Fimepinostat is a dual inhibitor of tumor and angiogenesis in glioblastoma and synergizes with temozolomide through suppressing MYC

Abstract

Glioblastoma, an aggressive brain tumor that largely depends on angiogenesis, has limited treatment options and poor prognosis. This study explores the therapeutic potential of fimepinostat, a dual HDAC/PI3K inhibitor, as a single agent alone and in combination of temozolomide in glioblastoma using preclinical tumor and angiogenesis models. We show that fimepinostat at nanomolar concentrations inhibited proliferation and induced apoptosis in a panel of glioblastoma cell lines. In addition, fimepinostat inhibited capillary network formation of microvascular endothelial cells derived from patients, indicating that fimepinostat inhibits glioblastoma angiogenesis. Combination index analysis indicates that fimepinostat and temozolomide is synergistic in inhibiting glioblastoma. Consistent with the in vitro findings, fimepinostat significantly inhibited glioblastoma growth in mice without causing any toxicity. The combination of fimepinostat and temozolomide significantly inhibited tumor growth and prolonged survival compared to monotherapy or control. Mechanism studies confirmed that fimepinostat acts on glioblastoma cells through suppressing Akt/MYC. Our findings suggest that dual targeting of tumor and angiogenesis by fimepinostat may provide an alternative approach for anti-glioblastoma therapy.

INTRODUCTION

Glioblastoma is the most common primary brain tumor and poses a significant challenge in neuro-oncology. The current standard treatment-comprising surgical resection followed by radiotherapy and Temozolomide chemotherapy-has remained largely unchanged for decades, offering a median survival of approximately 15 months, a one-year survival rate of around 41%, and a five-year survival rate of merely 7% [1]. As a highly vascular tumor, glioblastoma depends heavily on angiogenesis to sustain its rapid growth and aggressive behavior. Targeting angiogenesis in glioblastoma can effectively disrupt the tumor's blood supply, thereby impeding its growth and potential to metastasize [2]. Systemic therapy with bevacizumab, an angiogenesis inhibitor that targets vascular endothelial growth factor (VEGF), has been developed for the treatment of recurrent glioblastoma [3,4]. Thus, dual targeting of both the tumor and its angiogenic processes represents a promising therapeutic strategy for glioblastoma.

Fimepinostat (CUDC-907) is a potent, orally bioavailable, dual inhibitor targeting histone deacetylases (HDACs) and phosphoinositide 3-kinase (PI3K) [5]. By inhibiting both HDAC and PI3K pathways, fimepinostat exerts a multifaceted anti-cancer effect across malignancies, leading to the downregulation of oncogenic transcription factors like MYC and the disruption of key signaling pathways involved in tumor growth and resistance to apoptosis. In diffuse large B-cell lymphoma (DLBCL), fimepinostat has been shown to effectively suppress tumor growth by reducing MYC expression and inducing apoptosis [6]. Similarly, in multiple myeloma and solid tumors such as breast and colorectal cancers, fimepinostat has demonstrated the ability to inhibit cell proliferation and enhance the efficacy of other anti-cancer agents [7-14]. Phase I and II trials have further demonstrated its safety, tolerability, and preliminary efficacy in patients with relapsed or refractory lymphomas, including DLBCL and follicular lymphoma [15,16].

In this study, we explored the therapeutic potential of fimepinostat both as a single agent and in combination with temozolomide in glioblastoma, utilizing preclinical models of tumor growth and angiogenesis. Additionally, we aimed to elucidate the underlying mechanisms of fimepinostat’s action in glioblastoma.

METHODS

Cell culture, compounds and Western blotting

T98G, A172, U251, U87, and U373 glioblastoma cell lines were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified atmosphere containing 5% CO₂. Fimepinostat (Cat No.: S2759) and temozolomide (Cat No.: S1237) were obtained from Selleckchem and were reconstituted in dimethyl sulfoxide. Glioblastoma microvascular endothelial cells (GMECs) were isolated and enriched from glioblastoma tissues using fluorescence-activated cell sorting, following the protocol detailed in our previous studies [17,18]. GMECs were then cultured in Endothelial cell medium (Cell System, Cat No. 4Z0-500). Glioblastoma tissues were collected during surgical procedures from patients at the Affiliated Hospital of Southwest Medical University, following the acquisition of informed consent, as approved by the Institutional Review Board of Southwest Medical University (IRB No. 0156-202306). Western blotting was performed using the standard protocol and antibodies against phosphor and total Akt, MYC, Bcl-2, Bcl-xL and β-actin (Cell Signaling Inc.).

Proliferation and apoptosis assay

Cells were plated and exposed to fimepinostat at doses of 0, 5, 50, and 500 nM for a duration of 72 h. Following treatment, cell proliferation was assessed using the BrdU Proliferation Assay Kit (Abcam). To evaluate apoptosis, treated cells were detached and labeled with Annexin V-FITC and 7-AAD (Abcam). The proportion of Annexin V-positive cells was quantified through flow cytometry. Early apoptotic cells were defined as Annexin V_⁺/7-AAD_⁻, while late apoptotic cells were identified as Annexin V_⁺/7-AAD_⁺.

Caspase 3 activity assay

Cells were seeded in 96-well plates and treated with fimepinostat at 0, 5, 50, or 500 nM for 48 h. Caspase-3 activity was measured using the Caspase-Glo 3/7 Assay Kit (Promega), following the manufacturer’s instructions. Luminescence was recorded using a microplate reader, and relative activity was normalized to untreated controls.

Combination index (CI) analysis

Cells were seeded in 96-well plates and treated with fimepinostat, temozolomide and combination of both. The drug treatments were applied in a fixed-ratio combination based on dose-response curves for each agent. After treatment, cell proliferation was assessed using BrdU Proliferation Assay Kit (Abcam). The CI was calculated using the Compusyn software.

Capillary network formation assay

The capillary network formation assay was conducted utilizing Corning Matrigel matrix, following the procedure outlined in our prior studies [17,18]. GMECs at 10,000 cells per well were plated in 96-well plates pre-coated with Matrigel. Capillary tube formation was assessed after 8-h of incubation. Images were obtained with a phase-contrast microscope, and the total length of the capillary-like structures was measured using ImageJ software.

MYC activity assay

MYC transcriptional activity was assessed using the Human c-Myc Activity Assay Kit (RayBiotech). Briefly, cell lysates were prepared and incubated with an oligonucleotide containing the MYC consensus binding site. The DNA-protein complex was detected using a primary antibody specific to MYC. The signal was developed using a colorimetric substrate, and absorbance was recorded at 450 nm.

Glioblastoma xenograft growth in vivo

Female athymic nude mice, aged 4–6 weeks, were utilized to develop a glioblastoma xenograft model by subcutaneous injection of 5 × 10⁶ U87 cells into the flanks. When the tumors reached an average size of about 150 mm³, the mice were randomly assigned into four groups (n = 8 per group) and treated with vehicle (control), temozolomide (5 mg/kg), fimepinostat (10 mg/kg), or a combination of both drugs. The treatments were administered by oral gavage daily for 21 days. Tumor growth was monitored by measuring tumor size with calipers every 5 days, and tumor volumes were calculated. Tumor growth was monitored for 65 days, with survival recorded from the beginning of treatment until either death or the conclusion of the 80-day study. Kaplan–Meier survival curves were generated.

Statistical analyses

Statistical comparisons were conducted using the Student’s t-test for two groups, while one-way ANOVA followed by Tukey's post-hoc test was employed for comparisons across multiple groups. Each data point represents the average value obtained from five distinct mice. Statistical significance was defined as a p-value of less than 0.05.

RESULTS

Fimepinostat at nano-molar inhibits cell proliferation and apoptosis in multiple glioblastoma cell lines

We firstly evaluated the effects of fimepinostat on glioblastoma cell proliferation and survival to assess its potential as a therapeutic agent. We applied multiple glioblastoma cell lines that covers a wide range of cellular origin and genetic profiling and fimepinostat concentrations that are clinically achievable. BrdU incorporation assays revealed a dose-dependent inhibition of cell proliferation across all tested glioblastoma cell lines following fimepinostat treatment (p < 0.05), with IC50 at concentration < 50 nM in most cell lines (Fig. 1A). Furthermore, the induction of apoptosis by fimepinostat was evaluated using flow cytometry of Annexin V/7-AAD. The results demonstrated a dose-dependent increase in the percentage of Annexin V⁺/7-AAD^- (early apoptosis) and Annexin V⁺/7-AAD⁺ (late apoptosis) cells, suggesting enhanced apoptotic activity (Fig. 1B and Supplementary Fig. 1). This effect was most pronounced at 500 nM, where all cell lines exhibited a significant increase in apoptosis compared to untreated controls (p < 0.05).

Fig. 1. Fimepinostat inhibits proliferation and induces apoptosis in glioblastoma cells.

(A) BrdU incorporation assay was used to measure cell proliferation in T98G, A172, U251, U87, and U373 glioblastoma cell lines treated with increasing concentrations of fimepinostat. The data are presented as BrdU incorporation relative to the control (0 nM). (B) Apoptosis induction was assessed using an Annexin V assay in the same glioblastoma cell lines treated with fimepinostat at the indicated concentrations. The percentage of Annexin V-positive cells is shown. Data are expressed as mean ± SD from three independent experiments. *p < 0.05 compared to the control group.

To further characterize the pro-apoptotic effects of fimepinostat, we assessed caspase-3 activity and the expression of apoptosis-related proteins in glioblastoma cells. As shown in Fig. 2A, treatment with fimepinostat resulted in a dose-dependent increase in caspase-3 activity across all tested cell lines, with the most pronounced activation observed at 500 nM. Western blot analysis showed that fimepinostat treatment led to a marked reduction in Bcl-2 protein levels in all five glioblastoma cell lines tested (Fig. 2B). In contrast, the level of Bcl-xL remained largely unchanged following treatment. These results suggest that fimepinostat promotes apoptosis in glioblastoma cells by activating caspase-3 and downregulating anti-apoptotic Bcl-2 family proteins.

Fig. 2. Fimepinostat induces apoptosis in glioblastoma cells through caspase activation and Bcl-2 suppression.

(A) Caspase-3 activity was assessed in T98G, A172, U251, U87, and U373 following treatment with increasing concentrations of fimepinostat (0, 5, 50, and 500 nM) for 48 h. Caspase-3 activity was measured using the Caspase-Glo 3/7 assay and normalized to the untreated control. Data are presented as mean ± SD from three independent experiments. *p < 0.05 compared to the control group. (B) Western blot analysis of anti-apoptotic proteins Bcl-2 and Bcl-xL in glioblastoma cells treated with fimepinostat (0, 50, or 500 nM) for 48 h. β-actin was used as a loading control. Fimepinostat treatment resulted in a dose-dependent decrease in Bcl-2 expression, while Bcl-xL levels remained largely unchanged.

Combination of fimepinostat and temozolomide is synergistic in inhibiting proliferation and inducing apoptosis in glioblastoma cells

To evaluate the combinatory effects of fimepinostat in combination with chemotherapeutic drug temozolomide on proliferation, we performed a CI analysis across multiple glioblastoma cell lines. The CI values were plotted against the fraction of affected cells for each cell line, as shown in Fig. 3A–E. In all tested glioblastoma cell lines, the combination of fimepinostat with temozolomide resulted in CI values predominantly below 1, indicating a synergistic interaction. Notably, U251 and U373 cell lines demonstrated a pronounced synergy, as evidenced by the downward trend in CI values with increasing fractions of affected cells. The T98G, A172, and U87 cell lines also exhibited synergistic effects, though to a lesser extent, as their CI values remained below or around 1. These results suggest that fimepinostat, when used in combination with temozolomide, can enhance the therapeutic efficacy against glioblastoma. Consistently, the combination of fimepinostat and temozolomide resulted in a significantly greater induction of apoptosis in glioblastoma cells compared to either drug alone (Fig. 3F).

Fig. 3. Fimepinostat and temozolomide combination is synergistic in glioblastoma cells.

The combination index (CI) values were calculated for the cell lines (A) T98G, (B) A172, (C) U251, (D) U87, and (E) U373 treated with a combination of fimepinostat and temozolomide at varying concentrations. The CI values are plotted against the fraction of affected cells, with CI < 1 indicating synergy, CI = 1 indicating an additive effect, and CI > 1 indicating antagonism. (F) Combination treatment with fimepinostat and temozolomide significantly enhances apoptosis in glioblastoma cells. Each point represents the mean of three independent experiments. *p < 0.05 compared to the control group. ^#p < 0.05 compared to single arm.

Fimepinostat inhibits capillary network formation in glioblastoma-derived GMECs

To evaluate fimepinostat's effect on angiogenesis, we performed a Matrigel tube formation assay, an in vitro model for endothelial cell differentiation and tube formation. We used GMECs to replicate a clinically relevant glioblastoma angiogenesis model. In the control group, extensive tubule formation was observed within 6 h of plating GMECs onto Matrigel (Fig. 4A). Treatment with fimepinostat markedly disrupted the formation of capillary-like structures in GMECs from all three patients. Quantitative analysis revealed a dose-dependent reduction in capillary tube length relative to control-treated cells (Fig. 4B). At the highest concentration of 500 nM, capillary tube length was significantly reduced in all patient-derived GMECs (p < 0.05). Even at lower concentrations (5 and 50 nM), fimepinostat significantly inhibited tube formation. These results show that fimepinostat effectively inhibits angiogenesis in GMECs derived from patients with glioblastoma, suggesting its potential as an anti-angiogenic agent in the treatment of glioblastoma.

Fig. 4. Fimepinostat inhibits capillary tube formation in glioblastoma microvascular endothelial cells (GMECs) from patient-derived samples.

(A) Representative images of capillary tube formation in GMECs isolated from three different patients with glioblastoma (Patient #1, Patient #2, and Patient #3) treated with control (vehicle) or fimepinostat at 500 nM (10× magnification). (B) Quantification of capillary tube length relative to the control for GMECs from each patient treated with fimepinostat at increasing concentrations (0, 5, 50, and 500 nM). Data are expressed as mean ± SD from three independent experiments. *p < 0.05 compared to the control group.

Fimepinostat acts on glioblastoma cells through in a MYC-dependent manner

To investigate the mechanism by which fimepinostat exerts its anti-cancer effects in glioblastoma cells, we examined the AKT/c-Myc signaling pathway in T98G and U251 cells. Immunoblotting analysis demonstrated that treatment with fimepinostat led to a dose-dependent decrease in p-Akt and c-Myc protein levels, while total AKT levels remained unchanged (Fig. 5A). Consistently, MYC activity was significantly reduced in both T98G and U251 cells treated with fimepinostat (Fig. 5B, p < 0.05). Consistently, we further demonstrated that fimepinostat significantly decreased the mRNA expression levels of well-characterized MYC downstream targets-ODC1, CCND2, and CDK4-in T98G and U251 cells (Supplementary Fig. 2). MYC is a well-known transcription factor that directly or indirectly regulates the expression of several pro-angiogenic genes, including VEGF and angiopoietin-2 [19]. Since our study demonstrated that fimepinostat inhibits MYC activity in glioblastoma cells, we examined the expression level of VEGF-A and angiopoietin-2 in glioblastoma cells after fimepinostat treatment. Consistently, we revealed a dose-dependent downregulation of VEGF-A and angiopoietin-2 in T98G and U251 cells (Supplementary Fig. 3).

Fig. 5. Fimepinostat acts on glioblastoma cells.

via Akt/c-MYC pathway. (A) Western blot analysis of p-Akt, Akt, c-Myc, and β-actin in T98G and U251 glioblastoma cells treated with fimepinostat at concentrations of 0, 5, 50, and 500 nM. β-actin was used as a loading control. (B) MYC transcriptional activity was measured in T98G and U251 cells treated with increasing concentrations of fimepinostat. Data are presented as MYC activity relative to the control (0 nM fimepinostat). (C) Annexin V assay showing the percentage of apoptotic cells in T98G and U251 cells treated with 500 nM fimepinostat, with or without MYC overexpression. Data are expressed as mean ± SD from three independent experiments. *p < 0.05 compared to the control group.

Rescue experiments by overexpressing MYC in GMB cells followed by fimepinostat showed that overexpression of MYC significantly reversed cells from fimepinostat-induced apoptosis (Fig. 5C), suggesting that MYC downregulation contributes to the pro-apoptotic effects of fimepinostat. These results indicate that fimepinostat inhibits glioblastoma cells through the suppression of the AKT/c-Myc signaling pathway.

Combination of fimepinostat and temozolomide reduces tumor growth and extends survival in glioblastoma xenografts

We finally investigated the translational potential of fimepinostat in glioblastoma using mouse xenograft models. To evaluate potential systemic toxicity, a preliminary study was conducted in non–tumor-bearing mice treated with temozolomide, fimepinostat, or their combination. Body weight was monitored over a 60-day treatment period. As shown in Fig. 6A, all treatment groups exhibited comparable weight gain to the control group. No significant weight loss was observed, indicating that the treatments were well tolerated and did not induce overt systemic toxicity under the tested conditions. To assess the therapeutic efficacy of fimepinostat alone and in combination with temozolomide, we evaluated tumor growth and survival in a glioblastoma xenograft model. Fimepinostat alone significantly inhibited glioblastoma tumor growth compared to control (Fig. 6B). In addition, the combination of fimepinostat and temozolomide led to a significant and sustained suppression of tumor growth throughout the study period, with tumor volumes remaining nearly static even after day 65 (p < 0.05 compared to both monotherapy groups). Survival analysis demonstrated a significant improvement in survival in combination therapy compared to the control, temozolomide, and fimepinostat monotherapy groups (Fig. 6C, p < 0.05). The median survival was notably extended in the combination therapy group, with several mice surviving beyond the study's 60-day observation period, while all mice in the control group succumbed by day 45. These results are consistent with in vitro findings, indicating that the combination of fimepinostat and temozolomide offers a synergistic effect, significantly reducing tumor growth and improving survival in a glioblastoma xenograft model.

Fig. 6. Fimepinostat and temozolomide combination reduces tumor growth and prolongs survival in a glioblastoma xenograft model.

(A) Body weight monitoring in non–tumor-bearing mice treated with temozolomide (5 mg/kg), fimepinostat (10 mg/kg), or their combination. Mice were treated for 60 days, and body weight was indicated on Day 0 and Day 60. (B) Tumor growth curves of glioblastoma xenograft-bearing mice treated with control (vehicle), temozolomide (5 mg/kg), fimepinostat (10 mg/kg), or a combination of both drugs. Tumor size was measured every 5 days and is presented as mean tumor volume (mm³) ± SD (n = 8 per group). (C) Kaplan–Meier survival curves for glioblastoma xenograft-bearing mice treated with control (vehicle), temozolomide, fimepinostat, or the combination of both drugs. The probability of survival is plotted against time (days). *p < 0.05 compared to the control group. ^#p < 0.05 compared to the single drug alone.

DISCUSSION

Targeting the PI3K/Akt pathway and HDAC enzymes has emerged as an effective therapeutic strategy in glioblastoma [20,21]. Currently, five PI3K inhibitors and one Akt inhibitor have received clinical approval, primarily for the treatment of breast cancer and hematologic malignancies [22,23]. Additionally, four HDAC inhibitors have been approved by the Food and Drug Administration for treating haematological malignancies [24]. The ongoing development of novel inhibitors targeting PI3K/Akt and HDAC pathways remains a significant focus in oncology research, aiming to improve therapeutic selectivity and efficacy. A combination of PI3K/mTOR inhibitor BEZ235 and HDAC inhibitor panobinostat has been shown to synergistically inhibit proliferation and induces apoptosis in glioblastoma cells [25]. Fimepinostat, a dual inhibitor of PI3K and HDAC, offers advantages over the combination of PI3K inhibitor and HDAC inhibitor. In the present study, we show that fimepinostat is a promising candidate for the treatment of glioblastoma due to its efficacy as a single agent and its synergistic effects when combined with temozolomide.

In haematological malignancies, fimepinostat is administrated at a dose of 60 mg orally on a 5 days on/2 days off schedule, resulting in a maximum plasma concentration of 22 nM [16,26]. Our findings demonstrate that fimepinostat inhibits proliferation of all tested glioblastoma cell lines, with an IC50 of approximately 50 nM, suggesting that clinically achievable doses of fimepinostat could be effective in glioblastoma treatment. Additionally, capillary network formation analysis using endothelial cells isolated from patients with glioblastoma show that fimepinostat inhibits glioblastoma angiogenesis, indicating that fimepinostat targets not only tumor but also its angiogenic processes. While the anti-cancer activity of fimepinostat has been well studied [7,8,14], to our knowledge, we are the first to demonstrate its potential as an angiogenesis inhibitor. This is particularly significant because dual targeting of tumor and its angiogenesis has several advantages, including lower toxicity and greater therapeutic efficacy.

We employed a subcutaneous xenograft model for proof-of-concept studies, allowing precise tumor volume measurements, reproducible growth kinetics, and reliable drug response assessment. In an in vivo glioblastoma xenograft model, we demonstrated that fimepinostat, administered at non-toxic dose, significantly inhibited tumor growth and extended overall survival in mice. This aligns with the findings of Li et al. [27], where fimepinostat encapsulated in classical monocytes significantly improved survival rates by suppressing tumor growth and reversing immunosuppression in the tumor microenvironment. Our study further extends these findings by showing that the combination of fimepinostat and temozolomide is significantly more effective than either agent alone in inhibiting tumor growth and improving survival rates. Given the challenges associated with glioblastoma treatment, particularly blood-brain barrier penetration, further studies using orthotopic implantation models will be beneficial in confirming the in vivo efficacy of fimepinostat in the brain tumor microenvironment. Notably, a clinical trial is currently underway to evaluate how well fimepinostat works for the treatment of brain cancer, with the primary objective of confirming fimepinostat’s ability to penetrate the blood-brain barrier (NCT03893487).

Consistent with the previous studies showing that HDAC inhibition downregulates MYC mRNA expression and PI3K inhibition destabilizes MYC protein [28,29], we observed the remarkable reduction in MYC protein levels in fimepinostat-treated glioblastoma cells. This reduction in MYC was accompanied by a marked decrease in glioblastoma cell proliferation and survival, underscoring the critical role of MYC in these processes. Rescue experiments further confirmed that MYC inhibition is a key underlying mechanism of fimepinostat’s action in glioblastoma, as restoring MYC expression partially reversed the anti-tumor effects of fimepinostat. The strong correlation between c-Myc expression and tumor malignancy grade-being low in Grade I and II tumors and high in Grade III and IV-highlights its importance in the aggressive phenotype of glioblastoma. c-Myc is a master regulator of glioblastoma metabolism, survival, and growth [30,31], and its inhibition by fimepinostat disrupts these vital processes, thereby offering a targeted therapeutic approach for glioblastoma. This mechanism not only provides a deeper understanding of fimepinostat’s efficacy but also positions MYC as a crucial target in the treatment of glioblastoma. Of note, the synergistic effects observed with the combination treatment are mechanistically attributed to dual targeting of distinct oncogenic processes: temozolomide exerts its effect via DNA alkylation and methylation, while fimepinostat targets epigenetic and signaling pathways through MYC inhibition. These complementary mechanisms converge on critical survival pathways in glioblastoma, resulting in enhanced efficacy both in vitro and in vivo.

In conclusion, our study demonstrates that fimepinostat effectively targets glioblastoma by downregulating MYC expression, leading to reduced tumor cell proliferation, survival, and angiogenesis. The ability of fimepinostat to inhibit both tumor growth and the tumor’s angiogenic processes, particularly when combined with temozolomide, highlights its potential as a powerful therapeutic strategy for glioblastoma.

SUPPLEMENTARY MATERIALS

Supplementary data including three figures can be found with this article online at https://doi.org/10.4196/kjpp.25.056