SATB2 and USP10 in xenografts (Supplementary Fig. 12g–j). To confirm whether Wu-5 can penetrate the blood-brain barrier, we intraperitoneally administered Wu-5 or DMSO to BALB/c nude mice and subsequently detected Wu-5 in mouse brain tissues by LC-MS/MS. The results demonstrated that Wu-5 concentrations reached an average of 673 ng/g tissue in the brains of mice treated with Wu-5 for 2 hours, whereas Wu-5 levels were very low and nearly undetectable in the brains of DMSO-treated controls (Supplementary Fig. 12k). Together, these data suggest that pharmacological inhibition of USP10 significantly suppresses GSC self-renewal and tumor growth via promoting SATB2 destabilization, suggesting that that targeting USP10 may effectively improve GBM treatment.

Methods
Tissues and cells
Human GBM specimens in this study were collected from the Center for Neurological Disease Research at Taihe Hospital in accordance with an Institutional Research Ethics Committee-approved protocol. Informed consent was obtained from all subjects. GSCs and NSTCs were kindly provided by Dr. Jeremy N. Rich (University of Pittsburgh) and Dr. Shideng Bao (Cleveland Clinic). GSCs and NSTCs were isolated from primary GBM tumors or patient-derived xenografts and functionally characterized19,50,51. In brief, cells were dissociated from tumors using the Papain Dissociation Kit (Worthington Biochemical, Cat# LK003150) following the manufacturer’s instructions. The isolated cells were recovered overnight in the Neurobasal-A medium (Thermo Fisher, Cat# A2477501), supplemented with B27 (Thermo Fisher, Cat# 12587010), 20 ng/ml EGF (Gold biotechnology, Cat# 1150-04-1000), 20 ng/ml bFGF (PrimeGene, Cat# 104-02), 2 mM L-glutamine (Thermo Fisher, Cat# 35050061) and 1 mM sodium pyruvate (Thermo Fisher, Cat# 11360070). Cells were then sorted by FACs using a PE-conjugated anti-CD133 antibody (Miltenyi Biotec, Cat# 130-113-108) and a FITC-conjugated anti-CD15 antibody (BD Biosciences, Cat# 347423), to obtain the GSC population (CD15+/CD133+) and the NSTC population (CD15-/CD133-). The cancer stem cell characteristics of isolated GSCs were validated by expression of GSC markers, including SOX2, OLIG2, L1CAM, and CD133, and functional assays, including serial tumorsphere formation, induced differentiation and tumor propagation assays. GSCs were cultured in the Neurobasal-A medium with supplements as described above. NSTCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (VivaCell, Cat# C04001-500) to maintain the differentiation characteristics. HEK293T cells (ATCC, Cat# CRL-3216) were cultured in DMEM with 10% FBS. Information on patients’ sex was collected through self-reporting. Sex was not incorporated into the study design or analysis, as investigating potential sex-specific effects was outside the scope of this research. Patient information for the GBM specimens are listed in Supplementary Table 3.

Mass spectrometry analysis
SATB2 and its interacting proteins were co-immunoprecipitated from cell lysates of GSCs overexpressing SATB2-Myc or HEK293T cells overexpressing SATB2-Myc and His-ubiquitin. The precipitates were then analyzed by mass spectrometry at BGI-Tech to identify SATB2-interacting proteins and at Nanjing Newtop Biotech (Nanjing, China) to identify ubiquitinated sites on SATB2. Briefly, cell lysates were incubated with anti-Myc antibody (Proteintech, 60003-2-Ig) or IgG, along with protein A/G Plus agarose beads (Santa Cruz, sc-2003), overnight at 4°C. The SATB2-Myc protein complex captured by the beads is eluted, boiled and separated on SDS-PAGE, and stained by Coomassie blue staining. The visible band was cut, treated with acetonitrile, and then trypsinized.

For the identification of SATB2 binding proteins, peptides from both the experimental group and a control IgG group were extracted and separated. The peptides separated by liquid phase chromatography were ionized by a nano ESI source and then passed to a tandem mass spectrometer (Orbitrap Fusion Lumos, Thermo Fisher) for DDA (data-dependent acquisition) mode detection. The main parameters were set as follows: ion source voltage, 2 kV; MS1 scanning range, 350–1500 m/z; MS1 resolution, 60,000; MS2 starting m/z, 100; MS2 resolution, 15,000. The ion screening conditions for MS2 fragmentation were: charge 2+ to 6+, and the top 30 parent ions with the peak intensity exceeding 20,000. The ion fragmentation mode was HCD, and the fragment ions were detected in the Orbitrap. The dynamic exclusion time was set to 30 s. The AGC was set to 1E5 for MS1 and 2E4 for MS2. For protein identification, experimental MS/MS data were matched against theoretical MS/MS data from a protein database. The raw MS data were converted into a peak list and searched against the UniProt database using Mascot (version 2.3.02). The search was performed using an mgf file as input against a pre-constructed database. The Mascot parameters were set as follows: Enzyme: Trypsin; Peptide mass tolerance: 10 ppm; Fragment mass tolerance: 0.02 Da; Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Gln->pyro-Glu (N-term Q), Deamidated (NQ), Phospho (ST), Phospho (Y); Max missed cleavages: 1; Instrument type: ESI-FTICR. The search results were pre-processed and rescored using Percolator to improve the matching accuracy. The output was then filtered at a 1% false discovery rate (FDR) at the spectral level (PSM-level FDR ≤ 0.01) to obtain a list of significantly identified spectra and peptides.

For the identification of ubiquitinated sites on SATB2, peptides from both the experimental group and a control IgG group were extracted. The peptide samples were then separated and analyzed by a Q-Exactive HF-X mass spectrometer (Thermo Fisher) operating in DDA mode. The total analysis time was 60 min in positive ion mode. The full scan MS1 spectra were collected over the m/z range of 350–1800 with a resolution of 60,000 at 200 m/z, an AGC target of 3e6, and a maximum injection time of 50 ms. For MS2 acquisition, the top 20 most intense precursor ions from each full scan were selected for fragmentation. The MS2 spectra were acquired with a resolution of 15,000 at 200 m/z, an AGC target of 1e5, and a maximum injection time of 50 ms. HCD was used as the fragmentation mode with an isolation window of 1.6 m/z and a normalized collision energy of 28. For protein identification, experimental MS/MS data were matched against theoretical MS/MS data from a protein database. The raw MS data were searched against the UniProt database using MaxQuant (version 2.4.14.0). The MaxQuant parameters were set as follows: Enzyme: Trypsin; Precursor Tolerance (main search): 4.5 ppm; Precursor Tolerance (first search): 20 ppm; MS/MS tolerance: 20 ppm; Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Protein N-term), GlyGly (K). After database searching, the mass spectrometry data were filtered using the following criteria: PSM FDR ≤ 0.01, Site FDR ≤ 0.01, and Protein FDR ≤ 0.01 for peptide, modification site, and protein identification, respectively. To ensure the quality of modified peptide identifications, additional filters were applied, including a Modified Peptide Score ≥ 40 and the presence of at least one unmodified peptide (Unmodified peptides ≥ 0). A modification site was considered confidently localized when its localization probability was ≥ 0.75.

Intracranial tumor formation and treatment
BALB/c-Nude mice were purchased from Hunan Slack Jingda Experimental Animal (Changsha, China). Male or female BALB/c nude mice at 6–7 weeks were used and housed in the animal facility of the laboratory animal center at Huazhong Agricultural University. Mice were maintained at 22–24°C and 40–70% humidity with a 12 h light/12 h dark schedule. Sex was not incorporated into the study design or analysis, as investigating potential sex-specific effects was outside the scope of this research. 50,000 GSCs were implanted into the right cerebral cortex at a depth of 3.5 mm to establish intracranial xenografts. Mice were maintained until neurological signs appeared (survival analysis). Neurological signs included hunched posture, gait changes, lethargy, and weight loss. For the drug treatment, Wu-5 (7.5 mg/kg; TargetMol, Cat# T77763) was dissolved in DMSO (Santa Cruz, sc-358801). 7 days post-GSC transplantation, mice were treated with Wu-5 or vehicle control 6 days per week and maintained until the appearance of neurological signs (survival analysis). The maximum tumor burden permitted by the ethics committee is 1.2 cm in diameter for mice, and the tumor weight must not exceed 10% of the body weight. No animals exceeded these limits in this study.

Cell viability, tumorsphere formation, and in vitro limiting dilution assays
For the cell viability assay, cells were plated into a 96-well plate at a density of 1,500 cells/well. Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Cat# G7572) according to the manufacturer’s instructions. For the tumorsphere formation assay, 1,500 GSCs were seeded into each well of a 96-well plate. The number of tumorspheres in each well was recorded, and the diameter of tumorspheres was analyzed on the sixth day after cell seeding. Tumorsphere images were collected by using the ECHO Revolve RVL-100. For the in vitro limiting dilution assay, a gradient number of GSCs (0, 10, 20, 30, 40, 50 cells/well) with 24 replicates per number were plated into a 96-well plate. The presence of tumorspheres was recorded on day 6, and the self-renewal capacity of GSCs was analyzed using ELDA software available at http://bioinf.wehi.edu.au/software/elda/.

EdU incorporation assay
For the EdU incorporation assay, EdU solution was added to the GSC medium for 2 hours, then cells were fixed with 4% paraformaldehyde (PFA). EdU in cells was stained using the BeyoClick™ EdU-594 kit (Beyotime, Cat# C0078) following the manufacturer’s protocol. The percentage of EdU-positive cells to total cells was obtained to indicate proliferation.

DNA constructs and lentiviral production
Lentiviral constructs expressing shRNAs against human USP10, DTX3L, YY2, and a non-targeting shRNA were cloned into pLKO.1-puro vector (Addgene, Cat# 8453). The shRNA sequences are listed in Supplementary Table 4 and were synthesized by GeneCreate (Wuhan, China). USP10-Flag, DTX3L-Flag, DTX3L-HA, and SATB2-Myc were generated by cloning their open reading frame (ORF) into the pLVX-EF1α-IRES-Neo (MiaoLing Bio, Cat# P23885) or pLVX-EF1α-IRES-Puro vector (MiaoLing Bio, Cat# P0405). SATB2 (Full length) and its truncated mutants were generated by cloning the corresponding sequences into pcDNA3.1-Myc vector (Thermo Fisher, Cat# V85520). USP10 (Full length) and its truncated mutants were generated by cloning the corresponding sequences into pcDNA3.1-Flag vector, which was modified from pcDNA3.1-Myc vector. DTX3L (Full length) and its truncated mutants were generated by cloning the corresponding sequences into the pcDNA3.1-Flag vector. Point mutations were generated by site-directed mutagenesis. The SATB2 (232-437aa) mutants K233R, K266R, K350R, K373R and K400R were generated using SATB2 (232-437aa) as the template and cloned into pcDNA3.1-Myc vector. The SATB2 K266R-Myc was generated using SATB2-Myc as the template and cloned into the pLVX-EF1α-IRES-Puro vector. The USP10 CA-Flag was generated where C424 was mutated to A and cloned into the pLVX-EF1α-IRES-Neo vector. The DTX3L C561/564S-HA was generated, where C561 and C564 were mutated to S and cloned into the pLVX-EF1α-IRES-Neo vector. The USP10 ORF vector for cloning was kindly provided by Dr. Bo Yang and Dr. Hong Zhu (Zhejiang University). The pCDH-His-Ub-MSCV, pCDH-His-K48Ub-MSCV, and pCDH-His-K63Ub-MSCV plasmids were kindly provided by Dr. Xiuxing Wang (Nanjing Medical University). The pCDH-His-K6 Ub-MSCV, pCDH-His-K11 Ub-MSCV, pCDH-His-K27 Ub-MSCV, pCDH-His-K29 Ub-MSCV, and pCDH-His-K33 Ub-MSCV were generated using His-Ub as the template. Lentiviral particles were generated in HEK293T cells by co-transfection of lentiviral vector, pCL-VSVG (Addgene, Cat# 1733) and psPAX2 (Addgene, Cat# 12260) using a standard calcium phosphate transfection method. The viruses collected from the media were then concentrated through ultracentrifugation.

Quantitative Real-Time PCR (qPCR)
Total cellular RNA was extracted using RNAiso Plus (Takara, Cat# 9109) and reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Takara, Cat# RR036A). qPCR was conducted on a 7500 Fast Real-Time PCR System (Applied Biosystems) by using TaqPro Universal SYBR qPCR Master Mix (Vazyme, Cat# Q712-02). Target RNA abundance was indicated by the cycle threshold (Ct) value and calculated using the 2−ΔΔCT method. qPCR primers were synthesized by GeneCreate (Wuhan, China). qPCR data were collected by using the Applied Biosystems 7500 Fast Real-Time PCR System. Sequences of qPCR primers were as follows: 18S rRNA, forward 5’-CTACCACATCCAAGGAAGGCA-3’, reverse 5’-TTTTTCGTCACTACCTCCCCG-3’; USP10, forward 5’-GAGGG CACAGCTACCAACG-3’, reverse 5’-AGGGGAGATATGGCGGGAG-3’; DTX3L, forward 5’-AGCACCAAATACTTGTTGACGA-3’, reverse 5’-CAC CAGACGGTGTTTCTGCTT-3’; SATB2, forward 5’-GGAGAACGACAGCG AGGAA-3’, reverse 5’-CCGATGTATTGCTTTGCCTAGT-3’; FOXM1, forward 5’-ATACGTGGATTGAGGACCACTA-3’, reverse 5’-TCCAATGT CAAGTAGCGGTTG-3’; YY2, forward 5’-GGTGGGCTATTGCGACTCAG-3’, reverse 5’-GCTCGTCTTCAATGCT ATCCG-3’; PBX2, forward 5’- GACAACTCCATCGAACACTCG-3’, reverse 5’-ACATGGGTCGTGAA CTCATTAC-3’; NFIL3, forward 5’-AGAACAAACTAATTGCACTGGGA-3’, reverse 5’-GCTCGTCCACAAATGAACTCAC-3’.

Immunoblot (IB) analysis and co-immunoprecipitation (CoIP)
For IB analysis, cells were lysed in RIPA lysis buffer containing protease inhibitors for 30 minutes and centrifuged at 15,000 g for 15 min. The protein samples were separated by SDS-PAGE gels, transferred to PVDF membranes, and blocked with 5% non-fat milk for 1 hour. Blots were incubated with primary antibodies overnight, followed by HRP-conjugated secondary antibodies. Immunoreactivity was detected by Ultra-sensitive ECL chemiluminescent substrate (Biosharp, Cat# BL523B) with the Bio-Rad ChemiDoc XRS+ Imaging System. For CoIP, cells were lysed in IP lysis buffer containing protease inhibitors for 30 minutes and centrifuged at 15,000 g for 15 min. Protein lysates were incubated with primary antibodies and protein A/G Plus agarose beads (Santa Cruz, Cat# sc-2003) overnight at 4°C. Then, the beads were washed with IP wash buffer 3 times. The precipitated proteins were boiled and subjected to IB analysis. Antibodies and dilutions are listed in Supplementary Table 5. Uncropped images are provided in the Source Data file.

Ubiquitination assay
Cells were treated with MG-132 (20µM; GLPBIO, Cat# GC10383) for 6 hours and then lysed in IP buffer containing protease inhibitors. CoIP was performed with the indicated antibody and protein A/G Plus agarose beads (Santa Cruz, Cat# sc-2003) overnight at 4°C. The precipitated proteins were denaturized and released from beads by boiling in SDS loading buffer for 10 minutes. Protein lysates were detected by IB analysis with the anti-ubiquitin antibody (Biolegend, Cat# 646302).

In vitro ubiquitination assay
His-SATB2, His-DTX3L, GST-USP10, and His-ubiquitin proteins were expressed and purified in BL21 (DE3) E. coli. In vitro ubiquitination assay was performed at 37°C for 2 hours in a 30-μl reaction buffer (40 mM Tris pH 7.5, 10 mM MgCl2, 2 mM DTT) containing 40 nM His/GST-UBA1 (E1, TargetMol, Cat# TMPY-02840), 300 nM GST-UBE2D1 (E2, TargetMol, Cat# TMPJ-00999), 4 μM His-ubiquitin, 120 nM His-SATB2, 120 nM His-DTX3L, 100 nM GST-USP10, and 5 mM ATP (Beyotime, Cat# D7378). The reactions were terminated by the addition of SDS loading buffer, and the reaction products were analyzed by IB analysis.

Immunofluorescent staining
Immunofluorescent staining was performed in tissues or cultured cells. GBM xenografts were collected from mice when neurological signs appeared after GSC transplantation. Human GBM samples were collected from patients through surgical resection. Briefly, tumor sections or cultured cells were fixed in 4% PFA for 10 minutes and permeabilized with 0.3% Triton X-100. The slides were then incubated with primary antibody at 4°C overnight, followed by fluorescent second antibody at room temperature for 2 hours. Nuclei were counterstained with DAPI for 10 minutes, and sections were subjected to microscopy using a Leica DMi8 microscope or an Andor confocal spinning disk on a Nikon inverted microscope Eclipse Ti-E. Antibodies and dilutions are listed in Supplementary Table 5.

Luciferase reporter and chromatin immunoprecipitation (ChIP) assays
The promoter region (−2000 to +100) of human USP10 was inserted into the pGL3-basic vector (Promega, Cat# E1751) to generate the USP10 reporter construct. The mutant USP10 reporter construct was generated, where GGC (+27 to +29) was mutated to AAT and cloned into the pGL3-basic vector. Flag-YY2 was generated by cloning its ORF into the pLVX-EF1α-IRES-Puro vector (MiaoLing Bio, Cat# P0405). For the luciferase reporter assay, cells were co-transfected with USP10 reporter plasmid or mutant plasmid, Flag-YY2 overexpression vector, and pRL internal control vector (Promega, Cat# E2231). Luciferase activities were detected 48 hours post-transfection using the Dual-Luciferase® Reporter Assay System (Promega, Cat# E1910) according to the manufacturer’s instructions, with the BioTek Cytation5 plate reader. Firefly luciferase activities were normalized to Renilla. For the ChIP assay, cells were fixed with 1% formaldehyde for 10 minutes, terminated with glycine, and washed 3 times with cold PBS. Then, chromatin lysates were sonicated to break the chromatin, and the supernatant was collected after centrifugation. The sonicated lysates were pre-cleared with protein A/G Plus agarose beads (Santa Cruz, sc-2003) and immunoprecipitated by the indicated antibody or control IgG in the presence of salmon sperm DNA and BSA overnight. Beads were washed and then decrosslinked at 65°C for 12 hours. DNA was purified with a DNA purification kit and analyzed by PCR analysis. ChIP primers were synthesized by GeneCreate (Wuhan, China). Gel images were acquired by using the Tanon MINI Space 2000 Imaging System (Shanghai, China). The primers used are detailed below: forward 5’-CCGATGCGAGTGTGTATGTG-3’; reverse 5’-TATACTGCGGGCTGTG GAG-3’. Antibodies are shown in Supplementary Table 5. Uncropped images are provided in the Source Data file.

Statistics and reproducibility
All data are presented as the mean ± SD from at least three samples or experiments per data point. Statistical analyses were conducted using GraphPad Prism 10 software. Two-tailed unpaired Student’s t-test was used to evaluate the statistical significance for two groups. One-way ANOVA analysis followed by Tukey’s test was used to examine the statistical significance for more than two groups. For quantification with two groups and the groups with subgroups, two-way ANOVA analysis followed by Sidak’s test was used to evaluate statistical significance. For quantification with more than two groups and the groups with subgroups, two-way ANOVA analysis followed by Tukey’s test was used to evaluate statistical significance. Data were normally distributed. A log-rank test was used to determine the statistical significance of Kaplan-Meier survival curves. For all statistical tests, p < 0.05 was considered statistically significant. Precise experimental details are described in the figure legends.

Ethics statement
All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experimental Ethics Committee of Huazhong Agricultural University. The use of clinical specimens was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Taihe Hospital (the Affiliated Hospital of Hubei University of Medicine, Shiyan, China).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dysregulation of protein homeostasis is a well-established driver of cancer. In this manuscript, Guo et al. uncover a\nregulatory mechanism involving an E3 ubiquitin ligase and a deubiquitinase (DUB) that modulates the stability of SATB2, a\nkey protein implicated in glioblastoma (GBM) progression. The authors demonstrate that in glioma stem cells (GSCs), the\nelevated expression of USP10 relative to DTX3L stabilizes SATB2. This stabilization promotes the downstream effects of\nSATB2, including increased FOXM1 expression, which supports GSC maintenance and GBM growth. Conversely, the\nknockdown of USP10 or overexpression of DTX3L reduces FOXM1 levels and GSC proliferation, an effect rescued by\nSATB2 overexpression. Finally, the study reports that the USP10 inhibitor, Wu-5, significantly inhibits GSC-driven tumor\ngrowth.\nWhile the manuscript presents some novel findings, its overall impact is moderate. Several key issues need to be addressed\nto substantiate the authors' claims and enhance the rigor of the study.\nMajor points:\n1.The authors rely solely on MG132 to assess proteasome-mediated degradation of SATB2. However, lysosomal\ndegradation cannot be excluded based on the data provided. To address this, additional experiments using lysosomal\ninhibitors such as Bafilomycin A1 should be performed. This will help delineate the relative contributions of proteasomal and\nlysosomal pathways to SATB2 degradation.\n2.The manuscript proposes that \"cytoplasmic SATB2 may be translocated to the nucleus after deubiquitination.\" However,\nthis claim is inadequately supported. Figures 2e and 2f indicate that while USP10 and DTX3L are cytoplasmic, SATB2 is\nprimarily in nucleus, suggesting that deubiquitinated SATB2 might enter the nucleus. To substantiate this hypothesis, the\nauthors should conduct additional experiments: treating GSCs with MG132 to inhibit the degradation of ubiquitinated\nSATB2 and assess its localization through immunofluorescence; Isolating cytoplasm and nucleus fractions following MG132\ntreatment, perform SATB2 immunoprecipitation, and probe with an anti-ubiquitin antibody to determine its ubiquitination\nstatus in each compartment.\n3.The ubiquitination assays presented in Figures 5g and 5k have design flaws that hinder the interpretation of results. The\nauthors used myc-antibody to perform immunoprecipitation and then blot with His antibody to detect ubiquitinated SATB2.\nConsidering that many E3 ligases co-expressing with Ub show strong auto-ubiquitination, especially here SATB2 interacts\nwith DTX3L, thus this result couldn’t differentiate the ubiquitinated SATB2 and auto-ubiquitinated DTX3L which binds to\nSATB2. In addition, DTX3L and SATB2 share similar molecular weight. Western Blot with anti-SATB2 could solve this\nproblem. The authors should show the intact Ub blot, not the part above 95 kDa only. This specific point applies to the rest of\nubiquitination blot.\n4.Identifying the key ubiquitinated sites of SATB2 using mass spectrometry analysis helps strength the conclusion that K266\nis the ubiquitination/deubiquitination site of DTX3L and USP10. Regarding the identification of the ubiquitin chain type on\nSATB2 by DTX3L, K48 and K63 ubiquitin mutations are not enough as they can’t excluded other 5 types of Ub chains. The\nauthors should include all types of lysine mutated ubiquitin variants to provide a comprehensive understanding of the\nubiquitin chain architecture.\n5.The molecular mechanism underlying SATB2 degradation by Wu-5 treatment remains unclear. Both USP10 inhibition and\nthe downregulation of USP10 protein level could lead to increased ubiquitination of SATB2 by DTX3L, ultimately resulting in\nits degradation. The authors should provide further clarification to distinguish between these possibilities.\nMinor points:\n1. The manuscript contains numerous typos that affect its readability. Examples include:\nPage 5, Line 96: \"……refgulatory mechanism……\" Page 6, Line 126: \"……SATB2-interating……\"\nThe authors should carefully proofread the manuscript.\n2. It is unclear which shUSP10 was used in Figure 5C-D. The authors should clearly indicate the shUSP10 used in the\nexperiments.\n3. In Figure S2a, \"shUSP10-1\" appeared twice. It is unclear whether the authors performed the immunofluorescence\nexperiments twice using the same construct or if this is a labeling error.\n4. Misuse of tenses detracts from the quality of the manuscript.\nFor instance: Page 6, Line 119: \"……first check……\" should be revised to \"……first checked…….\"\n5. The abbreviation \"NSTCs\" (Non-stem tumor cells) is introduced without explanation. The authors should define this term\nupon its first use in the manuscript to ensure clarity.", "reviewer_2": "In this manuscript, Guo et al. investigated the mechanisms regulating SATB2 expression in glioma stem cells (GSCs) and,\nfor the first time, identified USP10 as a deubiquitinase and DTX3L as an E3 ligase for SATB2 in GSCs. The authors also\ndetermined that K266 is the lysine residue regulated by these two proteins. Furthermore, the authors demonstrated that\nUSP10 is essential for GSC self-renewal and tumorigenesis in vivo, with its effects being rescued by the overexpression of\nSATB2. The authors identified YY2 as the upstream transcription factor mediating USP10 upregulation in GSCs. Lastly, they\nassessed the therapeutic potential of the USP10 inhibitor Wu-5 in a glioma mouse xenograft model, highlighting the\ntranslational promise of this project. Overall, this study is well-designed with appropriate controls. The data are of high\nquality and robust in supporting the main conclusions, though additional evidence is needed in certain areas, as outlined\nbelow. The manuscript is well-prepared, with clear descriptions and detailed information. Moreover, the authors need to\naddress the following comments to further strengthen this study. The current enthusiasm for this manuscript is high.\n1. Fig. 2h-j and Fig. S1c-f: The authors showed that USP10 and DTX3L are expressed in more than 80% of SOX2+ or\nSATB2+ cells in clinical GBM samples. However, this does not establish that these genes are preferentially expressed in\nSOX2+ cells, as SOX2- cells were not included for comparison. Moreover, in Fig. S1c-f, only three replicates were included\nfor each group. Were these replicates derived from different fields of the same patient sample or from different patients? The\nauthors should include data from more fields (typically >10) as well as all analyzed patient samples. The same question\napplies to Fig. S7f-i.\n2. To support their conclusion that USP10, DTX3L, and SATB2 are preferentially expressed in GSCs, the authors should\nanalyze published single-cell RNA-seq datasets of clinical glioblastoma (GBM) samples and compare the expression of\nthese genes across different GBM cellular states. Additionally, they could perform a correlation analysis to examine the\nrelationships between the expression levels of each pair of these three genes in GBM cells.\n3. The authors concluded that USP10 expression is significantly higher than DTX3L in GSCs based on the qPCR results in\nFig. 2d. However, relative quantification in qPCR requires similar primer efficiencies for valid comparisons between different\nprimers. Did the authors test primer efficiency?\n4. Fig. 3i: In the H&E-stained coronal sections with indicated GSC tumor xenografts, no tumor cells or areas were observed\nin the shUSP10 groups in these two GSC xenograft models. Is this result representative? From the survival data and IF\nstaining in Fig. 3j, there should be tumor xenograft mass in the coronal brain sections in the shUSP10 groups. Were these\nthree each H&E-stained brain sections from the mice that were euthanized at similar time points? The authors should also\nconfirm USP10 knockdown and decreased SATB2 expression in the brain slices. The same concern applies to Figs. 4 and\n6.\n5. The authors reported decreased SOX2+ tumor cells in xenografts following USP10 knockdown, DTX3L overexpression,\nor 5-WU treatment. Did they observe an increase in differentiated cells by IF staining of some neuronal or astrocyte markers?\nWhat is the potential mechanism? Additionally, did SOX2 expression change after these treatments in vitro?\n6. An in vitro ubiquitination assay using recombinant proteins and ubiquitin should be performed to confirm USP10 and\nDTX3L as the deubiquitinase and E3 ligase for SATB2, respectively.\n7. According to the DepMap Portal, USP10 is categorized as a common essential gene. Do the authors have evidence that\nGSCs are more dependent on USP10 compared to differentiated counterparts or normal cells?\n8. In Fig 6, do the authors have data showing that 5-WU can penetrate the blood-brain barrier? They should also\ndemonstrate a decrease in SATB2 and USP10 levels in 5-WU-treated tumors.\n9. Can SATB2 overexpression rescue the effects of 5-WU treatment? In Fig 6, the authors have already established ectopic\nexpression of SATB2 GSC lines for this validation experiment.\n10. Although the authors stated in the Methods section that they used both male and female animals, they did not stratify\ntheir analysis by sex or mention whether similar results were observed in both sexes.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "SATB2 is a transcription factor shown to play a role in many types of cancer. In glioblastoma, SATB2 is\noverexpressed compared to low-grade gliomas; however, its role in GBM development and progression is\npoorly understood. In the manuscript titled USP10 promotes glioma stem cell maintenance and glioblastoma growth by antagonizing DTX3L-\nmediated SATB2 ubiquitination, the authors identify the deubiquitinase USP10 and the E3 ligase DTX3L as key mediators\nof SATB2 stability. Using an IP strategy, the authors show that USP10 and DTX3L directly interact with SATB2.The authors\ndemonstrate that USP10 and DTX3L are preferentially expressed in GBM GSCs, where USP10 can reverse DTX3L-\nmediated ubiquitination of SATB2 and rescue it from proteasomal degradation. Furthermore, shRNA-mediated knockout of\nUSP10 inhibits GSC proliferation, reduces tumor growth, and significantly extends survival time in a xenograft model.\nConversely, forced overexpression of DTX3L inhibits GSC formation and proliferation, as well as the growth of GBM tumors\nin vivo. Taken together, the authors effectively show that USP10 and DTX3L regulate SATB2 stability and GBM\nproliferation.The mechanism of SATB2 regulation was subsequently shown to be due to K48-mediated ubiquitination at a\nconserved lysine site (K266), leading to degradation. Building upon this, the authors utilize a novel inhibitor of USP10 to\ninduce SATB2 ubiquitination and degradation. Treatment of tumor-bearing xenografts with this inhibitor resulted in a\nreduction in tumor burden and an increase in survival time. The authors propose a model in which USP10 overexpression in\nGBM rescues SATB2, whereas USP10 inhibition promotes SATB2 degradation, ultimately reducing GBM growth.\nOverall, this is a well-constructed study that strongly supports the authors' model. They follow a stepwise and logical\napproach in confirming their hypothesis, and the data presented is of very high quality. The paper is well-written, will be of\nbroad interest, and I commend the authors on their work.\nSome minor comments\nIn Figure 8, the authors show that WU5 can inhibit GBM growth. Do the authors know if WU5 induces apoptosis?\nSticking with Figure 8, the authors show that WU5 effectively induces ubiquitination of SATB2, as expected. However, the\nobserved reduction in USP10 is potentially interesting. The authors mention that this was observed in a previous study, but\nthe mechanism remains unclear. Since MG132 is added to the media, proteasomal degradation cannot be the cause. Do the\nauthors know if WU5 affects USP10 transcription? Is SATB2 a transcriptional activator of USP10? Is USP10 also\nubiquitinated by DTX3L? It would be interesting to hear the authors’ perspective.\nMinor typo on line 119: It should read \"We first checked SATB2 protein levels.\""}, "author_response": "Responses to Reviewers’ Comments\nRe: NCOMMS-24-81157\nWe are grateful to the reviewers for their critical evaluation of our manuscript. We highly value the\ninsightful comments and helpful suggestions they provided. In response, we have conducted a substantial\nnumber of additional experiments and thoroughly revised the manuscript to address the major concerns\nraised. We are confident that the revised manuscript has been significantly enhanced as a result. Below,\nwe provide a detailed point-by-point response to the reviewers’ comments. We trust that our responses\nhave effectively addressed the important issues raised by the reviewers.\nReviewer #1 (Remarks to the Author):\nDysregulation of protein homeostasis is a well-established driver of cancer. In this manuscript, Guo et al.\nuncover a regulatory mechanism involving an E3 ubiquitin ligase and a deubiquitinase (DUB) that\nmodulates the stability of SATB2, a key protein implicated in glioblastoma (GBM) progression. The authors\ndemonstrate that in glioma stem cells (GSCs), the elevated expression of USP10 relative to DTX3L\nstabilizes SATB2. This stabilization promotes the downstream effects of SATB2, including increased\nFOXM1 expression, which supports GSC maintenance and GBM growth. Conversely, the knockdown of\nUSP10 or overexpression of DTX3L reduces FOXM1 levels and GSC proliferation, an effect rescued by\nSATB2 overexpression. Finally, the study reports that the USP10 inhibitor, Wu-5, significantly inhibits\nGSC-driven tumor growth.\nWhile the manuscript presents some novel findings, its overall impact is moderate. Several key issues\nneed to be addressed to substantiate the authors' claims and enhance the rigor of the study.\nResponse: We thank the reviewer for the time and effort to review our manuscript. We appreciate the\nconcerns and suggestions raised by the reviewer. In response, we have performed additional experiments\nto address these important concerns.\nReviewer #1-Major points: 1. The authors rely solely on MG132 to assess proteasome-mediated\ndegradation of SATB2. However, lysosomal degradation cannot be excluded based on the data provided.\nTo address this, additional experiments using lysosomal inhibitors such as Bafilomycin A1 should be\nperformed. This will help delineate the relative contributions of proteasomal and lysosomal pathways to\nResponse: We appreciate the reviewer for raising this important point. To investigate whether SATB2\nundergoes lysosomal degradation, we treated GSCs with the lysosome inhibitor Bafilomycin A1. Our\nresults revealed that Bafilomycin A1 had no impact on the expression of SATB2 (Please see Figure R1\nbelow). Thus, we concluded that the degradation of SATB2 is not mediated through the lysosomal\ndegradation pathway.\nFigure R1. Immunoblot (IB) analysis of SATB2 expression in GSCs treated with Bafilomycin A1 (20nM) for\nindicated hours (h).\nWe have added the new data in Supplementary Fig. 1c and described the results in our revised manuscript\nat Line 124 on Page 6: “However, the protein levels of SATB2 remained unchanged after treatment with\nthe lysosome inhibitor Bafilomycin A1.”.\nReviewer #1-Major points: 2. The manuscript proposes that \"cytoplasmic SATB2 may be translocated to\nthe nucleus after deubiquitination.\" However, this claim is inadequately supported. Figures 2e and 2f\nindicate that while USP10 and DTX3L are cytoplasmic, SATB2 is primarily in nucleus, suggesting that\ndeubiquitinated SATB2 might enter the nucleus. To substantiate this hypothesis, the authors should\nconduct additional experiments: treating GSCs with MG132 to inhibit the degradation of ubiquitinated\nSATB2 and assess its localization through immunofluorescence; Isolating cytoplasm and nucleus fractions\nfollowing MG132 treatment, perform SATB2 immunoprecipitation, and probe with an anti-ubiquitin antibody\nto determine its ubiquitination status in each compartment.\nResponse: We thank the reviewer for raising this critical issue. We have performed immunofluorescent\nstaining of SATB2 in GSCs after treatment with MG-132 or DMSO. The results showed that MG-132\ntreatment induced nuclear export of SATB2 in GSCs (Please see Figure R2a below). In addition, the\nubiquitination assay detected ubiquitination of SATB2 in the cytoplasm but not in the nucleus (Please see\nFigure R2b below).\nDMSO for 12 hours before fixation. Scale bar, 20 μm. b, Cytoplasmic and nuclear extracts prepared from\nGSCs were subjected to IP with SATB2 antibody or IgG followed by IB with ubiquitin (Ub) antibody. Cells\nwere treated with 20μM MG-132 for 6 hours before harvesting. The expression of marker proteins for the\ncytoplasm (GAPDH) and nucleus (Histone H3) was also shown.\nWe have added the new data in Supplementary Fig. 1d,e and described the results in our revised\nmanuscript at Line 162 on Page 8: “To verify this hypothesis, we first performed immunofluorescent\nstaining of SATB2 in GSCs after treatment with MG-132 or DMSO……These results confirm that the\ncytoplasmic SATB2 is translocated to the nucleus following deubiquitination.”.\nReviewer #1-Major points: 3. The ubiquitination assays presented in Figures 5g and 5k have design flaws\nthat hinder the interpretation of results. The authors used myc-antibody to perform immunoprecipitation\nand then blot with His antibody to detect ubiquitinated SATB2. Considering that many E3 ligases\nco-expressing with Ub show strong auto-ubiquitination, especially here SATB2 interacts with DTX3L,\nthus this result couldn’t differentiate the ubiquitinated SATB2 and auto-ubiquitinated DTX3L which binds to\nSATB2. In addition, DTX3L and SATB2 share similar molecular weight. Western Blot with anti-SATB2\ncould solve this problem. The authors should show the intact Ub blot, not the part above 95 kDa only. This\nspecific point applies to the rest of ubiquitination blot.\nResponse: We appreciate the reviewer's concern and constructive suggestions. As suggested, we\nreexamined the immunoprecipitated samples shown in Figures 5g and 5k for both SATB2 and the Myc tag.\nspecies were below the detection limit (Please see Figure R3a,b below). This observation is consistent\nwith numerous published studies using similar Co-IP methodologies, where typically only the unmodified\nform of the bait protein is observed1-6. We therefore speculate that ubiquitinated SATB2 represents a minor\nfraction under overexpression conditions and thus escapes detection. To address the reviewer’s concern,\nwe performed additional experiments. HEK293T cells were co-transfected with SATB2-Myc, His-ubiquitin,\nand either HA-tagged wild-type DTX3L (WT) or the catalytically inactive mutant (C561S/C564S, 2CS).\nNotably, WT DTX3L markedly enhanced SATB2 ubiquitination, whereas the 2CS mutant had only a weak\npromoting effect (Please see Figure R3c below). Subsequent analysis confirmed that WT DTX3L\npromoted K48-linked polyubiquitination of SATB2, with the 2CS mutant similarly exhibiting slight promotion\nof this modification (Please see Figure R3d below). Taken together, these data confirm that the observed\nubiquitinated species are primarily ubiquitinated SATB2, rather than auto-ubiquitinated DTX3L. Additionally,\nwe have replaced all Ub immunoblots with larger-frame images showing extended gel regions in the\nrevised manuscript, and the corresponding uncropped blots are provided in Supplementary Fig. 13.\ncell lysates were subjected to IP with Myc antibody followed by IB with indicated antibodies. Cells were\ntreated with MG-132 (20 µM) for 6 hours before harvesting. b, HEK293T cells were co-transfected with\nSATB2-Myc, DTX3L-Flag, and the indicated His-tagged WT Ub, Lys48-only (K48) Ub, or Lys63-only (K63)\nUb. Thereafter, cell lysates were subjected to IP with Myc antibody followed by IB with indicated\nantibodies. Cells were treated with MG-132 (20 µM) for 6 hours before harvesting. c, HEK293T cells were\nco-transfected with SATB2-Myc, His-Ub, and either HA-tagged DTX3L WT or DTX3L 2CS, and then cell\nlysates were subjected to IP with Myc antibody followed by IB with indicated antibodies. Cells were treated\nwith MG-132 (20 µM) for 6 hours before harvesting. d, HEK293T cells were co-transfected with\nSATB2-Myc, His-K48 Ub, and either HA-tagged DTX3L WT or DTX3L 2CS, and then cell lysates were\nsubjected to IP with Myc antibody followed by IB with indicated antibodies. Cells were treated with MG-132\n(20 µM) for 6 hours before harvesting.\nWe have updated the data in Fig. 5g and 5k, added new data in Supplementary Fig. 5f, g, and added the\ncorresponding description in our revised manuscript at Lines 235-237 on Page 11.\nReviewer #1-Major points: 4. Identifying the key ubiquitinated sites of SATB2 using mass spectrometry\nanalysis helps strength the conclusion that K266 is the ubiquitination/deubiquitination site of DTX3L and\nUSP10. Regarding the identification of the ubiquitin chain type on SATB2 by DTX3L, K48 and K63\nubiquitin mutations are not enough as they can’t excluded other 5 types of Ub chains. The authors should\ninclude all types of lysine mutated ubiquitin variants to provide a comprehensive understanding of the\nubiquitin chain architecture.\nResponse: We thank the reviewer for the important suggestion. To identify the ubiquitination site of\nSATB2, we performed mass spectrometry to analyze the immunoprecipitated SATB2 proteins from\nHEK293T cells co-expressing SATB2-Myc and His-ubiquitin. The results showed that ubiquitin moieties\nwere exclusively detected on residue K266 (Please see Figure R4 below), suggesting that the K266 site of\nSATB2 may be regulated by USP10 or DTX3L.\nat K266. HEK293T cells were transfected with SATB2-Myc and His-Ub constructs. Cell lysates were\nimmunoprecipitated with anti-Myc antibody, and the captured SATB2-Myc was analyzed by mass\nspectrometry to identify ubiquitination sites.\nWe have added the new data in Fig. 7f and described the results in our revised manuscript at Line 331 on\nPage 15: “To confirm the ubiquitination site of SATB2……confirming that the K266 site is the ubiquitination\nsite of SATB2.”.\nWe agree with the reviewer that the K48 and K63 ubiquitin mutants are insufficient to elucidate the\narchitecture of the ubiquitin chain. In response to the reviewer’s suggestion, we generated ubiquitin\nvectors with intact K6, K11, K27, K29, or K33 linkages to examine whether these types of linkages also\nparticipate in the DTX3L/USP10-modulated ubiquitination of SATB2. Although DTX3L efficiently promoted\nK48-linked polyubiquitination (Figure 5k), it failed to catalyze polyubiquitin chain formation with these\nlinkages (Please see Figure R5a below). Furthermore, USP10 was also unable to disassemble\npolyubiquitin chains of these linkage types (Please see Figure R5b below). Therefore, we concluded that\nDTX3L predominantly promotes K48-linked polyubiquitination of SATB2, whereas USP10 inhibits this\nmodification.\nHis-tagged WT Ub, Lys6-only (K6) Ub, Lys11-only (K11) Ub, Lys27-only (K27) Ub, Lys29-only (K29) Ub, or\nLys33-only (K33) Ub. Thereafter, cell lysates were subjected to IP with Myc antibody followed by IB with\nHis antibody. Cells were treated with MG-132 (20µM) for 6 hours before harvesting. b, HEK293T cells\nwere co-transfected with SATB2-Myc, USP10-Flag, and the indicated His-tagged WT Ub, Lys6-only (K6)\nUb, Lys11-only (K11) Ub, Lys27-only (K27) Ub, Lys29-only (K29) Ub, or Lys33-only (K33) Ub. Thereafter,\ncell lysates were subjected to IP with Myc antibody followed by IB with His antibody. Cells were treated\nwith MG-132 (20µM) for 6 hours before harvesting.\nWe have added the new data in Fig. 5l,n and described the results in our revised manuscript at Line 268\non Page 12: “Polyubiquitination can occur through seven different Lys residues on ubiquitin……Further\nanalysis revealed that USP10 specifically disassembled K48-linked polyubiquitination without affecting\nother polyubiquitin chain types.”.\nReviewer #1-Major points: 5. The molecular mechanism underlying SATB2 degradation by Wu-5 treatment\nremains unclear. Both USP10 inhibition and the downregulation of USP10 protein level could lead to\nincreased ubiquitination of SATB2 by DTX3L, ultimately resulting in its degradation. The authors should\nprovide further clarification to distinguish between these possibilities.\nResponse: We thank the reviewer for this insightful question. Previous studies have demonstrated that 1–\n5 μM Wu-5 had no effect on USP10 protein levels, while 10 μM markedly reduced USP10 protein levels in\nacute myeloid leukemia (AML) and breast cancer cells7, 8. To clarify its effect in GSCs, we performed time-\nand dose-dependent experiments. Our results revealed that both USP10 and SATB2 levels decreased in a\ntime-dependent manner following treatment with 16 μM Wu-5, with an initial reduction at 12 hours and\n7\nfurther progressive declines at 24, 36, and 48 hours (Please see Figure R6a below). Moreover, treatment\nwith 8 and 16 μM Wu-5 for 24 hours dose-dependently reduced USP10 and SATB2 protein levels,\nwhereas lower concentrations (2 and 4 μM) had no significant impact on either protein (Please see Figure\nR6b below). These findings indicate that USP10 and SATB2 protein levels decrease in parallel in a dose-\nand time-dependent manner upon Wu-5 treatment in GSCs. Furthermore, cell viability assay revealed that\n8 and 16 μM Wu-5 also dose-dependently inhibited GSC growth, whereas lower concentrations (2 and 4\nμM) showed no significant effect (Please see Figure R6c below). Previous study has demonstrated that\nWu-5 exhibits inhibitory activity at low micromolar concentrations (1.5-3 μM)7. However, the lower\nconcentrations tested (2 and 4 μM) proved ineffective in our GSC model, likely because USP10 protein\nlevels showed no reduction under these conditions. Taken together, these results suggest that the\nreduction in USP10 protein levels, rather than mere enzymatic inhibition without affecting protein\nabundance, is likely the primary mechanism driving SATB2 degradation and the subsequent inhibition of\nGSC growth induced by effective concentrations of Wu-5.\nFigure R6. a, IB analysis of SATB2 and USP10 protein levels in GSCs treated with 16 μM Wu-5 at the\nindicated time points. b, IB analysis of SATB2 and USP10 protein levels in GSCs treated with the indicated\ndoses of Wu-5 or the vehicle control for 24 hours. c, Cell viability assay of GSCs treated with the indicated\ndoses of Wu-5 or the vehicle control. n=4 biological independent samples.\nData are represented as mean ± s.d. ****p<0.0001, two-way ANOVA analysis followed by Tukey’s test (c).\nn.s., not significant.\ncorresponding description in our revised manuscript at Lines 374 and 379 on Page 17: “Cell viability assay\nrevealed that 8 and 16 μM Wu-5 dose-dependently inhibited GSC growth, whereas lower concentrations\n(2 and 4 μM) showed no significant effect.” and “As expected, Wu-5 treatment reduced SATB2 protein\nlevels in a dose- and time-dependent manner. We also observed that Wu-5 treatment reduced USP10\nprotein levels in a dose- and time-dependent manner.”.\nThese results are further discussed in the “Discussion” section at Line 468 on Page 21: “Previous studies\nhave demonstrated that 1–5 μM Wu-5 had no effect on USP10 protein levels……is likely the primary\nmechanism driving SATB2 degradation and the subsequent inhibition of GSC growth induced by effective\nconcentrations of Wu-5.”.\nReviewer #1-Minor points: 1. The manuscript contains numerous typos that affect its readability. Examples\ninclude:\nPage 5, Line 96: \"……refgulatory mechanism……\" Page 6, Line 126: \"……SATB2-interating……\"\nThe authors should carefully proofread the manuscript.\nResponse: Thank you for pointing out the typos. We have corrected the typing errors (Lines 96 and 128 in\nthe revised manuscript and carefully proofread the manuscript.\nReviewer #1-Minor points: 2. It is unclear which shUSP10 was used in Figure 5C-D. The authors should\nclearly indicate the shUSP10 used in the experiments.\nResponse: Thank you for your comment. We have now clarified that the shUSP10 used in Figure 5c,d is\nshUSP10-1.\nReviewer #1-Minor points: 3. In Figure S2a, \"shUSP10-1\" appeared twice. It is unclear whether the\nauthors performed the immunofluorescence experiments twice using the same construct or if this is a\nlabeling error.\nResponse: We appreciate the reviewer’s careful observation. This was a labeling error, and we have\ncorrected it in the revised Figure S2c (Figure S2a in the original manuscript).\nReviewer #1-Minor points: 4. Misuse of tenses detracts from the quality of the manuscript.\nFor instance: Page 6, Line 119: \"……first check……\" should be revised to \"……first checked…….\"\nand we have carefully reviewed the manuscript to ensure proper tense usage throughout.\nReviewer #1-Minor points: 5. The abbreviation \"NSTCs\" (Non-stem tumor cells) is introduced without\nexplanation. The authors should define this term upon its first use in the manuscript to ensure clarity.\nResponse: We appreciate the reviewer's comment. We have now defined NSTCs as non-stem tumor\ncells upon its first use in the revised manuscript (Line 151, Page 7) for clarity.\nIn this manuscript, Guo et al. investigated the mechanisms regulating SATB2 expression in glioma stem\ncells (GSCs) and, for the first time, identified USP10 as a deubiquitinase and DTX3L as an E3 ligase for\nSATB2 in GSCs. The authors also determined that K266 is the lysine residue regulated by these two\nproteins. Furthermore, the authors demonstrated that USP10 is essential for GSC self-renewal and\ntumorigenesis in vivo, with its effects being rescued by the overexpression of SATB2. The authors\nidentified YY2 as the upstream transcription factor mediating USP10 upregulation in GSCs. Lastly, they\nassessed the therapeutic potential of the USP10 inhibitor Wu-5 in a glioma mouse xenograft model,\nhighlighting the translational promise of this project. Overall, this study is well-designed with appropriate\ncontrols. The data are of high quality and robust in supporting the main conclusions, though additional\nevidence is needed in certain areas, as outlined below. The manuscript is well-prepared, with clear\ndescriptions and detailed information. Moreover, the authors need to address the following comments to\nfurther strengthen this study. The current enthusiasm for this manuscript is high.\nResponse: We are grateful for the reviewer’s positive comments and appreciate the concerns and\nsuggestions raised. In response, we have conducted extensive additional experiments to address these\nconcerns. We believe that the current manuscript has been significantly improved through these efforts\nand the constructive feedback provided.\nReviewer #2: 1. Fig. 2h-j and Fig. S1c-f: The authors showed that USP10 and DTX3L are expressed in\nmore than 80% of SOX2+ or SATB2+ cells in clinical GBM samples. However, this does not establish that\nthese genes are preferentially expressed in SOX2+ cells, as SOX2- cells were not included for comparison.\nMoreover, in Fig. S1c-f, only three replicates were included for each group. Were these replicates derived\nfrom different fields of the same patient sample or from different patients? The authors should include data\nfrom more fields (typically >10) as well as all analyzed patient samples. The same question applies to Fig.\nS7f-i.\nResponse: We appreciate the comments and suggestions from the reviewer. In the original versions of\nFigures S1c-f and S7f-i, we analyzed eight randomly selected fields from each of three independent GBM\nspecimens and calculated the mean value per specimen (n=3 biological replicates). Following the\nreviewer’s suggestion, we reanalyzed the data by including three additional independent GBM specimens\nand increasing the number of fields analyzed per sample. Specifically, twelve randomly selected fields\nfrom each of six independent GBM specimens were analyzed, the mean value per specimen was\n11\ncalculated (n=6 biological replicates), and the results are presented in Figure R7 below.\nWe agree with the reviewer that our initial statistical analyses did not sufficiently demonstrate the\npreferential expression of USP10 and DTX3L in SOX2+ or SATB2+ cells. To address this concern, we\nperformed additional quantitative analyses to determine the proportions of USP10+ cells within SOX2- cells\nand within SATB2- cells, as well as the proportions of DTX3L+ cells within SOX2- cells and within SATB2-\ncells. Our results revealed that both USP10 and DTX3L were expressed in over 80% of SOX2+ cells and in\nover 80% of SATB2+ cells, whereas staining for both markers was rarely detected in SOX2- cells and in\nSATB2- cells (Please see Figure R7a-d below). Subsequent analyses demonstrated that YY2 was\nexpressed in more than 80% of SOX2+ cells and in more than 85% of USP10+ cells, compared to less than\n10% in SOX2- cells and in SATB2- cells. (Please see Figure R7e, f below). Taken together, these findings\nconfirm that both USP10 and DTX3L are preferentially expressed in SOX2+ cells and in SATB2+ cells, and\nthat YY2 is significantly enriched in SOX2+ cells and in USP10+ cells.\nFigure R7. a, Quantification of the fraction of USP10+ cells in SOX2- cells and in SOX2+ cells in human\nGBMs. b, Quantification of the fraction of DTX3L+ cells in SOX2- cells and in SOX2+ cells in human GBMs.\nc, Quantification of the fraction of USP10+ cells in SATB2- cells and in SATB2+ cells in human GBMs. d,\nQuantification of the fraction of DTX3L+ cells in SATB2- cells and in SATB2+ cells in human GBMs. e,\nQuantification of the fraction of YY2+ cells in SOX2- cells and in SOX2+ cells in human GBMs. f,\nQuantification of the fraction of YY2+ cells in USP10- cells and in USP10+ cells in human GBMs.\nData are represented as mean ± s.d. n=6 independent GBM samples per group. For each sample, the\nmean value was calculated from 12 randomly selected fields and used for statistical analysis.\n****p<0.0001, two-tailed unpaired t-test.\nFig. 1c-f and 7g, i in the original manuscript) and described the statistical method in the corresponding\nfigure legends.\nReviewer #2: 2. To support their conclusion that USP10, DTX3L, and SATB2 are preferentially expressed\nin GSCs, the authors should analyze published single-cell RNA-seq datasets of clinical glioblastoma (GBM)\nsamples and compare the expression of these genes across different GBM cellular states. Additionally,\nthey could perform a correlation analysis to examine the relationships between the expression levels of\neach pair of these three genes in GBM cells.\nResponse: We thank the reviewer for raising this point. Given the absence of a standardized approach to\ndefining GSCs in scRNA-seq data9-11, we believe that using scRNA-seq data to determine the preferential\nexpression of USP10, DTX3L, and SATB2 in GSCs may not be reliable. Nevertheless, our study has\nexperimentally confirmed that USP10, DTX3L, and SATB2 proteins are preferentially expressed in GSCs,\nand we sincerely appreciate the reviewer's suggestion to further strengthen this conclusion.\nAs suggested, we analyzed the TCGA_GBM dataset to examine the pairwise correlations in expression\nlevels among the three genes. The results revealed a weak positive correlation between USP10 and\nSATB2 expression (Please see Figure R8a below), while no significant correlation was observed between\nDTX3L expression and either SATB2 or USP10 (Please see Figure R8b,c below). It is well established that\nmRNA expression levels do not always correlate directly with protein levels, owing to post-transcriptional\nand post-translational regulation. Consequently, even if the mRNA-level correlations were not as expected,\nthis does not imply weak or absent correlations at the protein level. Therefore, we believe these data do\nnot contradict the findings in this manuscript.\nFigure R8. a-c, Pearson correlation analysis between USP10 and SATB2 (a), DTX3L and SATB2 (b), and\nDTX3L and USP10 expression (c) in TCGA_GBM dataset (Agilent-4502A).\nbased on the qPCR results in Fig. 2d. However, relative quantification in qPCR requires similar primer\nefficiencies for valid comparisons between different primers. Did the authors test primer efficiency?\nResponse: We thank the reviewer for raising this question. We calculated the amplification efficiency for\nthese two primer pairs using the slopes of their corresponding standard curves. As shown in Figure R9a,b,\nthe qPCR primer efficiency for USP10 was similar to that for DTX3L (T387 GSCs: USP10\nefficiency=96.1%, DTX3L efficiency=97.9%; H2S GSCs: USP10 efficiency=95.3%, DTX3L efficiency=\n96.4%). Therefore, these two primer pairs were used to compare USP10 and DTX3L expression levels by\nqPCR.\nFigure R9. a,b, cDNA samples were prepared from GSCs (T387, a, or H2S, b) and subjected to 5-fold\nserially dilution for qPCR amplification. The amplification efficiency for each primer pair was determined by\nplotting the threshold cycle (Ct) values against the logarithm of the cDNA concentrations and calculating\nthe slope. Standard curves were generated from five dilution points for each primer pair.\nReviewer #2: 4. Fig. 3i: In the H&E-stained coronal sections with indicated GSC tumor xenografts, no\ntumor cells or areas were observed in the shUSP10 groups in these two GSC xenograft models. Is this\nresult representative? From the survival data and IF\nstaining in Fig. 3j, there should be tumor xenograft mass in the coronal brain sections in the shUSP10 groups. Were these\nthree each H&E-stained brain sections from the mice that were euthanized at similar time points? The authors should also\nconfirm USP10 knockdown and decreased SATB2 expression in the brain slices. The same concern applies to\nResponse: We thank the reviewer for raising this important issue. For the H&E experiments, mice were\neuthanized at consistent time point for each experiment, with representative H&E images presented in the\nprevious versions of the figures. Specifically, in Figs. 3i, 4k, 6k,l, and Fig. S9c,d, mouse brains were\ncollected at day 28 (T387 GSCs) or day 22 (H2S GSCs) after intracranial transplantation with GSCs.\nAlthough no obvious tumor areas were observed in the shUSP10 or DTX3L-Flag groups, this does not\nimply that there were no tumor cells in the brains of mice transplanted with GSCs expressing shUSP10 or\nDTX3L-Flag.GSCs expressing shUSP10 or DTX3L-Flag proliferate slowly and require extended periods to\nform tumors in mouse brains. Consequently, mice bearing xenografts from GSCs expressing shUSP10 or\nDTX3L-Flag ultimately succumbed, albeit after a prolonged period compared to controls (Figs. 3h, 4j, 6i,j,\nand Fig. S9a,b). The collection time of the mouse brains has been described in the corresponding figure\nlegends in our previous manuscript.\nWe agree with the reviewer that it is important to determine the expression of USP10, SATB2 and DTX3L\nin brain slices. Given that USP10-silencing or DTX3L-overexpressing GSCs exhibited significantly slower\ntumor growth compared to control GSCs, it was very difficult to collect tumors derived from the\nUSP10-silencing/DTX3L-overexpressing GSCs and the control GSCs at the same time point. Therefore,\nwe typically collected the brains bearing GBM xenografts from mice when neurological signs appeared.\nThis endpoint typically occurred 2-3 days prior to mortality, by which time tumors had reached substantial\nsize. The collection time of the mouse brains has been described in the corresponding figure legends in\nour revised manuscript. Subsequently, we performed immunofluorescent staining of USP10 and SATB2 in\nGBM xenografts and found that the expression levels of USP10 and SATB2 were significantly decreased\nin xenografts derived from USP10-silencing GSCs (Please see Figure R10a,b below). Additionally,\nimmunofluorescent staining revealed that DTX3L expression was elevated and SATB2 levels were\nreduced in xenografts derived from DTX3L-overexpressing GSCs (Please see Figure R10c,d below).\nGSCs expressing shNT or shUSP10. GBM xenografts were collected from mice when neurological signs occur after GSC transplantation. Scale bar, 50 μm. b, Quantification of USP10 or SATB2 intensity in\nxenografts derived from GSCs expressing shNT or shUSP10. n=5 tumors per group. c,\nImmunofluorescence of DTX3L (green) or SATB2 (red) in tumor xenografts derived from GSCs expressing\nvector control or DTX3L-Flag. GBM xenografts were collected from mice when neurological signs occur\nafter GSC transplantation. Scale bar, 50 μm. d, Quantification of DTX3L or SATB2 intensity in\nxenografts derived from GSCs expressing vector control or DTX3L-Flag. n=5 tumors per group.\nData are represented as mean ± s.d. ****p<0.0001, one-way ANOVA analysis followed by Tukey’s test (b)\nand two-tailed unpaired t-test (d).\nWe have added the new data in Supplementary Fig. 4a,b and 6a,b and described the results in our revised\nmanuscript at Lines 216 and 245: “Furthermore, immunofluorescent staining confirmed a marked\nreduction in USP10 and SATB2 expression in xenografts expressing shUSP10.” and “Immunofluorescent\n16\nanalysis further confirmed that DTX3L expression was elevated and SATB2 levels were reduced in\nxenografts derived from DTX3L-overexpressing GSCs.”\nReviewer #2: 5. The authors reported decreased SOX2+ tumor cells in xenografts following USP10\nknockdown, DTX3L overexpression, or 5-WU treatment. Did they observe an increase in differentiated\ncells by IF staining of some neuronal or astrocyte markers? What is the potential mechanism? Additionally,\ndid SOX2 expression change after these treatments in vitro?\nResponse: We thank the reviewer for raising the issue. As suggested, we performed immunofluorescent\nstaining for neuronal markers (MAP2, TUBB3) and the astrocyte marker (GFAP) in GSC-derived GBM\nxenografts. Our results demonstrated that either USP10 disruption or DTX3L overexpression significantly\nincreased the population of MAP2-, TUBB3-, and GFAP-positive cells in the xenografts (Please see Figure\nR11a-h below).\n(T387, a, or H2S, b) expressing shNT or shUSP10. GBM xenografts were collected from mice when\nneurological signs occur after GSC transplantation. Scale bar, 50 μm. c, d, Quantification of MAP2+,\nTUBB3+, and GFAP+ cells in xenografts derived from GSCs (T387, c, or H2S, d) expressing shNT or\nshUSP10. n=5 tumors per group. e, f, Immunofluorescence of MAP2, TUBB3, or GFAP in tumor\nxenografts derived from GSCs (T387, e, or H2S, f) expressing vector control or DTX3L-Flag. GBM\nxenografts were collected from mice when neurological signs occur after GSC transplantation. Scale bar,\n50 μm. g, h, Quantification of MAP2+, TUBB3+, and GFAP+ cells in xenografts derived from GSCs (T387,\ng, or H2S, h) expressing vector control or DTX3L-Flag. n=5 tumors per group.\nData are represented as mean ± s.d. ****p<0.0001, one-way ANOVA analysis followed by Tukey’s test (c\nand d), two-tailed unpaired t-test (g and h).\nSimilarly, Wu-5 treatment markedly increased MAP2-, TUBB3-, and GFAP-positive cells in GBM\nxenografts (Please see Figure R12a-d below). Furthermore, all three interventions (USP10 disruption,\nDTX3L overexpression, or Wu-5 treatment) consistently reduced SOX2 expression in GSCs (Please see\nFigure R12e-g below). Collectively, these data indicate that USP10 inhibition, DTX3L upregulation, or\nWu-5 treatment effectively induces GSC differentiation.\nSOX2 is known to play a critical role in maintaining GSC stemness, and its downregulation has been\nshown to promote GSC differentiation12, 13. Our previous work showed that silencing SATB2 reduces SOX2\nprotein levels in GSCs14. Therefore, we speculate that USP10 inhibition or DTX3L overexpression may\npromote GSC differentiation by reducing SATB2 protein levels, although the underlying mechanism\nwarrants further investigation.\nxenografts from mice treated with Wu-5 or the vehicle control. GBM xenografts were collected from mice\nwhen neurological signs occur after GSC transplantation. Scale bar, 50 μm. c, d, Quantification of MAP2+,\nTUBB3+, and GFAP+ cells in GSC (T387, c, or H2S, d)-derived xenografts from mice treated with Wu-5 or\nthe vehicle control. n=5 tumors per group. e, Immunoblot (IB) analysis of SOX2 and USP10 expression in\nGSCs transduced with shNT or shUSP10. f, IB analysis of SOX2, DTX3L, and Flag expression in GSCs\ntransduced with vector control or DTX3L-Flag. g, IB analysis of SOX2 expression in GSCs treated with the\nindicated doses of Wu-5 or the vehicle control for 24 hours.\nData are shown as mean ± s.d. ****p<0.0001, two-tailed unpaired t-test (c and d).\nWe have added the new data in Supplementary Fig. 2j, 4a,b, 5d, 6a,b, 11a, and 12c-f and described the\nresults in our revised manuscript at Lines 194, 212, 230, 243, 375, and 397: “Consistently, silencing\nneuronal markers (MAP2 and TUBB3) and the astrocyte marker (GFAP) revealed that disruption of USP10\nsignificantly increased the number of MAP2-, TUBB3-, and GFAP-positive cells. This finding demonstrates\nthat USP10 disruption promotes the differentiation of GSCs in GBM xenografts.”, “Consistently, DTX3L\noverexpression also decreased the expression of SOX2.”, “Furthermore, DTX3L overexpression markedly\npromoted the differentiation of GSCs in GBM xenografts, as indicated by immunofluorescence of MAP2,\nTUBB3, and GFAP.”, “Wu-5 treatment also impaired GSC self-renewal and downregulated SOX2\nexpression in a dose-dependent manner (8 and 16 μM).”, and “Moreover, immunofluorescent staining for\nMAP2, TUBB3, and GFAP revealed that Wu-5 robustly promoted GSC differentiation in GBM xenografts.”.\nThese results are further discussed in the “Discussion” section at Line 445 on Page 20: “SOX2 is known to\nplay a critical role in maintaining GSC stemness……although the underlying mechanism warrants further\ninvestigation.”.\nReviewer #2: 6. An in vitro ubiquitination assay using recombinant proteins and ubiquitin should be\nperformed to confirm USP10 and DTX3L as the deubiquitinase and E3 ligase for SATB2, respectively.\nResponse: We thank the reviewer for the important point. As suggested, we performed in vitro\nubiquitination assays. Our results demonstrated that DTX3L directly promoted the ubiquitination of SATB2\n(Please see Figure R13a below), whereas USP10 antagonized DTX3L-mediated SATB2 ubiquitination\n(Please see Figure R13b below).\nFigure R13. a, His-SATB2 was co-incubated with His-DTX3L, along with E1 (His/GST-UBA1), E2\n(GST-UBE2D1), and Ub (His-ubiquitin) at 37 °C for 2 hours, followed by IB analysis. b, His-SATB2 was\nco-incubated with His-DTX3L and GST-USP10, along with E1 (His/GST-UBA1), E2 (GST-UBE2D1), and\nUb (His-ubiquitin) at 37 °C for 2 hours, followed by IB analysis.\nmanuscript at Line 290 on Page 13: “In vitro ubiquitination assay demonstrated that DTX3L directly\npromoted the ubiquitination of SATB2, whereas USP10 antagonized DTX3L-mediated SATB2\nubiquitination.”. The corresponding experimental procedure is detailed in the Methods section (Line 666,\nPage 29).\nReviewer #2: 7. According to the DepMap Portal, USP10 is categorized as a common essential gene. Do\nthe authors have evidence that GSCs are more dependent on USP10 compared to differentiated\ncounterparts or normal cells?\nResponse: We thank the reviewer for the important question. We examined the effect of USP10 disruption\non the growth of non-stem tumor cells (NSTCs) and found that USP10 disruption had little effect on NSTC\ngrowth (Please see Figure R14a, b below). Consistently, USP10 is preferentially expressed in GSCs\nrelative to matched NSTCs (Figure 2a). Therefore, USP10 is critically required for GSC maintenance,\nwhereas its disruption has a minor impact on NSTCs.\nFigure R14. a, IB analysis of USP10 expression in NSTCs transduced with shNT or shUSP10. b, Cell\nviability assay of NSTCs transduced with shNT or shUSP10. n= 4 biological independent samples.\nData are shown as mean ± s.d. Two-tailed unpaired t-test (b).\nWe have added the new data in Supplementary Fig. 2h,i and described the results in our revised\nmanuscript at Line 191 on Page 9: “In contrast, silencing USP10 had little effect on the growth of NSTCs.”.\nReviewer #2: 8. Fig 6, do the authors have data showing that 5-WU can penetrate the blood-brain barrier?\nThey should also demonstrate a decrease in SATB2 and USP10 levels in 5-WU-treated tumors.\nResponse: We thank the reviewer for raising these important issues. To determine whether Wu-5 can\npenetrate the blood-brain barrier, we intraperitoneally administered Wu-5 or DMSO to BALB/c nude mice\nand subsequently detected Wu-5 in mouse brain tissues by LC-MS/MS. The results demonstrated that\nhours, whereas Wu-5 levels were extremely low and nearly undetectable in the brains of DMSO-treated\ncontrols (Please see Figure R15a below). We also examined the expression of SATB2 and USP10 in GBM\nxenografts of mice treated with Wu-5. Immunofluorescent staining revealed that Wu-5 treatment reduced\nSATB2 and USP10 in GBM xenografts (Please see Figure R15b-e below).\nFigure R15. a, BALB/c nude mice were intraperitoneally injected with Wu-5 (7.5 mg/kg) and euthanized\nafter 2 hours. The concentrations of Wu-5 in mouse brain tissues were measured by LC-MS/MS. The\nrepresentative MS/MS spectrum of Wu-5 in mouse brain tissue is shown on the left. The Concentrations of\nWu-5 in mouse brain tissues are shown on the right. n=5 mice per group. b, c, Immunofluorescence of\nSATB2 (red) or USP10 (green) in GSC (T387, b, or H2S, c)-derived xenografts from mice treated with\nWu-5 or the vehicle control. GBM xenografts were collected from mice when neurological signs occur after\nGSC transplantation. Scale bar, 50 μm. d, e, Quantification of SATB2 and USP10 intensity in GSC (T387,\nd, or H2S, e)-derived xenografts from mice treated with Wu-5 or the vehicle control. n=5 tumors per group.\nData are represented as mean ± s.d. ****p<0.0001, two-tailed unpaired t-test (a, d, and e).\nmanuscript at Line 399 on Page 18: “Additionally, Wu-5 treatment reduced SATB2 and USP10 in\nxenografts. To confirm whether Wu-5 can penetrate the blood-brain barrier……whereas Wu-5 levels were\nextremely low and nearly undetectable in the brains of DMSO-treated controls.”.\nReviewer #2: 9. Can SATB2 overexpression rescue the effects of 5-WU treatment? In Fig 6, the authors\nhave already established ectopic expression of SATB2 GSC lines for this validation experiment.\nResponse: We thank the reviewer for the important question. To determine whether SATB2\noverexpression can rescue the effects of Wu-5 treatment on GSC maintenance, we transduced GSCs with\nSATB2-Myc and treated them with Wu-5 (16 μM). IB analysis showed that ectopic SATB2 expression\nrestored SATB2 to endogenous levels, which had been reduced by Wu-5 treatment (Please see Figure\nR16a below). Importantly, restoring SATB2 to endogenous levels significantly rescued the Wu-5-induced\nimpairment of GSC proliferation and self-renewal (Please see Figure R16b-f below). These data indicate\nthat Wu-5 treatment inhibits GSC maintenance via the USP10-SATB2 axis.\ncontrol followed by treatment with Wu-5 (16 μM) or vehicle for 48 hours. b, Cell viability assay of GSCs\ntransduced with SATB2-Myc or vector control followed by treatment with Wu-5 (16 μM) or vehicle at the\nindicated time points. n=4 biological independent samples. c, Tumorsphere images of GSCs transduced\nwith SATB2-Myc or vector control followed by treatment with Wu-5 (16 μM) or vehicle for 6 days. Scale bar,\n100 μm. d, e, Quantification of the number (d) and diameter (e) of tumorspheres formed by GSCs\nexpressing SATB2-Myc or vector control after treatment with Wu-5 (16 μM) or vehicle for 6 days. n=4\nbiological independent cell cultures. f, In vitro limiting dilution analysis of tumorsphere formation of GSCs\nexpressing SATB2-Myc or vector control treated with Wu-5 (16 μM) or vehicle.\nData are shown as mean ± s.d. ****p<0.0001, two-way ANOVA analysis followed by Tukey’s test (b),\none-way ANOVA analysis followed by Tukey's test (d and e), and ELDA analysis for differences in stem\ncell frequencies (f).\nWe have added the new data in Supplementary Fig. 11e-j and described the results in our revised\nmanuscript at Line 386 on Page 17: “To determine whether SATB2 overexpression can rescue the effects\nof Wu-5 treatment on GSC maintenance……These data indicate that Wu-5 treatment inhibits GSC\nmaintenance via the USP10-SATB2 axis.”.\nReviewer #2: 10. Although the authors stated in the Methods section that they used both male and female\nanimals, they did not stratify their analysis by sex or mention whether similar results were observed in both\nsexes.\nResponse: We thank the reviewer for raising this important point. Our study design did not incorporate\nsex as a biological variable, as investigating potential sex-specific effects was outside the scope of this\nresearch. We have added this information to the Methods section (Line 548 on Page 24). Both male and\nfemale mice were included in the study to enhance generalizability to the broader population. We fully\nacknowledge the importance of sex as a biological variable. However, the specific hypotheses driving this\nstudy were focused on the mechanisms underlying aberrant SATB2 expression, and our analysis plan was\ndesigned accordingly. We are confident that the conclusions drawn from the aggregated data remain valid\nand robustly address the primary research questions of this study.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: Thank you for your participation and valuable contributions to the manuscript review.\nSATB2 is a transcription factor shown to play a role in many types of cancer. In glioblastoma, SATB2 is\noverexpressed compared to low-grade gliomas; however, its role in GBM development and progression is\npoorly understood. In the manuscript titled USP10 promotes glioma stem cell maintenance and\nglioblastoma growth by antagonizing DTX3L-mediated SATB2 ubiquitination, the authors identify the\ndeubiquitinase USP10 and the E3 ligase DTX3L as key mediators of SATB2 stability. Using an IP strategy,\nthe authors show that USP10 and DTX3L directly interact with SATB2.The authors demonstrate that\nUSP10 and DTX3L are preferentially expressed in GBM GSCs, where USP10 can reverse\nDTX3L-mediated ubiquitination of SATB2 and rescue it from proteasomal degradation. Furthermore,\nshRNA-mediated knockout of USP10 inhibits GSC proliferation, reduces tumor growth, and significantly\nextends survival time in a xenograft model. Conversely, forced overexpression of DTX3L inhibits GSC\nformation and proliferation, as well as the growth of GBM tumors in vivo. Taken together, the authors\neffectively show that USP10 and DTX3L regulate SATB2 stability and GBM proliferation.The mechanism\nof SATB2 regulation was subsequently shown to be due to K48-mediated ubiquitination at a conserved\nlysine site (K266), leading to degradation. Building upon this, the authors utilize a novel inhibitor of USP10\nto induce SATB2 ubiquitination and degradation. Treatment of tumor-bearing xenografts with this inhibitor\nresulted in a reduction in tumor burden and an increase in survival time. The authors propose a model in\nwhich USP10 overexpression in GBM rescues SATB2, whereas USP10 inhibition promotes SATB2\ndegradation, ultimately reducing GBM growth.\nOverall, this is a well-constructed study that strongly supports the authors' model. They follow a stepwise\nand logical approach in confirming their hypothesis, and the data presented is of very high quality. The\npaper is well-written, will be of broad interest, and I commend the authors on their work.\nResponse: We are very grateful for the positive comments from the reviewer. We also greatly appreciate\nthe helpful suggestions provided by the reviewer. We have revised our manuscript according to the\nreviewer’s suggestions.\nReviewer #4: 1. In Figure 8, the authors show that WU5 can inhibit GBM growth. Do the authors know if\nWU5 induces apoptosis?\nResponse: We thank the reviewer for raising this important question. To address the issue, we examined\nthe levels of apoptotic markers Cleaved Caspase-7 and Cleaved PARP following Wu-5 treatment. Wu-5\ndose-dependently induced GSC apoptosis, as demonstrated by the elevated levels of Cleaved Caspase-7\n26\nand Cleaved PARP (Please see Figure R17 below).\nFigure R17. Immunoblot (IB) analysis of Cleaved Caspase-7 and Cleaved PARP levels in GSCs treated\nwith the indicated doses of Wu-5 or the vehicle control for 24 hours.\nWe have added the new data in Supplementary Fig. 11b and described the results in our revised\nmanuscript at Line 377 on Page 17: “Furthermore, Wu-5 (8 and 16 μM) dose-dependently induced GSC\napoptosis…… apoptotic markers Cleaved Caspase-7 and Cleaved PARP.”.\nReviewer #4: 2. Sticking with Figure 8, the authors show that WU5 effectively induces ubiquitination of\nSATB2, as expected. However, the observed reduction in USP10 is potentially interesting. The authors\nmention that this was observed in a previous study, but the mechanism remains unclear. Since MG132 is\nadded to the media, proteasomal degradation cannot be the cause. Do the authors know if WU5 affects\nUSP10 transcription? Is SATB2 a transcriptional activator of USP10? Is USP10 also ubiquitinated by\nDTX3L? It would be interesting to hear the authors’ perspective.\nResponse: We thank the reviewer for these valuable suggestions. To evaluate whether Wu-5 influences\nUSP10 transcription, we performed qPCR and found that Wu-5 treatment did not alter USP10 mRNA\nlevels (Please see Figure R18a below). Next, we investigated whether SATB2 acts as a transcriptional\nactivator of USP10. Disruption of SATB2 in GSCs had no effect on USP10 mRNA and protein levels\n(Please see Figure R18b,c below). Consistent with our previous findings showing that DTX3L\noverexpression had no significant effect on USP10 expression (Supplementary Figure 5a,b), further\nexperiments revealed that DTX3L overexpression did not promote USP10 ubiquitination in GSCs (Please\nsee Figure R18d below).\nthe vehicle control. n=3 biological independent samples. b, qPCR analysis of USP10 and SATB2 mRNA\nexpression in GSCs transduced with shNT or shSATB2. n=3 biological independent samples. c, IB\nanalysis of USP10 and SATB2 expression in GSCs transduced with shNT or shSATB2. d, GSCs were\ntransduced with vector control or DTX3L-Flag, and then cell lysates were subjected to IP with USP10\nantibody or IgG followed by IB with Ub antibody. Cells were treated with MG-132 (20 µM) for 6 hours\nbefore harvesting.\nData are represented as mean ± s.d. ****p<0.0001, one-way ANOVA analysis followed by Tukey’s test (a\nand b).\nWe have added the new data in Supplementary Fig. 2a,b, 5c, and 11d and described the results in our\nrevised manuscript at Lines 180, 224, and 383: “In contrast, SATB2 disruption did not alter USP10 mRNA\nor protein levels.”, “In addition, the overexpression of DTX3L led to a reduction in FOXM1 expression,\nwhereas it had no significant impact on USP10 expression and ubiquitination.”, and “Nonetheless,\ntreatment with 8 and 16 μM Wu-5 had no impact on SATB2 and USP10 mRNA levels.”.\nReviewer #4: 3. Minor typo on line 119: It should read \"We first checked SATB2 protein levels.\"\nResponse: We thank the reviewer for pointing this out. The tense has been corrected (Line 119, Page 6),\nand we have carefully reviewed the manuscript to ensure proper tense usage throughout.\n1. Dai, W.W. et al. Overcoming therapeutic resistance in oncolytic herpes virotherapy by targeting\nIGF2BP3-induced NETosis in malignant glioma. Nat Commun 15,131 (2024).\n2. Sun, X.X. et al. Loss of the receptors ER, PR and HER2 promotes USP15-dependent stabilization of\nPARP1 in triple-negative breast cancer. Nat Cancer 4, 716-733 (2023).\n3. Tang, J.N. et al. Targeting USP8 Inhibits O-GlcNAcylation of SLC7A11 to Promote Ferroptosis of\nHepatocellular Carcinoma via Stabilization of OGT. Adv Sci 10, e2302953 (2023).\n4. Xie, P. et al. Neddylation of PTEN regulates its nuclear import and promotes tumor development. Cell\nRes 31, 291-311 (2021).\n5. Zhu, G.X., Herlyn, M. & Yang, X.L. TRIM15 and CYLD regulate ERK activation via lysine-63-linked\npolyubiquitination. Nat Cell Biol 23, 978-991 (2021).\n6. Zhang, L. et al. USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-β type I\nreceptor. Nat Cell Biol 14, 717-726 (2012).\n7. Yu, M. et al. Wu-5, a novel USP10 inhibitor, enhances crenolanib-induced FLT3-ITD-positive AML cell\ndeath via inhibiting FLT3 and AMPK pathways. Acta Pharmacol Sin 42, 604-612 (2021).\n8. Zeng, L.D. et al. Tuning Immune-Cold Tumor by Suppressing USP10/B7-H4 Proteolytic Axis\nReinvigorates Therapeutic Efficacy of ADCs. Adv Sci 11, e2400757 (2024).\n9. Wu, X.J. et al. Threonine fuels glioblastoma through YRDC-mediated codon-biased translational\nreprogramming. Nat Cancer 5, 1024-1044 (2024).\n10. Bhaduri, A. et al. Outer Radial Glia-like Cancer Stem Cells Contribute to Heterogeneity of Glioblastoma.\nCell Stem Cell 26, 48-63 (2020).\n11. Lu, C.F. et al. Combined targeting of glioblastoma stem cells of different cellular states disrupts\nmalignant progression. Nat Commun 16, 2974 (2025).\n12. Abdelrahman, A.E., Ibrahim, H.M., Elsebai, E.A., Ismail, E.I. & Elmesallamy, W. The clinicopathological\nsignificance of CD133 and Sox2 in astrocytic glioma. Cancer Biomark 23, 391-403 (2018).\n13. Fang, X.G. et al. Inhibiting DNA-PK induces glioma stem cell differentiation and sensitizes\nglioblastoma to radiation in mice. Sci Transl Med 13, eabc7275 (2021).\n14. Tao, W.W. et al. SATB2 drives glioblastoma growth by recruiting CBP to promote FOXM1 expression in\nglioma stem cells. EMBO Mol Med 12, e12291 (2020).\n29"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the points I raised in my previous review. I have no further concerns, and the\nmanuscript is ready for publication.", "reviewer_2": "The authors have thoroughly addressed all my previous comments with extensive new data and detailed\nclarifications. The manuscript has been significantly improved in both quality and clarity. I have no further\nconcerns.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.", "reviewer_4": "I have no additional comments. The authors have done a good job of addressing my queries."}, "author_response": "Responses to Reviewers’ Comments\nRe: NCOMMS-24-81157A\nREVIEWERS' COMMENTS:\nReviewer #1 (Remarks to the Author):\nThe authors have addressed the points I raised in my previous review. I have no further concerns, and the\nmanuscript is ready for publication.\nResponse: We are grateful for the reviewer’s positive comments.\nReviewer #2 (Remarks to the Author):\nThe authors have thoroughly addressed all my previous comments with extensive new data and detailed\nclarifications. The manuscript has been significantly improved in both quality and clarity. I have no further\nconcerns.\nResponse: We appreciate the reviewer's positive comments.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: Thank you for your participation and valuable contributions to the manuscript review.\nReviewer #4 (Remarks to the Author):\nI have no additional comments. The authors have done a good job of addressing my queries.\nResponse: We sincerely appreciate the reviewer's positive feedback."}]

Decision letter