Journal: Signal Transduction and Targeted Therapy

Article Title: Dual-targeting pharmacological UFMylation inhibition reprograms tumor and immune microenvironments to achieve long-term glioblastoma regression

doi: 10.1038/s41392-026-02819-w

Figure Lengend Snippet: Identification of Osimertinib and CP-24 as potent disruptors of the DDRGK1-UFL1 interaction. a Schematic representation of the experimental design. b Cherry-picked compounds from virtual screening. Molecules with docking scores < −7.5 and molecular weights < 600 Da were collected as the cherry-picked library for further NanoBRET screening. c High-throughput NanoBRET screening to identify compounds that inhibit the DDRGK1-UFL1 interaction. d Chemical structure of Osimertinib. e Chemical structure of CP-24. f Osimertinib inhibited the NanoBRET signal from the DDRGK1-UFL1 interaction in a dose-dependent manner ( N = 6). Data are presented as mean ± s.d. g CP-24 inhibited the NanoBRET signal from the DDRGK1-UFL1 interaction in a dose-dependent manner ( N = 3). Data are presented as mean ± s.d

Article Snippet: To quantify the effects of Osimertinib, CP-24, Afatinib, Erlotinib, Gefitinib, Tinodasertib, Tomiversotib, WZ4002, Temozolomide (MedChemExpress, HY-17364), and radiation (10 Gy) on GBM cell growth, untreated, TMZ-resistant, or radiation-treated GBM cells were seeded in opaque-walled 96-well plates at a density of 1000 cells per well.

Techniques: High Throughput Screening Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual-targeting pharmacological UFMylation inhibition reprograms tumor and immune microenvironments to achieve long-term glioblastoma regression

doi: 10.1038/s41392-026-02819-w

Figure Lengend Snippet: Binding of Osimertinib and CP-24 to DDRGK1. ITC analysis showing heat release upon Osimertinib ( a , b ) or CP-24 ( c , d ) binding to purified DDRGK1 Δ216–314 . ITC analysis showing heat release from purified UFL1 Δ1–212 binding to purified DDRGK1 Δ216–314 , which was pre-incubated with DMSO ( e , f ), Osimertinib ( g , h ), or CP-24 ( i , j ). co-IP showing interaction of DDRGK1 with UFL1 when cells were treated with DMSO, Osimertinib, or CP-24 ( k ), with quantification shown in ( l – n ). Statistical significance was determined using t -tests; N = 3. For m , Osimertinib vs . DMSO: p = 0.0313, CP-24 vs . DMSO: p = 0.0272. For n , Osimertinib vs . DMSO: p = 0.0206, CP-24 vs . DMSO: p = 0.0348. Other comparisons: ns. Data are presented as mean ± s.d. Molecular weight changes of DDRGK1 Δ216–314 after incubation with Osimertinib ( o ) or CP-24 ( p ) compared to DMSO. q Workflow for MS analysis to identify Osimertinib binding sites on DDRGK1 Δ216–314 . r MS analysis identified peptides generated by trypsin digestion of DDRGK1 Δ216–314 . Lysine (K) and Arginine (R) served as trypsin digestion sites. s – t Osimertinib binding sites on DDRGK1 Δ216–314 identified by MS. u Molecular docking model of Osimertinib binding to Ser226 of DDRGK1 Δ216–314 . v Molecular docking model of Osimertinib binding to Lys227 of DDRGK1 Δ216–314 . w Molecular docking model of CP-24 binding to DDRGK1 Δ216–314

Article Snippet: To quantify the effects of Osimertinib, CP-24, Afatinib, Erlotinib, Gefitinib, Tinodasertib, Tomiversotib, WZ4002, Temozolomide (MedChemExpress, HY-17364), and radiation (10 Gy) on GBM cell growth, untreated, TMZ-resistant, or radiation-treated GBM cells were seeded in opaque-walled 96-well plates at a density of 1000 cells per well.

Techniques: Binding Assay, Purification, Incubation, Co-Immunoprecipitation Assay, Molecular Weight, Generated

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual-targeting pharmacological UFMylation inhibition reprograms tumor and immune microenvironments to achieve long-term glioblastoma regression

doi: 10.1038/s41392-026-02819-w

Figure Lengend Snippet: Osimertinib and CP-24 inhibit ER-phagy and induce ER stress as potent UFMylation inhibitors. a – e Osimertinib and CP-24 dose-dependently decreased global UFMylation levels and DDRGK1 protein levels in A172, LN229, U251, and U87 cells. Data in ( b – e ) are presented as mean + s.d. f Schematic illustration showing the ER-phagy detection system. g Osimertinib and CP-24 significantly inhibited starvation-induced ER-phagy, as visualized by confocal imaging. Scale bar: 20 μm. h Quantification of ER-phagy puncta per cell using ImageJ software. Seven images from the control (DMEM + DMSO) group and eight images from each other group were randomly selected for quantification. Statistical significance was determined by one-sided t -test. EBSS + DMSO vs . DMEM + DMSO: p < 0.0001; EBSS + Osimertinib vs . EBSS + DMSO: p < 0.0001; EBSS + CP-24 vs . EBSS + DMSO: p = 0.004. Data are presented as mean ± s.d. i Osimertinib and CP-24 significantly inhibited starvation-induced ER-phagy as revealed by FACS analysis and analyzed by one-sided t -test ( N = 5). EBSS + DMSO vs . DMEM + DMSO: p < 0.0001; EBSS + Osimertinib vs . EBSS + DMSO: p = 0.0006; EBSS + CP-24 vs . EBSS + DMSO: p < 0.0001. Data are presented as mean ± s.d. j Osimertinib and CP-24 reduced the size of DDRGK1-UFL1 aggregation puncta and induced the formation of ER whorl-like structures. Arrows indicate ER whorl-like structures. Scale bar: 20 μm. k Average DDRGK1-UFL1 aggregation puncta size quantified using ImageJ. 6 images per group were analyzed by two-sided t tests. DMSO vs . Osimertinib: p < 0.0001, DMSO vs . CP-24: p = 0.0152. Data are presented as mean ± s.d. l Heatmap showing the upregulation of ER stress-related genes after Osimertinib and CP-24 treatment, as measured by bulk RNA-seq. The heatmap is colored according to z-score-normalized gene transcription levels. m qRT-PCR analysis of the transcription of ER stress-related genes after Osimertinib and CP-24 treatment in A172 cells. Data were analyzed by one-sided t -test ( N = 3). For HSPA5: A172 + Osimertinib vs . A172 + DMSO: p = 0.0003, A172 + CP-24 vs . A172 + DMSO: p < 0.0001; for sXBP1: A172 + Osimertinib vs . A172 + DMSO: p = 0.0098, A172 + CP-24 vs . A172 + DMSO: p < 0.0075; for CANX: A172 + Osimertinib vs . A172 + DMSO: p = 0.0003, A172 + CP-24 vs . A172 + DMSO: p < 0.0001; for ERN1: A172 + Osimertinib vs . A172 + DMSO: p = 0.019; for PERK: A172 + Osimertinib vs . A172 + DMSO: p = 0.0002, A172 + CP-24 vs . A172 + DMSO: p < 0.0001. Other comparisons: not significant (ns). Data are presented as mean ± s.d. n qRT-PCR analysis of the transcription of ER stress-related genes after Osimertinib and CP-24 treatment in U87 cells. Data were analyzed by one-sided t -test ( N = 3). For sXBP1: U87 + CP-24 vs . U87 + DMSO: p < 0.0001; for CANX: U87 + Osimertinib vs . U87 + DMSO: p = 0.0006, U87 + CP-24 vs . U87 + DMSO: p = 0.0003; for PERK: U87 + Osimertinib vs . U87 + DMSO: p = 0.0002, U87 + CP-24 vs . U87 + DMSO: p < 0.0001. Other comparisons: ns. Data are presented as mean ± s.d

Article Snippet: To quantify the effects of Osimertinib, CP-24, Afatinib, Erlotinib, Gefitinib, Tinodasertib, Tomiversotib, WZ4002, Temozolomide (MedChemExpress, HY-17364), and radiation (10 Gy) on GBM cell growth, untreated, TMZ-resistant, or radiation-treated GBM cells were seeded in opaque-walled 96-well plates at a density of 1000 cells per well.

Techniques: Imaging, Software, Control, RNA Sequencing, Quantitative RT-PCR

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual-targeting pharmacological UFMylation inhibition reprograms tumor and immune microenvironments to achieve long-term glioblastoma regression

doi: 10.1038/s41392-026-02819-w

Figure Lengend Snippet: Osimertinib and CP-24 exhibit pleiotropic anti-tumor effects in vitro. a DDRGK1 expression level negatively correlates with patient survival, as analyzed by log-rank test (data from the CGGA database). b DDRGK1 expression level negatively correlates with patient survival, as analyzed by log-rank test (data from the GEPIA database). c DDRGK1 expression level negatively correlates with patient survival, as analyzed by log-rank test (data from the TCGA database). d DDRGK1 expression level negatively correlates with patient survival in IDH wild type patients, as analyzed by log-rank test (data from the CGGA database). e Relationship between DDRGK1 expression level and patient survival in IDH mutant patients, as analyzed by log-rank test (data from the CGGA database). f DDRGK1 expression level positively correlates with glioma WHO malignancy grade, as analyzed by ANOVA test (data from the CGGA database). g DDRGK1 expression level positively correlates with patient age, as analyzed by two-sided t -test (data from the CGGA database). Osimertinib ( h ) and CP-24 ( i ) induced cell death in A172, LN229, U251, and U87 GBM cells ( N = 3). Data are presented as mean ± s.d. Osimertinib ( j ) and CP-24 ( k ) induced cell death in GBM 322, GBM324, GBM326, and GBM375 GBM patient-derived cells ( N = 3). Data are presented as mean ± s.d. l Heatmap showing the upregulation of apoptosis-related genes after Osimertinib and CP-24 treatment. The heatmap is colored according to z-score-normalized gene transcription levels. m Heatmap showing the downregulation of cell proliferation-related genes after Osimertinib and CP-24 treatment. The heatmap is colored according to z-score-normalized gene transcription levels. n TMZ-resistant A172 cells (TMZ + A172) were more sensitive to Osimertinib than parental A172 cells, as analyzed by one-sided t -test ( N = 6). A172 + TMZ vs . A172: p = 0.0472; U87 + TMZ vs . U87: p = 0.1244. Data are presented as mean ± s.d. o TMZ-resistant A172 (A172 + TMZ) and U87 (U87 + TMZ) cells were more sensitive to CP-24 than parental A172 and U87 cells, as analyzed by one-sided t -test ( N = 6). A172 + TMZ vs . A172: p = 0.0073; U87 + TMZ vs . U87: p = 0.0096. Data are presented as mean ± s.d. p Irradiated A172 (A172 + IR) and U87 (U87 + IR) cells were more sensitive to Osimertinib than parental A172 and U87 cells, as analyzed by one-sided t -test ( N = 3). A172 + IR vs . A172: p = 0.0267; U87 + IR vs . U87: p = 0.0361. Data are presented as mean ± s.d. q Irradiated A172 and U87 cells were more sensitive to CP-24 than parental A172 and U87 cells, as analyzed by one-sided t -test ( N = 3). A172 + IR vs . A172: p = 0.0010; U87 + IR vs . U87: p = 0.0006. Data are presented as mean ± s.d. r Heatmap showing the downregulation of DNA repair-related genes following Osimertinib and CP-24 treatment. The heatmap is colored according to z-score-normalized gene transcription levels. s qRT-PCR results showing the effects of Osimertinib and CP-24 in THP-1-derived macrophages, as analyzed by t -tests ( N = 3). Osimertinib vs . DMSO: CD68: p = 0.0001, CD86: p = 0.0072, CD80: p = 0.0330, ITGAX: p = 0.0099, CXCL16: p = 0.0004. CP-24 vs . DMSO: CD68: p = 0.0003, CD86: p = 0.0001, CD80: p = 0.0002, ITGAX: p = 0.0006, CXCL16: p = 0.0002. Other comparisons: ns. Data are presented as mean ± s.d. t qRT-PCR results showing the effects of Osimertinib and CP-24 on human primary bone marrow-derived macrophages, as analyzed by t -tests ( N = 3). Osimertinib vs . DMSO: CD68: p = 0.0008, CD80: p = 0.0335, ITGAX: p < 0.0001, CXCL16: p = 0.0043. CP-24 vs . DMSO: CD68: p = 0.0003, CD86: p = 0.0009, CD80: p = 0.0236, ITGAX: p = 0.0008, CXCL16: p = 0.0001. Other comparisons: ns. Data are presented as mean ± s.d

Article Snippet: To quantify the effects of Osimertinib, CP-24, Afatinib, Erlotinib, Gefitinib, Tinodasertib, Tomiversotib, WZ4002, Temozolomide (MedChemExpress, HY-17364), and radiation (10 Gy) on GBM cell growth, untreated, TMZ-resistant, or radiation-treated GBM cells were seeded in opaque-walled 96-well plates at a density of 1000 cells per well.

Techniques: In Vitro, Expressing, Mutagenesis, Derivative Assay, Irradiation, Quantitative RT-PCR

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual-targeting pharmacological UFMylation inhibition reprograms tumor and immune microenvironments to achieve long-term glioblastoma regression

doi: 10.1038/s41392-026-02819-w

Figure Lengend Snippet: Osimertinib exhibits pleiotropic anti-tumor effects in vivo. a Schematic illustration of the experimental protocol used to evaluate the anti-tumor effects of Osimertinib in NCG orthotopic U87 xenograft GBM model. b Tumor burden in vehicle- (CTR) or Osimertinib-treated NCG mice bearing orthotopic GBM xenograft was monitored by whole-body bioluminescence imaging (BLI). Data were analyzed by two-sided t -test ( N = 12), p = 0.0372. Data are presented as mean + s.d. c Survival of vehicle- or Osimertinib-treated NCG mice bearing orthotopic GBM xenograft was monitored. Data were analyzed by log-rank test ( N = 12), p = 0.0395. d Schematic illustration of the experimental protocol used to evaluate the anti-tumor effects of Osimertinib in an orthotopic GBM375 patient-derived xenograft (PDX) GBM model established in BALB/c nude mice. e Tumor burden in CTR or Osimertinib-treated BALB/c nude mice bearing orthotopic GBM PDX tumors was monitored by whole-body bioluminescence imaging (BLI). Data were analyzed by two-sided t -test ( N = 10), p = 0.0470. Data are presented as mean + s.d. f Survival of CTR or Osimertinib-treated BALB/c nude mice bearing orthotopic PDX GBM tumors was monitored. Data were analyzed by log-rank test ( N = 10), p = 0.0480. g Schematic illustration of the experimental protocol used to evaluate the anti-tumor effects of Osimertinib a syngeneic orthotopic GL261 GBM model established in C57BL/6J mice. h Tumor burden in CTR or Osimertinib-treated C57BL/6J mice bearing orthotopic GL261 GBM tumors monitored by whole-body bioluminescence imaging (BLI). Data were analyzed by Mann–Whitney test ( N = 14), p = 0.0091. i Survival of CTR or Osimertinib-treated C57BL/6J mice bearing orthotopic GL261 GBM tumors was monitored. Data were analyzed by log-rank test ( N = 14), p = 0.0002. j PD-1 expression pattern in tumor-infiltrating CD3 + CD8 + T cells. k Quantitative analysis of ( j ) showing that Osimertinib treatment decreased the proportion of PD-1 + CD3 + CD8 + T cells among tumor-infiltrating CD3 + CD8 + T cells. Data were analyzed by one-sided t -test ( N = 5), p < 0.0001. l Quantitative analysis of ( j ) showing that Osimertinib treatment decreased PD-1 expression level in tumor-infiltrating CD3 + CD8 + T cells. Data were analyzed by one-sided t -test ( N = 5), p = 0.0076. MFI Mean fluorescence intensity. m PD-1 expression pattern in tumor-infiltrating CD3 + CD4 + T cells. n Quantitative analysis of ( m ) showing that Osimertinib treatment decreased the proportion of PD-1 + CD3 + CD4 + T cells among tumor-infiltrating CD3 + CD4 + T cells. Data were analyzed by one-sided t -test ( N = 5), p = 0.0114. o Quantitative analysis of ( m ) showing the effects of Osimertinib treatment on PD-1 expression level in tumor-infiltrating CD3 + CD4 + T cells. Data were analyzed by one-sided t -test ( N = 5), not significant (ns). MFI: Mean fluorescence intensity. p , q Global UFMylation level in tumor cells ( p ). Data were analyzed by two-sided t test ( q , N = 5). p < 0.0001. Data are presented as mean ± s.d. r , s Global UFMylation level in tumor-infiltrating CD45 + CD3 - immune cells ( r ). Data were analyzed by two-sided t test ( s , N = 5). p = 0.0493. Data are presented as mean ± s.d. t , u Global UFMylation level in tumor-infiltrating CD45 + CD3 + CD4 + T cells ( t ). Data were analyzed by two-sided t test ( u , N = 5). p = 0.0016. Data are presented as mean ± s.d. v , w Global UFMylation level in tumor-infiltrating CD45 + CD3 + CD8 + T cells ( v ). Data were analyzed by two-sided t test ( w , N = 5). p = 0.0002. Data are presented as mean ± s.d

Article Snippet: To quantify the effects of Osimertinib, CP-24, Afatinib, Erlotinib, Gefitinib, Tinodasertib, Tomiversotib, WZ4002, Temozolomide (MedChemExpress, HY-17364), and radiation (10 Gy) on GBM cell growth, untreated, TMZ-resistant, or radiation-treated GBM cells were seeded in opaque-walled 96-well plates at a density of 1000 cells per well.

Techniques: In Vivo, Imaging, Derivative Assay, MANN-WHITNEY, Expressing, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Dual-targeting pharmacological UFMylation inhibition reprograms tumor and immune microenvironments to achieve long-term glioblastoma regression

doi: 10.1038/s41392-026-02819-w

Figure Lengend Snippet: Long-term immune memory and prevention of glioblastoma recurrence. a Schematic illustration of the experimental protocol used to assess the anti-GBM immune memory in Osimertinib-treated tumor-free C57BL/6J mice. b Tumor burden in control (CTR) or Osimertinib-treated tumor-free mice, monitored by whole-body bioluminescence imaging (BLI). Percentage of naïve, effector, and memory CD3 + CD8 + T cells ( c ) and CD3 + CD4 + T cells ( d ) after 2 days of tumor rechallenge in CTR or Osimertinib-treated tumor-free mice. e Expansion of CD3 + CD8 + effector T cells after tumor rechallenge in CTR or Osimertinib-treated tumor-free mice. Data were analyzed by one-sided t -test ( N = 6), p = 0.0060. Data are presented as mean ± s.d. f Expansion of CD3 + CD8 + memory T cells after tumor rechallenge in CTR or Osimertinib-treated tumor-free mice. Data were analyzed by one-sided t -test ( N = 6), p < 0.0001. Data are presented as mean ± s.d. g Percentage of CD3 + CD8 + naïve T cells decreased after tumor rechallenge in CTR or Osimertinib-treated tumor-free mice. Data were analyzed by one-sided t -test ( N = 6), p < 0.0001. Data are presented as mean ± s.d. h Expansion of CD3 + CD4 + effector T cells after tumor rechallenge in tumor-free mice. Data were analyzed by one-sided t -test ( N = 6), p < 0.0001. Data are presented as mean ± s.d. i No significant change in the percentage of CD3 + CD4 + memory T cells after tumor rechallenge in CTR or Osimertinib-treated tumor-free mice. Data were analyzed by one-sided t -test ( N = 6), ns. Data are presented as mean ± s.d. j Percentage of CD3 + CD4 + naïve T cells decreased after tumor rechallenge in CTR or Osimertinib-treated tumor-free mice. Data were analyzed by one-sided t -test ( N = 6), p = 0.0147. Data are presented as mean ± s.d

Article Snippet: To quantify the effects of Osimertinib, CP-24, Afatinib, Erlotinib, Gefitinib, Tinodasertib, Tomiversotib, WZ4002, Temozolomide (MedChemExpress, HY-17364), and radiation (10 Gy) on GBM cell growth, untreated, TMZ-resistant, or radiation-treated GBM cells were seeded in opaque-walled 96-well plates at a density of 1000 cells per well.

Techniques: Control, Imaging