Fig 2: (A) Proliferation assay in 72 hours, (B) Cell cycle assay, (C) Migration assay and (D) Invasion assay at 72 hours of Con, sh-B4GalT1 and control-shB4GalT1 group. (E) Western blot assay of IDH1, MGMT and ki-67 expression in vitro. (F) Quantification of IDH1, MGMT and ki-67 expression. Protein levels were normalized to GAPDH. (sh-B4GalT1 vs. other group, *p < 0.05, n = 6 per group).

Fig 3: Proposed model of TMZ resistance mechanisms that can be overcome by co-treatment with NRH and PARGi. (A) TMZ generates multiple DNA lesions, including O6-methylguanine (O6-MeG, ~9%), N7-methylguanine (N7-MeG, >70%), and N3-methyladenine (N3-MeA, ~10%); (B) The O6-MeG DNA lesion can be directly repaired by MGMT leading to TMZ resistance. If the O6-MeG/T mis-pair is acted upon by proteins of the MMR pathway, this leads to cell death. However, when the MMR pathway is defective, due to loss of expression or mutations in genes of the MMR pathway, then the O6-MeG/T mis-pair is tolerated and leads to TMZ resistance. Finally, the N7-MeG or N3-MeA DNA lesions are repaired by proteins of the BER pathway; (C) (i) TMZ induces PARP1/PARP2 activation, inducing the synthesis of PAR; (ii) NRH increases NAD+, which enhances PARP1/PARP2 activation and promotes an increase in PAR formation; (iii) Inhibition of PARG blocks degradation of PAR; (iv) Co-treatment of TMZ, NRH, and PARGi results in the hyperaccumulation of PAR, trapping DNA repair factors and leading to an increase in unrepaired DNA damage, a decrease in cell survival signaling, and an increase in cell death signaling.

Fig 4: MGMT-mediated resistance to TMZ is overcome by a co-treatment with NRH and PARGi. (A) Immunoblot analysis of MGMT in LN428 cells (WT) modified to express human MGMT (LN428/MGMT). Tubulin was used as the loading control. (B) The relative number of LN428/MGMT cells after treatment with TMZ or NRH (100 μM) + PARGi (10 μM) plus TMZ for 7 days (TMZ doses as indicated), normalized to DMSO. (C) The relative number of LN428/MGMT cells after treatment with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (125μM), T + N, T + P, or T + N + P for 7 days, normalized to DMSO (**** p < 0.0001, one-way ANOVA). (D) Immunoblot analysis of MGMT in U87MG, T98G, or U2OS cells. β-Actin was used as the loading control. (E) The relative number of U87MG, T98G, or U2OS cells after treatment with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (125 μM), T + N, T + P, or T + N + P for 7 days, normalized to DMSO (**** p < 0.0001 for each DMSO control; #### p < 0.0001 referred to TMZ treatment of U87MG cells, two-way ANOVA). (F) Immunoblot analysis of total cell lysates for PAR, p-AKT, γ-H2AX, and cleaved caspase 3, 24 h after treatment of LN428/MGMT cells with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (250 μM), T + N, T + P, or T + N + P. β-Actin was used as the loading control. (G) Immunoblot analysis of total cell lysates for PAR, p-AKT, γ-H2AX, and cleaved caspase 3, after 24 h treatment of U2OS cells with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (125 μM), T + N, T + P, or T + N + P. β-Actin was used as the loading control.

Fig 5: MGMT as a potential biomarker of MRT response and the therapy effect on DDR. (A) Western blot analysis of SMARCB1 (46 kDa), PARP1 (116 kDa)/cleaved PARP1 (cPARP1; 89 kDa), MGMT (21 kDa), and GAPDH (37 kDa) proteins. (B) Quantification of band intensities in (A) normalized to GAPDH loading control. One-way ANOVA was performed across 6 xenograft tumors. Significant change was reported with p = 0.0038; Tukey’s HSD further identified significant change only between G401 and WT-16 (p = 0.0022). Data are presented as mean ± SEM of 3 independent tests. Asterisks *** indicate a statistical difference of p ≤ 0.001 (t-test). (C) Inverse correlation between MGMT protein expression and the antitumor activity of PEG~TLZ+TMZ in MRT xenografts. Axis Y shows tumor response categories: 1-MCR (maintained complete response), 2-CR (complete response), 3-PR (partial response), 4-SD (stable disease), and 5-PD (progressive disease). Axis X: MGMT protein expression normalized to GAPDH. This resulted in Pearson correlation coefficient R = 0.88 with a significant p-value of 0.019. (D) G401SMARCB1−/− cell potentiation to TLZ+TMZ by MGMT inhibition. Cells were treated with increasing concentrations of TMZ (0–1 mM), TLZ IC50 (176 nM), and O6BG (25 μM). Alamar Blue was added to the plates at 72 h and incubated for 4 h; fluorescence was measured at 590 nm (excitation 530 nm) on a Clariostar microplate reader. (E) Protein array of basal levels of DDR proteins in G401SMARCB1−/− and G401SMARCB1+/+ cell lines. Blot was processed 24 h after treatment with DOX (1 μg/mL). The spliced fragments of the same blot are marked with thin white lines. (F) Plot quantitating the DDR protein expression (from the DDR protein array assay as in (E)) in G401SMARCB1−/− and G401SMARCB1+/+ cells treated with TMZ (IC50 = 446 μM for G401SMARCB1+/+ and 395 μM for G401SMARCB1−/−), TLZ (IC50 = 95 nM G401SMARCB1+/+ and 176 nM for G401SMARCB1−/−), and TLZ+TMZ for 72 h. Original western blots are presented in Supplementary File S1.