A Promising Way to Overcome Temozolomide Resistance through Inhibition of Protein Neddylation in Glioblastoma Cell Lines

There is no effective therapy for the lately increased incidence of glioblastoma multiforme (GBM)—the most common primary brain tumor characterized by a high degree of invasiveness and genetic heterogeneity. Currently, DNA alkylating agent temozolomide (TMZ) is the standard chemotherapy. Nevertheless, TMZ resistance is a major problem in the treatment of GBM due to numerous molecular mechanisms related to DNA damage repair, epigenetic alterations, cellular drug efflux, apoptosis-autophagy, and overactive protein neddylation. Low molecular weight inhibitors of NEDD8-activating enzyme (NAE), such as MLN4924, attenuate protein neddylation and present a promising low-toxicity anticancer agent. The aim of our study was to find an effective combination treatment with TMZ and MLN4924 in our TMZ-resistant GBM cell lines and study the effect of these combination treatments on different protein expressions such as O6-methylguanine methyltransferase (MGMT) and p53. The combination treatment successfully decreased cell viability and sensitized TMZ-resistant cells to TMZ, foreshadowing a new treatment strategy for GBM.


Introduction
The World Health Organization (WHO) has introduced an international grading scheme for the classification and diagnosis of gliomas [1]. The most aggressive WHO grade 4 glioblastoma multiforme (GBM) is also the most common primary malignant brain tumor in the central nervous system (CNS), accounting for 50% of all primary brain gliomas [2,3]. The prognosis for patients with GBM is very poor, with a median survival from diagnosis of about 15 months [4].
The standard therapy includes the surgical resection of the majority of the tumor mass followed by radiotherapy and chemotherapy [4]. Despite several international efforts, its treatment faces huge challenges, such as the presence of the blood-brain barrier (BBB) which prevents~98% of small molecule drugs from entering the CNS [5], and the diffuse invasiveness of the tumor cells [6]; therefore, surgical excision does not prevent tumor recurrence or increase survival [7]. To increase the effectiveness of treatments, chemotherapeutic agents have been tested to improve the survival of patients, including DNA alkylating agents such as temozolomide (TMZ), bis-chloroethylnitrosourea (BCNU), and lomustine (CCNU) [8]. Of these agents, TMZ is the drug of choice for standard chemotherapy [8] since it was approved by the US Food and Drug Administration (FDA) in 2005 [9]. TMZ is a second-generation imidazotetrazine lipophilic prodrug that is able to cross the BBB and can therefore be administered orally [10]. The active metabolite pression of NEDD8 and several neddylation enzymes [19]. Furthermore, overexpression of these enzymes is associated with disease progression; therefore, inhibition of protein neddylation became an attractive anticancer strategy [17]. In order to suppress neddylation, a drug called MLN4924 (also known as pevonedistat) was discovered by Millennium Pharmaceuticals [17]. MLN4924 is a small molecule NAE inhibitor and can bind to active sites of NAE, therefore, inhibiting the progress of the next enzyme reaction [19]. This inhibition results in suppressed cullin neddylation and causes the accumulation of CRL-substrates leading to DNA damage, cell cycle arrest, senescence, and apoptosis in different cancer species [19]. Moreover, increasing experimental evidence suggests that MLN4924 suppresses glioblastoma cell growth in vitro and in vivo [19]. According to preclinical studies, MLN4924 has potent antitumor activity against several solid tumors and hematological malignancies [17]. Moreover, in phase I/II clinical trials, MLN4924 showed good clinical efficiency and low toxicity [19]. Furthermore, preclinical studies proved that MLN4924 in combination with chemotherapy increased antitumor activity in solid tumor cell lines and xenograft models [17].

Determination of the Methylation Status of the MGMT Promoter in GBM Cell Lines
Resistance to chemotherapy is a major problem in the treatment of GBM. The methylation status of the MGMT promoter region is used as a marker for TMZ resistance, the hypermethylation of the MGMT promoter is associated with longer survival in GBM patients treated with TMZ. Both DNA methylation-mediated silencing of the MGMT gene and high expression of ABC transporters could be reasons for ineffective TMZ treatment. Therefore, we explored the DNA methylation profile of the MGMT promoter region and investigated the expression level of GBM-implicated transporters in GBM cell lines. To evaluate the methylation status of the MGMT promoter region in untreated GBM cell lines, the COBRA assay was used (Figure 1). Almost complete methylation of the MGMT promoter was detected in U-118 MG (99%), SNB78 (98%), A172(98%), SF539 (95%), and SNB75 (86%) cell lines. The MGMT promoters of SNB19 (73%) and SF268 (70%) cell lines were moderately methylated, while the MGMT promoter of the H4 (14%) cell line was the least methylated. proves that protein modification by neddylation is overactivated in numerous human cancers (lung cancer, intrahepatic cholangiocarcinoma, liver cancer, colorectal cancer, glioblastoma, nasopharyngeal carcinoma, esophageal squamous cell carcinoma) [17] due to the overexpression of NEDD8 and several neddylation enzymes [19]. Furthermore, overexpression of these enzymes is associated with disease progression; therefore, inhibition of protein neddylation became an attractive anticancer strategy [17]. In order to suppress neddylation, a drug called MLN4924 (also known as pevonedistat) was discovered by Millennium Pharmaceuticals [17]. MLN4924 is a small molecule NAE inhibitor and can bind to active sites of NAE, therefore, inhibiting the progress of the next enzyme reaction [19]. This inhibition results in suppressed cullin neddylation and causes the accumulation of CRL-substrates leading to DNA damage, cell cycle arrest, senescence, and apoptosis in different cancer species [19]. Moreover, increasing experimental evidence suggests that MLN4924 suppresses glioblastoma cell growth in vitro and in vivo [19]. According to preclinical studies, MLN4924 has potent antitumor activity against several solid tumors and hematological malignancies [17]. Moreover, in phase I/II clinical trials, MLN4924 showed good clinical efficiency and low toxicity [19]. Furthermore, preclinical studies proved that MLN4924 in combination with chemotherapy increased antitumor activity in solid tumor cell lines and xenograft models [17].

Determination of the Methylation Status of the MGMT Promoter in GBM Cell Lines
Resistance to chemotherapy is a major problem in the treatment of GBM. The methylation status of the MGMT promoter region is used as a marker for TMZ resistance, the hypermethylation of the MGMT promoter is associated with longer survival in GBM patients treated with TMZ. Both DNA methylation-mediated silencing of the MGMT gene and high expression of ABC transporters could be reasons for ineffective TMZ treatment. Therefore, we explored the DNA methylation profile of the MGMT promoter region and investigated the expression level of GBM-implicated transporters in GBM cell lines. To evaluate the methylation status of the MGMT promoter region in untreated GBM cell lines, the COBRA assay was used (Figure 1). Almost complete methylation of the MGMT promoter was detected in U-118 MG (99%), SNB78 (98%), A172(98%), SF539 (95%), and SNB75 (86%) cell lines. The MGMT promoters of SNB19 (73%) and SF268 (70%) cell lines were moderately methylated, while the MGMT promoter of the H4 (14%) cell line was the least methylated. Genomic DNA was extracted and treated with bisulfite; the MGMT promoter region was amplified by PCR, digested with BstUI, and visualized by agarose gel electrophoresis as described in Section 4. For semiquantitative evaluation, the percentage of cleaved (methylated) DNA was determined using ImageJ software (version 1.53t). M = molecular weight marker. Genomic DNA was extracted and treated with bisulfite; the MGMT promoter region was amplified by PCR, digested with BstUI, and visualized by agarose gel electrophoresis as described in Section 4. For semiquantitative evaluation, the percentage of cleaved (methylated) DNA was determined using ImageJ software (version 1.53t). M = molecular weight marker.

Determination of Expression of ABC Transporters Using Quantitative Real-Time PCR (qPCR)
Since ABC transporters play an important role in the development of chemoresistance, the expressions of ABCC1 and ABCG2 transporters were studied in untreated GBM cell lines using qPCR ( Figure 2). All cell lines expressed both ABCC1 ( Figure 2a) and ABCG2 (Figure 2b) transporters. The highest expression of ABCC1 and ABCG2 was observed in U-118MG. The lowest expression of both transporters was detected in SF539 cells. The difference in expression was significant between U-118MG and SF539; however, there was no significant difference in expression compared to other cell lines.
Since ABC transporters play an important role in the development of chemoresistance, the expressions of ABCC1 and ABCG2 transporters were studied in untreated GBM cell lines using qPCR ( Figure 2). All cell lines expressed both ABCC1 ( Figure 2a) and ABCG2 (Figure 2b) transporters. The highest expression of ABCC1 and ABCG2 was observed in U-118MG. The lowest expression of both transporters was detected in SF539 cells. The difference in expression was significant between U-118MG and SF539; however, there was no significant difference in expression compared to other cell lines.

Evaluation of the Sensitivity of GBM Cell Lines to TMZ and MLN4924
To determine the sensitivity of GBM cell lines to TMZ, cells were treated with different TMZ concentrations for 48 h and ATP content was measured using the CellTiter-Glo 2.0 Assay to determine cell viability, as described in Section 4. For the treatment, 1 µM, 1000 µM, and 3000 µM of TMZ were used. Relying on the results of previous experiments, these concentrations differentiate well between GBM cell lines based on their TMZ sensitivity. Data were normalized to the solvent containing control values for each cell line, and three independent experiments were performed. Based on the results of these experiments, the half-maximal inhibitory concentrations (IC50) of TMZ were calculated in Excel. IC50 values ranged from 346 to 3407 µM (Figure 3a), including the most TMZ-resistant SNB19 (3407 µM) and the most sensitive SF539 (346 µM) cells. Based on the IC50 values and the methylation status of the MGMT promoter, three cell lines (SNB19, H4, SF539) were selected for subsequential analysis. SNB19 cells are 9.8-fold more resistant to TMZ than SF539 cells. The H4 cell line is resistant to TMZ within limits; according to the IC50 values, it is 5.7-fold more resistant to TMZ than the SF539 cell line and 1.7-fold more sensitive to TMZ than the resistant SNB19 cells.
Targeted inhibition of NEDD8-activating enzyme E1 (NAE1) might increase the treatment efficacy of GBM alone or in combination with TMZ.
To determine the half-maximal inhibitory concentrations (IC50) of MLN4924, an inhibitor of the NEDD8-activating enzyme E1, GBM cell lines were treated with different concentrations of MLN4924 (0.01 µM, 0.1 µM, 1 µM) for 48 h and ATP content was measured to determine cell viability. Three independent experiments were performed and data were normalized to the solvent-containing controls for each cell line. IC50 concentrations were calculated relying on the results of the above treatments. IC50 values ranged from

Evaluation of the Sensitivity of GBM Cell Lines to TMZ and MLN4924
To determine the sensitivity of GBM cell lines to TMZ, cells were treated with different TMZ concentrations for 48 h and ATP content was measured using the CellTiter-Glo 2.0 Assay to determine cell viability, as described in Section 4. For the treatment, 1 µM, 1000 µM, and 3000 µM of TMZ were used. Relying on the results of previous experiments, these concentrations differentiate well between GBM cell lines based on their TMZ sensitivity. Data were normalized to the solvent containing control values for each cell line, and three independent experiments were performed. Based on the results of these experiments, the half-maximal inhibitory concentrations (IC 50 ) of TMZ were calculated in Excel. IC 50 values ranged from 346 to 3407 µM (Figure 3a), including the most TMZ-resistant SNB19 (3407 µM) and the most sensitive SF539 (346 µM) cells. Based on the IC 50 values and the methylation status of the MGMT promoter, three cell lines (SNB19, H4, SF539) were selected for subsequential analysis. SNB19 cells are 9.8-fold more resistant to TMZ than SF539 cells. The H4 cell line is resistant to TMZ within limits; according to the IC 50 values, it is 5.7-fold more resistant to TMZ than the SF539 cell line and 1.7-fold more sensitive to TMZ than the resistant SNB19 cells.
Targeted inhibition of NEDD8-activating enzyme E1 (NAE1) might increase the treatment efficacy of GBM alone or in combination with TMZ.
To determine the half-maximal inhibitory concentrations (IC 50 ) of MLN4924, an inhibitor of the NEDD8-activating enzyme E1, GBM cell lines were treated with different concentrations of MLN4924 (0.01 µM, 0.1 µM, 1 µM) for 48 h and ATP content was measured to determine cell viability. Three independent experiments were performed and data were normalized to the solvent-containing controls for each cell line. IC 50 concentrations were calculated relying on the results of the above treatments. IC 50 values ranged from 0.39 to 2.45 µM (Figure 3b), including the most MLN4924-resistant SNB78 (2.45 µM) and the most sensitive SF539 (0.39 µM) cells. Based on TMZ sensitivity, three cell lines (SNB19, H4, SF539) were selected for further analysis, including the most TMZ-resistant SNB19, the moderately resistant H4, and the most sensitive SF539 cell line; therefore, only their MLN4924 sensitivity was compared below. The second most ML4924-resistant cell line is the TMZ-resistant SNB19, which is 3.7-fold more resistant to MLN4924 than SF539 cells. The H4 cell line is resistant to MLN4924 within limits; according to the IC 50 values, it is 1.7 times more resistant to MLN4924 than the SF539 cell line and 2.0 times more sensitive to MLN4924 than the resistant SNB19 cells. Interestingly, the SF539 cell line is the most sensitive to both TMZ and MLN4924 treatment.
the most sensitive SF539 (0.39 µM) cells. Based on TMZ sensitivity, three cell lines (SNB19, H4, SF539) were selected for further analysis, including the most TMZ-resistant SNB19, the moderately resistant H4, and the most sensitive SF539 cell line; therefore, only their MLN4924 sensitivity was compared below. The second most ML4924-resistant cell line is the TMZ-resistant SNB19, which is 3.7-fold more resistant to MLN4924 than SF539 cells. The H4 cell line is resistant to MLN4924 within limits; according to the IC50 values, it is 1.7 times more resistant to MLN4924 than the SF539 cell line and 2.0 times more sensitive to MLN4924 than the resistant SNB19 cells. Interestingly, the SF539 cell line is the most sensitive to both TMZ and MLN4924 treatment.

Effect of TMZ and MLN4924 Combination Treatments on Cell Viability
We next examined whether the neddylation inhibitor MLN4924 alters TMZ-induced cytotoxicity. Since MLN4924 induces DNA damage, it was hypothesized that MLN4924 may sensitize cells to TMZ in a way where DNA damage overwhelms DNA repair mechanisms. Previously selected cell lines (SNB19, H4, SF539) were treated with the appropriate IC50 concentrations of each drug alone or in different combinations for 48 h:24 h MLN4924 pretreatment followed by 24

Effect of TMZ and MLN4924 Combination Treatments on Cell Viability
We next examined whether the neddylation inhibitor MLN4924 alters TMZ-induced cytotoxicity. Since MLN4924 induces DNA damage, it was hypothesized that MLN4924 may sensitize cells to TMZ in a way where DNA damage overwhelms DNA repair mechanisms. Previously selected cell lines (SNB19, H4, SF539) were treated with the appropriate IC 50 concentrations of each drug alone or in different combinations for 48 h:24 h MLN4924 pretreatment followed by 24

Effect of TMZ and MLN4924 Combination Treatments on Apoptosis
We next sought to determine whether the decrease in cell viability is caused by increased cell apoptosis. Previously selected GBM cell lines (SNB19, H4, SF539) were treated with TMZ and MLN4924 separately and with the most effective combination treatment (48 h TMZ + 48 h MLN4924). Cells were treated with the appropriate IC50 concentrations of each drug alone, or in combination for 48 h. Solvent-containing control and treated (48 h TMZ, 48 h MLN4924, and 48 h TMZ + 48 h MLN4924) samples were studied by fluorescence microscopy, and apoptotic cells were counted by nuclear morphology after staining with Hoechst 33342 dye ( Figure 5).
In control samples, 4-8% of cells in each cell line were apoptotic. TMZ induced apoptosis in 45% of SNB19 cells (Figure 5a), 48% of H4 cells (Figure 5b), and 57% of cells in SF539 cells (Figure 5c). MLN4924 induced apoptosis in 53% of SNB19 cells, 51% of H4 cells, and 45% of SF539 cells. The combination treatments (48 h TMZ + 48 h MLN4924) were significantly more effective than TMZ or MLN4924 treatments alone in the TMZresistant SNB19 (67%) and H4 (84%). In the TMZ-sensitive SF539 cells, the combination treatment is significantly more effective than the MLN4924 treatment alone; however, Box plots have box boundaries ranging from the 25-75% percentiles, the center line representing the median, and whiskers illustrating the minimum and maximum of the data points. Significant results are presented as * p < 0.05, ** p < 0.01, *** p < 0.001.

Effect of TMZ and MLN4924 Combination Treatments on Apoptosis
We next sought to determine whether the decrease in cell viability is caused by increased cell apoptosis. Previously selected GBM cell lines (SNB19, H4, SF539) were treated with TMZ and MLN4924 separately and with the most effective combination treatment (48 h TMZ + 48 h MLN4924). Cells were treated with the appropriate IC 50 concentrations of each drug alone, or in combination for 48 h. Solvent-containing control and treated (48 h TMZ, 48 h MLN4924, and 48 h TMZ + 48 h MLN4924) samples were studied by fluorescence microscopy, and apoptotic cells were counted by nuclear morphology after staining with Hoechst 33342 dye ( Figure 5).
In control samples, 4-8% of cells in each cell line were apoptotic. TMZ induced apoptosis in 45% of SNB19 cells (Figure 5a), 48% of H4 cells (Figure 5b), and 57% of cells in SF539 cells (Figure 5c). MLN4924 induced apoptosis in 53% of SNB19 cells, 51% of H4 cells, and 45% of SF539 cells. The combination treatments (48 h TMZ + 48 h MLN4924) were significantly more effective than TMZ or MLN4924 treatments alone in the TMZ-resistant SNB19 (67%) and H4 (84%). In the TMZ-sensitive SF539 cells, the combination treatment is significantly more effective than the MLN4924 treatment alone; however, there is no significant difference between the effect of combination treatment and TMZ treatment in the case of apoptosis. there is no significant difference between the effect of combination treatment and TMZ treatment in the case of apoptosis.

Analysis of Expression of Proteins Involved in the Neddylation Pathway and TMZ Resistance
Next, Western blot analysis was used to study the expression of proteins involved in the neddylation pathway and TMZ resistance. As the most effective combination treatment was 48 h TMZ combined with 48 h MLN4924 (48 h TMZ + 48 h MLN4924), this treatment combination was used in addition to single treatments (48 h TMZ, 48 h MLN4924) in SNB19 (Figure 6a), H4 (Figure 6b) and SF539 (Figure 6c) cells before analyzing different protein expressions. NAE1 expression levels were analyzed to evaluate the status of the neddylation pathway. All cell lines expressed the NAE1 protein, indicating that this pathway is active. The hallmark of NAE1 inhibition is the accumulation of CRL substrates (e.g., cyclin D1, Mdm2, and p21) and, therefore, these proteins were examined to demonstrate the effectiveness of MLN4924 treatment. All three MLN4924-treated cell lines expressed cyclin D1 relatively highly compared to their controls. In H4 and SF539 The rate of apoptosis was calculated as the percentage of the counted cells. At least 5 independent experiments were performed. Box plots have box boundaries ranging from the 25-75% percentiles, the center line represents the median, and the whiskers illustrate the minimum and maximum of the data points. Significant results are presented as * p < 0.05, ** p < 0.01, *** p < 0.001.

Analysis of Expression of Proteins Involved in the Neddylation Pathway and TMZ Resistance
Next, Western blot analysis was used to study the expression of proteins involved in the neddylation pathway and TMZ resistance. As the most effective combination treatment was 48 h TMZ combined with 48 h MLN4924 (48 h TMZ + 48 h MLN4924), this treatment combination was used in addition to single treatments (48 h TMZ, 48 h MLN4924) in SNB19 (Figure 6a), H4 (Figure 6b) and SF539 (Figure 6c) cells before analyzing different protein expressions. NAE1 expression levels were analyzed to evaluate the status of the neddylation pathway. All cell lines expressed the NAE1 protein, indicating that this pathway is active. The hallmark of NAE1 inhibition is the accumulation of CRL substrates (e.g., cyclin D1, Mdm2, and p21) and, therefore, these proteins were examined to demonstrate the effectiveness of MLN4924 treatment. All three MLN4924-treated cell lines expressed cyclin D1 relatively highly compared to their controls. In H4 and SF539 cells the cyclin D1 expression also increased due to the combination treatment. The relative amount of Mdm2 in SNB19 and H4 is higher as a result of the MLN4924 treatment; however, in SF539, there is no difference in Mdm2 expression in MLN4924-treated cells. In SNB19 and H4 cells, TMZ and combination treatment increased the Mdm2 expression compared to their controls. p21 accumulated in MLN4924-treated SNB19 and SF539 cells but not in MLN4924-treated H4 cells. Combination treatment increased p21 expression in SNB19 and SF539 cells compared to control samples. Taken together, 48 h of MLN4924 and combination treatment increased the amount of CRL substrate proteins (Mdm2, p21, cyclin D1). Since p53 is an important regulator of the cell cycle and repair processes and also a CRL substrate, we examined its expression in treated cells. The p53 protein is expressed in SNB19 cells; its expression is increased during TMZ and combination treatment and decreased during MLN4924 treatment. H4 control cells expressed p53, whose expression is increased during TMZ treatment and decreased during MLN4924 and combination treatment. In the SF539 cell line, p53 protein expression was increased during MLN4924 treatment and decreased during TMZ and combination treatment. The expression of MGMT protein in treated cells was also analyzed by Western blotting. The TMZ-resistant SNB19 and H4 cells express MGMT; however, its expression is decreased due to the combination treatment. The TMZsensitive SF539 expresses MGMT protein in a very low amount. Interestingly, its expression is induced during MLN4924 treatment. ). Since p53 is an important regulator of the cell cycle and repair processes and also a CRL substrate, we examined its expression in treated cells. The p53 protein is expressed in SNB19 cells; its expression is increased during TMZ and combination treatment and decreased during MLN4924 treatment. H4 control cells expressed p53, whose expression is increased during TMZ treatment and decreased during MLN4924 and combination treatment. In the SF539 cell line, p53 protein expression was increased during MLN4924 treatment and decreased during TMZ and combination treatment. The expression of MGMT protein in treated cells was also analyzed by Western blotting. The TMZ-resistant SNB19 and H4 cells express MGMT; however, its expression is decreased due to the combination treatment. The TMZ-sensitive SF539 expresses MGMT protein in a very low amount. Interestingly, its expression is induced during MLN4924 treatment.

Analysis of MGMT Protein Expression in Response to Combination Treatments by Immunocytochemistry
To determine if 48 h TMZ, 48 h MLN4924, and combination (48 h TMZ + 48 h MLN4923) treatment alters MGMT protein expression in SNB19 (Figure 7a), H4 (Figure 7b), and SF539 (Figure 7c) cell lines, immunocytochemistry was performed using a specific MGMT antibody. Figure 7 shows the quantitative analysis of the immunocytochemistry performed with different treatments, and Figure 8 shows representative confocal microscopic images of the result of MGMT immunocytochemistry in SNB19 cells. In SNB19 cells, a strong MGMT expression was found in TMZ-and MLN4924-treated cells, however, the MGMT expression is significantly decreased due to the combination treatment. The expression of MGMT is significantly increased during MLN4924 treatment in H4 cells and significantly decreased compared to the combination treatment. There was no significant difference between the TMZ and combination treatment's effects on MGMT expression. In SF539 cells, MGMT expression is the highest during MLN4924 treatment and it is significantly higher than that detected during 48 h TMZ or combination treatment.

Analysis of MGMT Protein Expression in Response to Combination Treatments by Immunocytochemistry
To determine if 48 h TMZ, 48 h MLN4924, and combination (48 h TMZ + 48 h MLN4923) treatment alters MGMT protein expression in SNB19 (Figure 7a), H4 (Figure  7b), and SF539 (Figure 7c) cell lines, immunocytochemistry was performed using a specific MGMT antibody. Figure 7 shows the quantitative analysis of the immunocytochemistry performed with different treatments, and Figure 8 shows representative confocal microscopic images of the result of MGMT immunocytochemistry in SNB19 cells. In SNB19 cells, a strong MGMT expression was found in TMZ-and MLN4924-treated cells, however, the MGMT expression is significantly decreased due to the combination treatment. The expression of MGMT is significantly increased during MLN4924 treatment in H4 cells and significantly decreased compared to the combination treatment. There was no significant difference between the TMZ and combination treatment's effects on MGMT expression. In SF539 cells, MGMT expression is the highest during MLN4924 treatment and it is significantly higher than that detected during 48 h TMZ or combination treatment.

Discussion
A number of signaling pathways, the expression level, and post-translational status of regulatory factors are significantly altered in GBM [20]. Due to this genetic and proteomic heterogeneity, the prognosis of GBM and the response of patients to therapy is mainly based on genetic variations and the epigenetic environment [21]. Methylation of the DNA repair enzyme MGMT gene promoter region leads to its epigenetic silencing that results in the reduction in DNA repair and increases chemosensitivity to TMZ [22]. We tested the sensitivity of several GBM cell lines to TMZ and selected three cell lines for further investigation. Although 73% of the MGMT promoter region was methylated in the SNB19 cell line, we detected expression of the MGMT protein, and this cell line showed the highest resistance to TMZ treatment. Of the cell lines tested, the MGMT promoter in the H4 cell line was the least methylated, and the expression of MGMT protein showed that this cell line was resistant to TMZ treatment. In the SF539 cell line, the MGMT promoter was highly methylated, MGMT protein expression was undetectable, and this cell line was the most sensitive to TMZ treatment. SF539 expressed ABCC1 and ABCG2 transporters in the lowest level, which can contribute to TMZ sensitivity. Previous studies have reported that the neddylation pathway is overactivated in several types of human cancers, including GBM, playing an important role in their development and progression and, therefore, the inhibition of this pathway could be utilized as a potential treatment for GBM [19]. We wanted to investigate whether we could overcome TMZ resistance by using a combination treatment with a drug that inhibits the overactive neddylation pathway in these cell lines. Inhibition of neddylation results in the accumulation of several intracellular proteins leading to DNA damage, induction of cell cycle arrest, and apoptosis [23]. Many proteins involved in the pathomechanism of GBM are neddylated; therefore, we used MLN4924, an

Discussion
A number of signaling pathways, the expression level, and post-translational status of regulatory factors are significantly altered in GBM [20]. Due to this genetic and proteomic heterogeneity, the prognosis of GBM and the response of patients to therapy is mainly based on genetic variations and the epigenetic environment [21]. Methylation of the DNA repair enzyme MGMT gene promoter region leads to its epigenetic silencing that results in the reduction in DNA repair and increases chemosensitivity to TMZ [22]. We tested the sensitivity of several GBM cell lines to TMZ and selected three cell lines for further investigation. Although 73% of the MGMT promoter region was methylated in the SNB19 cell line, we detected expression of the MGMT protein, and this cell line showed the highest resistance to TMZ treatment. Of the cell lines tested, the MGMT promoter in the H4 cell line was the least methylated, and the expression of MGMT protein showed that this cell line was resistant to TMZ treatment. In the SF539 cell line, the MGMT promoter was highly methylated, MGMT protein expression was undetectable, and this cell line was the most sensitive to TMZ treatment. SF539 expressed ABCC1 and ABCG2 transporters in the lowest level, which can contribute to TMZ sensitivity. Previous studies have reported that the neddylation pathway is overactivated in several types of human cancers, including GBM, playing an important role in their development and progression and, therefore, the inhibition of this pathway could be utilized as a potential treatment for GBM [19]. We wanted to investigate whether we could overcome TMZ resistance by using a combination treatment with a drug that inhibits the overactive neddylation pathway in these cell lines. Inhibition of neddylation results in the accumulation of several intracellular proteins leading to DNA damage, induction of cell cycle arrest, and apoptosis [23]. Many proteins involved in the pathomechanism of GBM are neddylated; therefore, we used MLN4924, an inhibitor of the neddylation pathway, to determine the effect of the combination treatment. Combination treatment with TMZ + MLN4924 significantly reduced the viability compared to single TMZ treatment in all three selected cell lines, including the highly TMZ-resistant SNB19 cell line. Interestingly, the amount of MGMT protein is decreased in cells that are both treated with TMZ alone and in combination with MLN4924, indicating that this combination of drugs somehow intervenes in MGMT expression or degradation. The expression of MGMT in treated cells is inversely proportional to the expression of p53. Dora Bocangel et al. reported that MGMT expression is downregulated by p53 in human tumor cells [24]. Dimitris P. Xirodimas et al. reported that neddylation of p53 inhibits its transcriptional activity [25]. Therefore, inhibition of the overactivated neddylation pathway in GBM by MLN4924 may increase the transcriptional activity of p53, consequently downregulating MGMT. This effect was not pronounced in single MLN4924 treatments, however, it was in some cases of the combination treatments. MGMT protein expression is significantly decreased in combination-treated cells compared to temozolomide-treated cells in the highly TMZ-resistant SNB19 cell line. Due to this treatment p53 protein expression is increased in addition to decreased MGMT protein expression suggesting that TMZ in combination with MLN4924 is able to inhibit MGMT-mediated DNA repair through the enhancement of p53-mediated MGMT inhibition and could be the background of increased chemosensitivity. However, in the moderately TMZ-resistant H4 cells and the TMZ-sensitive SF539 cells, both p53 and MGMT protein expression is decreased according to combination treatment implying that another signaling pathway is involved in the TMZ sensitization in these cells.
Despite several novel therapeutic targets in the past decades, monotherapy has failed in clinical trials; therefore, combination therapy is becoming a key element of present-day antitumor therapy [26]. Other studies showed that NAE1 inhibitor MLN4924 may function as a novel chemosensitizer in various types of cancer such as esophageal squamous cell carcinoma, ovarian cancer, cervical carcinoma, and urothelial carcinoma [23,[27][28][29]. A previous study demonstrated that MLN4924 in combination with cisplatin is an efficient strategy to target cisplatin resistance in ovarian cancer since MLN4924 enhances DNA damage and oxidative stress [28]. MLN4924 sensitizes leukemia cells to retinoic acid-induced apoptosis by inducing the accumulation of c-Jun and NOXA [30]. Liang Zhou et al., reported that MLN4924 in combination with belinostat reciprocally disables the DNA damage response in acute myelogenous leukemia [31]. Moreover, C Paiva et al., reported that MLN4924 in combination with alkylating agents (bendamustine, chlorambucil) sensitizes chronic lymphocytic leukemia B cells to alkylating agents through enhanced DNA damage and apoptosis [32].
Our study revealed that TMZ in combination with the NAE1 inhibitor MLN4924 can successfully decrease cell viability and induce apoptosis even in highly TMZ-resistant glioblastoma cell lines. However, the mechanism through which TMZ in combination with MLN4924 induces apoptosis requires further investigation. Further in vivo experiments are needed to confirm the effectiveness and clinical applicability of the TMZ and MLN4924 combination treatments. at room temperature. Non-specific antibody binding sites were blocked by 3% bovine serum albumin (BSA, Sigma-Aldrich Chemical Co.) in 1× PBS at room temperature for 1 h on a rotator. Anti-MGMT primary antibody (Thermo Fisher Scientific) was added in a final dilution of 1:20 dissolved in 3% BSA in 1× PBS and then incubated overnight at 4 • C. The next day, samples were washed three times with 1× PBS for 5 min. Secondary Cy3-conjugated antibody (Jacksons ImmunoResearch Laboratories, West Grove, PA, USA) was added to the samples in a final dilution of 1:2000 dissolved in 3% BSA in 1× PBS and then cells were gently shaken overnight in the dark at 4 • C on a rotator. Non-specifically bound secondary antibodies were removed by washing the cells three times with 1× PBS. For negative control, cells were incubated only with the secondary antibody. Cell nuclei were stained by Hoechst 33342 (Calbiochem) fluorescent DNA dye in the final concentration of 0.5 µg/mL for 10 min. Samples were mounted onto coverslips using Vectashield (Vector Laboratories) anti-fading mounting medium and visualized by an Olympus FluoView 1000 confocal laser scanning fluorescence microscope (Olympus). The signal intensity for each cell was quantified using ImageJ software (version 1.53t).

Statistical Analysis
Statistical analysis was conducted using Statistica 10 software (Statsoft, Tulsa, OK, USA). Determination of normal distribution was conducted by the Shapiro-Wilk test. Statistical significance was confirmed by the Kruskal-Wallis test followed by Dunn post hoc test if significance was observed. p < 0.05 value was considered statistically significant.