Targeting chaperone-mediated autophagy inhibits properties of glioblastoma stem cells and restores anti-tumor immunity

Abstract

Chaperone-mediated autophagy (CMA) is a selective autophagic process essential for maintaining cellular quality and responding to stress. Dysregulation of the CMA pathway is increasingly recognized in various cancers, yet the mechanisms behind CMA hyperactivation in cancer cells remain unclear. Here, we show that CMA is upregulated in patient-derived glioblastoma stem cells (GSCs), indicated by a significant increase in the lysosomal abundance of the CMA receptor, lysosome-associated membrane protein 2 A (LAMP2A). This increase results from MST4-mediated phosphorylation of LAMP2A, enhancing its stability and promoting homotrimer formation while inhibiting degradation by Cathepsin A. CMA supports GSC proliferation and self-renewal by activating mTORC1 through the selective degradation of its negative regulators, TSC1 and TSC2. Additionally, CMA is involved in epigenetic silencing of the cGAS-STING pathway, promoting tumor immune escape via lysosomal degradation of the DNA demethylase TET3. Inhibition of CMA synergizes with immune checkpoint therapy in glioblastoma models, highlighting a potential therapeutic target.

Fig. 1. The elevated CMA activity in GSCs correlates with lysosomal LAMP2A abundance.

a Representative images (left) and quantitative analysis (right) of CMA flux, indicated by the number of KFERQ puncta, in GSC and non-GSC cells stably expressing PAmCherry-KFERQ or PAmCherry-KFSDA (a mutant KFERQ sequence that cannot be recognized by HSC70). n = 40 randomly selected cells. Scale bar, 10 μm. b In vitro binding and uptake assay of GAPDH by the lysosome in GSC and non-GSC cells. n = 3 independent experiments. c Immunoblotting (IB) analyses for indicated proteins after treating with 10 μM MG132, 5 mM 3-MA, or LN (10 μM leupeptin and 20 mM NH₄Cl) in GSC cells (M83 and 23) and non-GSC cells (LN229 and U251), respectively. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin.n = 3 independent experiments. d Representative images (left) and quantification (right) of CMA activity detection by KFERQ puncta numbers in GSCs (M83 and 456) and their corresponding DGCs. n = 40 randomly selected cells. Scale bar, 10 μm. e IB analyses for LAMP2A and CMA-associated chaperone proteins (HSC70, HSP40 and HSP90) in lysosomes isolated from the indicated GSCs and DGCs, with LAMP1 serving as a lysosomal marker. f Sphere-forming frequency of GSC M83 and 456 cells with indicated modifications. sh-C, control for knock down. Vec, a control vector. WT, wide-type. Representative H&E images of mouse brain sections (g) and quantitative analysis of tumor volume (h) from athymic (BALB/c Nude) mice intracranially implanted with the GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. i Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 4 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (a, b, d), two-sided likelihood ratio test (f), one-way ANOVA with Dunnett’s multiple comparisons test (h) or log-rank test (i). ns, not significant. Source data are provided as a Source Data file.

Fig. 2. MST4 enhances the lysosomal abundance of the CMA regulator LAMP2A.

Assessment of LAMP2A stability in lysosomes isolated from indicated GSCs and DGCs (a), as well as GSC M83 cells expressing sh-C or sh-MST4 (h). Lysosomes were incubated at 0 °C for 10 min with or without protease inhibitor cocktail (PI) to inhibit lysosomal protease activity, followed by pelleting at indicated time points, and subsequent IB analysis for LAMP2A and LAMP1 densitometric quantification. b, i Densitometric quantification of LAMP2A protein level is shown. n = 3 independent experiments. c IP-IB analyses for indicated proteins in GSCs and DGCs. WCL, whole-cell lysates. Immunoglobulin G (IgG) was used as an isotype control. d A list of top LAMP2A-associated proteins identified through LAMP2A immunoprecipitation and mass spectrometric analysis. We marked the protein of interest in red font. e Identification of MST4 peptide among LAMP2A-interacting proteins precipitated from GSCs via mass spectrometry. y-ions generated from C-terminal (red), b-ions generated from N-terminal (blue), other fragments (black). f, j, l IP-IB analyses in GSC M83 cells with indicated modifications. g, m IP-IB analyses for indicated proteins in WCL and lysosomal fractions of GSCs with indicated modifications. β-actin and LAMP1 served as loading controls for WCL and lysosomal samples, respectively. k Purified lysosomal membranes from the indicated GSC M83 cells were analyzed using native continuous gel electrophoresis and subjected to IB detection for LAMP2A. n, o Representative images (n) and quantification (o) of CMA activity detection by KFERQ puncta numbers in GSC M83 and 456 cells with indicated modifications. n = 40 randomly selected cells. Scale bar, 10 μm. p In vitro binding and uptake assay of GAPDH by the lysosome in GSC M83 and 456 cells with indicated modifications. Protease inhibitor cocktail (PI) was used to inhibit lysosomal protease activity. n = 3 independent experiments. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using two-way ANOVA with Bonferroni’s multiple comparisons test (b, i) or one-way ANOVA with Dunnett’s multiple comparisons test (o, p). Source data are provided as a Source Data file.

Fig. 3. MST4 phosphorylates LAMP2A through direct interaction.

a Mass-spectrometric analysis of p-LAMP2A in HEK293T cells co-overexpressing Flag-LAMP2A and HA-MST4. Asterisk at the top, threonine 40, serine 97 and threonine 136 residues that were found phosphorylated determined by mass spectrometric analysis. y-ions generated from C-terminal (red), b-ions generated from N-terminal (blue), other fragments (black). b In vitro kinase assays utilizing wild-type (WT) MST4 with either LAMP2A-WT or indicated mutants as substrates, followed by SDS-PAGE analysis and IB detection with an anti-Flag antibody, anti-p-S/T antibody and veriBlot for IP Detection Reagent (HRP). c IP-IB analyses for the indicated proteins in HEK293T cells transduced with the indicated plasmids. d IP-IB analyses for the indicated proteins in GSC M83 cells expressing Myc-Cath A along with Flag-LAMP2A-WT or indicated mutants. e, f Cell proliferation (e) and sphere-forming frequency (f) of GSC M83 and 456 cells with indicated modifications. n = 3 independent experiments in (e). g, h Representative H&E images mouse brain sections (g) and quantitative analysis of tumor volume (h) from athymic (BALB/c Nude) mice intracranially implanted with GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. i Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 3 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (e), two-sided likelihood ratio test (f), one-way ANOVA with Dunnett’s multiple comparisons test (h) or log-rank test (i). ns, not significant. Source data are provided as a Source Data file.

Fig. 4. MST4-mediated phosphorylation of LAMP2A maintains CMA activity and GSC properties.

a, i IB analyses for indicated proteins in WCL and lysosomal fractions of GSC M83 cells (a) or GSC 23 cells (i) with indicated modifications. Representative images (left in b, or j) and quantification (right in b, or k) of CMA activity detection by KFERQ puncta numbers in GSC M83 cells (b) or GSC 23 cells (j-k) with indicated modifications. n = 40 randomly selected cells. Scale bar, 10 μm. c, l In vitro binding and uptake assay of GAPDH by the lysosome in GSC M83 cells (c) or GSC 23 cells (l) with indicated modifications. n = 3 independent experiments. d-e, m-n Cell proliferation (d, m) and sphere-forming frequency (e, n) of GSC M83 and 456 cells (d-e) and GSC 23 cells (m-n) with indicated modifications. n = 3 independent experiments. f, g Representative H&E images of mouse brain sections (f) and quantitative analysis of tumor volume (g) from athymic (BALB/c Nude) mice intracranially implanted with GSC M83 cells with indicated modifications. n = 5 mice per group. Scale bar, 1.0 mm. h Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 5 mice per group. o, p Representative H&E images of mouse brain sections (o) and quantitative analysis of tumor volume (p) from athymic (BALB/c Nude) mice intracranially implanted with GSC 23 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. q Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC 23 cells with indicated modifications. n = 5 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons test (b, c, d, g), two-sided likelihood ratio test (e, n), log-rank test (h, q) or one-way ANOVA with Tukey’s multiple comparisons test (k, l, m, p). ns, not significant. Source data are provided as a Source Data file.

Fig. 5. CMA regulates mTORC1 activity through lysosomal degradation of TSC1/2.

a, b Gene set enrichment analysis highlighted the main biological processes significantly altered due to LAMP2A KD in GSCs, derived from RNA-seq data (GSE181556, a) and proteomic data (PXD027069, b). These pathways of interest especially immune-related pathways are indicated in red. c IB analyses for indicated proteins in GSC M83 cells expressing sh-C, sh-MST4 or sh-LAMP2A. d, e IP-IB analyses for indicated proteins in HEK293T cells transduced with the indicated plasmids. f IB for TSC1 (top) and TSC2 (bottom) in HEK293T cells subjected to indicated modifications, subsequently treated with CHX (100 μg/mL) for the indicated time. g Quantification of TSC1 (left) and TSC2 (right) protein levels in HEK293T cells with indicated modifications and treatments. n = 3 independent experiments. h, i IB analyses for TSC1/2 protein levels in WCL and lysosomal fractions of GSC M83 cells, with or without LAMP2A KD (h) or HSC70 KD (i). j IB analyses for TSC1 and TSC2 in GSC M83 cells, either untreated or subjected to 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH₄Cl) for an additional 12 hours post-CHX (100 μg/mL) treatment for 12 h. k Representative H&E images of mouse brain sections (left) and quantification of tumor volume (right) from athymic (BALB/c Nude) mice intracranially implanted with GSC M83 cells with indicated modifications. n = 3 mice per group. Scale bar, 1.0 mm. l Kaplan-Meier survival curves of athymic (BALB/c Nude) mice intracranially transplanted with GSC M83 cells with indicated modifications. n = 5 mice per group. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-sided Fisher’s exact test (a, b), two-way ANOVA with Bonferroni’s multiple comparisons test (g), one-way ANOVA with Dunnett’s multiple comparisons test (k) or log-rank test (l). ns, not significant. Source data are provided as a Source Data file.

Fig. 6. CMA inhibition attenuates GBM tumorigenicity by augmenting anti-tumor immunity.

a Spearman correlation heatmap between CMA activity scores and immune infiltration levels in TCGA-GBM dataset. Color gradient represents correlation coefficients, with numerical values and statistical significance (p-values) annotated. GSC M83 cells with indicated modification were co-cultured for 24 hours with or without CD8⁺ T cells isolated from GBM patients’ peripheral blood. Flow cytometry quantified T cell-derived: TNF-α (b), IFN-γ (c), and GZMB (d) production. n = 3 independent experiments. MFI, mean fluorescence intensity. e IP-IB analyses for indicated proteins in GL261 and CT-2A cells expressing sh-C, sh-Mst4 or sh-Lamp2a. f Representative luciferase-based bioluminescence images of athymic (BALB/c Nude) mice (top) or C57BL/6 mice (bottom) intracranially transplanted with GL261 cells expressing sh-C, sh-Lamp2a or sh-Mst4. Colored scale bars represent photons/s/cm²/steradian. g Relative photon flux of athymic (BALB/c Nude) mice (left) or C57BL/6 mice (right) intracranially transplanted with GL261 cells with indicated modifications. n = 3 mice per group. h, i Representative H&E images of mouse brain sections (h) and quantification of tumor volume (i) from athymic (BALB/c Nude) mice or C57BL/6 mice intracranially implanted with GL261 cells expressing sh-C, sh-Lamp2a or sh-Mst4. n = 3 mice per group. Scale bar, 1.0 mm. j, k Kaplan-Meier survival curves of athymic (BALB/c Nude) mice (j) or C57BL/6 mice (k) intracranially transplanted with GL261 cells expressing sh-C, sh-Lamp2a or sh-Mst4. n = 5 mice per group. l Scheme representing the experimental procedure, created with BioRender.com. m Representative luciferase-based bioluminescence images of C57BL/6 mice intracranially transplanted with CT-2A cells with indicated modifications and treatments. Colored scale bars represent photons/s/cm²/steradian. n Kaplan-Meier survival curves of C57BL/6 mice intracranially transplanted with CT-2A cells with indicated modifications and treatments. n = 8 mice per group. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons test (b–d, g, i) or log-rank test (j, k, n). Source data are provided as a Source Data file.

Fig. 7. CMA regulates the cGAS/STING signaling pathway through targeting TET3.

GSEA enrichment plots for the cGAS/STING signaling pathway (a) and the interferon α/β signaling cascade (b) in GSCs lacking LAMP2A. c, d Impact of IR (6 Gy) on the activation of STING/TBK1 in GSC M83 cells, both with and without LAMP2A KD (c) or MST4 KD (d). e, f Quantitative real-time PCR (qRT-PCR) analysis of mRNA levels of CGAS (e) and STING (f) in GSC M83 cells transduced with sh-C, sh-MST4 or sh-LAMP2A. n = 3 independent experiments. Analysis of the relationship between mRNA levels and methylation status of CGAS (g) and STING (h) in GBM samples derived from TCGA datasets. i IB analyses for cGAS and STING in HEK293T cells transfected with Vec, TET1, TET2 or TET3. Band intensities of indicated proteins were quantified using Image J, normalized to β-actin. j IB analyses for indicated proteins in HEK293T cells with indicated modifications. k IP-IB for indicated proteins in HEK293T cells overexpressing Myc-TET3 wild-type (WT) or mutant variant (Mut). l IB analysis for TET3 in HEK293T cells overexpressing Myc-TET3-WT or -Mut, following treatment with CHX (100 μg/mL) for indicated time. m Quantification of TET3 protein level in HEK293T cells with indicated modifications and treatments. n = 3 independent experiments. IB analyses for indicated proteins in WCL and lysosomal fractions of GSC M83 cells with or without LAMP2A KD (n) or HSC70 KD (o). p IB analysis for TET3 in GSC M83 cells, either untreated or subjected to 10 μM MG132, 5 mM 3-MA or LN (10 μM leupeptin and 20 mM NH₄Cl) for an additional 12 hours post-CHX (100 μg/mL) treatment for 12 h. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using two-sided Kolmogorov-Smirnov test (a, b), one-way ANOVA with Dunnett’s multiple comparisons test (e, f), two-sided Pearson correlation test (g, h) or two-way ANOVA with Bonferroni’s multiple comparisons test (m). Source data are provided as a Source Data file.

Fig. 8. MST4 phosphorylation of LAMP2A suppresses cGAS-STING signaling by downregulating TET3 expression.

a, c IB analyses for indicated proteins in GSC M83 and 456 cells subjected to the indicated modifications after 6 Gy IR treatment. b, d qRT-PCR analysis of IFNB1 mRNA level in GSC M83 and 456 cells with indicated modifications after 6 Gy IR exposure. n = 3 independent experiments. IB analyses for indicated proteins in GSC M83 (e) and 456 (f) cells with or without TET3 KD, Polyphyllin D (PPD, 3.2 μM) treatment or 6 Gy IR exposure. g, i IB analyses for indicated proteins in GSC M83 (g) and 23 (i) cells with indicated modifications, with or without 6 Gy IR exposure. qRT-PCR analysis of IFNB1 mRNA level in GSC M83 (h) and 23 (j) cells with indicated modifications following 6 Gy IR exposure. n = 3 independent experiments. k The upper panel shows representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially implanted GL261 cells under specified conditions. The colored scale bars indicate photons/s/cm²/steradian. The lower panel displays representative IF staining images for CD8⁺ cells in tumor tissues, with a scale bar of 100 μm. l Kaplan-Meier survival curves of C57BL/6 mice intracranially transplanted with GL261 cells with indicated modifications. n = 5 mice per group. m Quantitative analysis of CD8⁺ cells in tumor tissues from C57BL/6 mice with GL261 cells under specified conditions, based on 10 randomly selected microscopic fields. Data are presented as the means ± S.E.M. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons test (b, d, m), one-way ANOVA with Tukey’s multiple comparisons test (h, j) and log-rank test (l). Source data are provided as a Source Data file.

Fig. 9. Inhibition of CMA potentiates the therapeutic efficacy of IR and ICT.

a, f Scheme representing the experimental procedure, created with BioRender.com. b Representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially transplanted CT-2A cells with indicated modifications and treatments. Colored scale bars represent photons/s/cm²/steradian. c-e Kaplan-Meier survival curves of C57BL/6 mice with intracranially transplanted with CT-2A cells with modifications and treatments. n = 5 mice per group. g Representative luciferase-based bioluminescence images of C57BL/6 mice with intracranially transplanted CT-2A cells with indicated treatments. Colored scale bars represent photons/s/cm²/steradian. h Kaplan-Meier survival curves of C57BL/6 mice with intracranially transplanted with CT-2A cells with indicated treatments. n = 5 mice per group. i Representative IF staining images for CD8⁺ cell staining in tumor tissues (left) and the corresponding quantification of CD8⁺ cells in C57/BL6 mice with intracranial GL261 cells transplants under various treatments (right). Data are presented as the mean ± S.E.M. n = 10 randomly selected microscopic fields. Scale bar, 100 μm. All the experiments showed consistent results in at least three independent biological replicates. Statistical significance was assessed using log-rank test (c-e, h) or one-way ANOVA with Dunnett’s multiple comparisons test (i). ns, not significant. Source data are provided as a Source Data file.

Fig. 10. The prognostic significance of the correlative expression levels of MST4, p-LAMP2A, TSC1/2, and TET3 in clinical glioma.

a Representative immunohistochemical (IHC) staining images of phosphorylated LAMP2A (LAMP2A pT136), MST4, TET3, TSC1 and TSC2 in clinical glioma specimens. Scale bar, 50 μm. b Scoring of IHC staining of human glioma samples using the specified antibodies, followed by correlation analysis utilizing a two-tailed Pearson correlation test. n = 83 glioma samples. Note that some sample scores may overlap. c Kaplan-Meier survival analyses for glioma patients categorized by high or low expression levels of LAMP2A pT136, MST4, TET3, TSC1 and TSC2. n = 83 glioma samples. IHC score ≥ 4 was considered as high expression samples, IHC score ≤ 3 was considered as low expression samples. Statistical significance was assessed using log-rank test. d Schematic representation of the MST4-LAMP2A signaling axis in modulating mTORC1 and cGAS/STING pathways, as well as its impact on GBM tumorigenesis, created with BioRender.com. Source data are provided as a Source Data file.