Additional file 1 of High cereblon expression in neuroendocrine cancer confers vulnerability to GSPT1 molecular glue degrader

Supplementary Material 1. Figure S1. Dose–response curves of CYRS381 in the Oncolines™ 102 cancer cell line panel. Dose–response curves for CYRS381 are shown for all 102 cell lines included in the panel. Figure S2. Correlation between N-MYC expression and CYRS381 sensitivity. (A) Protein–protein interaction network of positively correlated TFs with pIC50 of CYRS381. The network between the positively correlated genes with pIC50 of CYRS381 annotated in ‘regulation of transcription from RNA polymerase II promoter,’ from the gene ontology analysis as shown in (Fig. 1E) was generated using STRING DB. (B) The Markov clustering (MCL) of the protein–protein interaction shown in panel A. The MCL clustering analysis was performed using STRING DB at the default setting. (C) Correlation between mRNA expressions of MYC family genes and CYRS381 pIC50. Pearson's R correlation coefficient and corresponding P-values for co-variations are indicated in the figure. N-MYC or L-MYC mRNA expression has been tested in the clinical trial of GSPT1 MGD as putative predictive biomarkers [24]. (D) mRNA expression levels of N-MYC in lung cancer cell lines were obtained from DepMap portal. Statistically significant differences between comparison groups are noted in the panel: n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.00001. Figure S3. Differential in vitro efficacy of CYRS381 in neuroendocrine cancer versus non-neuroendocrine cancer cell lines. Cells were treated with CYRS381 at varying concentrations for 72 h in a 96-well plate. Following treatment, cell proliferation ratio and cell viability were assessed using the WST-8 assay kit by measuring absorbance. (A) The viability of each sample was calculated as a percentage of the corrected absorbance relative to the DMSO control group. Based on these values, a cell viability curve was generated, and the IC50 value for Fig. 1G was determined using GraphPad Prism. (B) The absorbance values at each time point (0, 24, 48, and 72 h) were measured, and the cell proliferation ratio was calculated by normalizing each value to the absorbance at 0 h. Figure S4. Mechanism of apoptosis induced by CYRS381. (A) Cell cycle analysis was performed in PC9 and H1155 cells treated with CYRS381 (300 nM or 1 μM) for 24 h. (B) Densitometric analysis of the western blot data presented in Fig. 1H was performed, and the degradation half-life (t₁/₂) of GSPT1 was estimated for each cell line. (C) GSPT1 degradation over an extended time course (0–72 h) following treatment with 300 nM or 1 μM CYRS381 was assessed by immunoblotting. (D) Time-dependent induction of integrated stress response (ISR) markers (ATF4 and CHOP) and apoptotic markers (cleaved caspase-3 and cleaved PARP) following CYRS381 treatment. (E) Densitometric analysis of GSPT1 immunoblotting signals normalized to GAPDH in Supplementary Figures S4C and S4D. Figure S5. In vivo efficacy of CYRS381 in H526 mouse xenograft model. A total of 1 × 107 H526 cells were subcutaneously injected into the right flank of nude mice. The control group received a vehicle, while the treatment groups were administered with CYRS381 at doses of 0.5, 1, 3, or 6 mg/kg daily. (A) Changes in average tumor volume in the H526 xenograft model (n = 5 per group). The data for each group at the endpoint are as follows: Vehicle: 1160.55 ± 246.43, 0.5 mg/kg: 789.48 ± 144.79, 1 mg/kg: 841.73 ± 162.13, 3 mg/kg: 331.88 ± 77.85, and 6 mg/kg: 82.01 ± 16.95. Data are presented as mean ± SEM. (B) Expression of GSPT1 was analyzed by immunoblotting using tumor lysates collected 8 h after the fifth daily dose of CYRS381. Significance was measured using one-way ANOVA with Dunnett's multiple comparisons test on tumor volume at the end-point (n.s. not significant, ** P < 0.01, and *** P < 0.001), compared to vehicle-treated group. Figure S6. Effect of GSPT1 overexpression on GSPT1 MGD sensitivity in the NCI-H1155 cell line. (A) Rescue of cell viability by FLAG-GSPT1 or FLAG-GSPT1G575N overexpression in the NCI-H1155 neuroendocrine cancer cell line upon CYRS381 treatment. (B) GSPT1 degradation induced by CYRS381 in parental versus FLAG-GSPT1 or FLAG-GSPT1G575N-overexpressing NCI-H1155 cells, assessed by immunoblotting. GSPT1G575N is a mutant form of GSPT1 containing a substitution at the key glycine residue within the G-loop, which is critical for CRBN recognition, rendering it resistant to degradation by CRBN-based MGDs. Figure S7. Sensitive in vitro and in vivo efficacy of MRT-2359 in neuroendocrine cancer cell lines and a PDX model. (A) Differential in vitro efficacy of MRT-2359 in neuroendocrine cancer versus non-neuroendocrine cancer cell lines. Cells were treated with MRT-2359 at varying concentrations for 72 h in 96-well plates. Following treatment, cell viability was assessed using the CellTiter-Glo™ assay. The viability of each sample was calculated as a percentage of the background-corrected luminescence relative to the DMSO control group. Based on these values, cell viability curves were generated using GraphPad Prism. (B) In vivo efficacy of MRT-2359 in a CTG-1713 SCLC PDX model. MRT-2359 administration (10 mg/kg, PO, QD) led to regression of tumor volume in tumor-bearing mice. Tumor growth curves represent the mean ± S.D. (n = 10 per group). Statistical significance was determined by two-way ANOVA (****P < 0.0001). Figure S8. The expression level of CRBN determines the efficacy of CYRS381. (A, B) H1155 cells were transfected with negative control siRNA (siCTR) or two different sets of siRNAs targeting CRBN or DDB1 for 48 h. After transfection, the cells were seeded into 96-well plates and treated with CYRS381 following overnight incubation. Cell viability was measured after 72 h using absorbance values obtained from the WST-8 assay. (C) Comparison of basal GSPT1 expression between Mock and CRBN-overexpressing (CRBN-OE) NSCLC cell lines. (D) Evaluation of CK1α and GSPT1 degradation following 24 h treatment with lenalidomide or pomalidomide in Mock and CRBN-OE NSCLC cell lines. Figure S9. Direct binding of NEC-driving transcription factors at the CRBN promoter. (A) ChIP-seq tracks for H3K27ac, N-MYC, Pol II, ASCL1, and NeuroD1 from Kelly (neuroblastoma) and DMS-53 (SCLC) cell lines. Publicly available datasets (GSE211953 and GSE179072) were downloaded from GEO ( https://www.ncbi.nlm.nih.gov/geo/ ) and visualized using Integrative Genomics Viewer (IGV) [59]. (B) CUT&RUN-qPCR analysis of N-MYC and ASCL1 binding at the CRBN promoter. Comparative enrichment was assessed between parental H2023 and H2023-RPMA 5C3 cells. Fold enrichment relative to the corresponding IgG control is shown. Data represent the mean ± SEM (n = 3 per group). Statistical significance was determined using Student’s t-test (**P < 0.01). (C) ChIP-seq tracks for H3K27ac and ASCL1 from the GI-ME-N neuroblastoma cell line, and ATAC-seq data from CRPC and NEPC cell lines. Publicly available ChIP-seq and ATAC-seq datasets (GSE214791 and GSE183199) were obtained from GEO. (D) CUT&RUN-qPCR analysis of H3K27ac binding at the CRBN promoter. Comparative enrichment was assessed between parental H2023 and H2023-RPMA 5C3 cells. Fold enrichment relative to the corresponding IgG control is shown. Data represent the mean ± SEM (n = 3 per group). Statistical significance was determined using Student’s t-test (**P < 0.01). Figure S10. CRBN mutation status and prognostic impact of CRBN expression in lung cancer patients. (A) Overall survival and disease-free survival of LUAD and LUSC patients stratified by CRBN expression levels. The analysis was performed using data from GEPIA2 ( https://gepia2.cancer-pku.cn/ ). (B) Genetic alterations in the CRBN gene were analyzed in tumor samples from SCLC patients using data from cBioPortal ( https://www.cbioportal.org/ ). Figure S11. Evaluation of the synergistic effect of combining a GSPT1 degrader with chemotherapeutic agents. (A) Cell viability of H1155 and H526 cells treated with CYRS381 in combination with etoposide or cisplatin for 72 h. (B) Assessment of synergy score for the combination treatments, based on cell viability data shown in (A). The analysis was performed using SynergyFinder ( https://synergyfinder.org/ ).