The Antitumor Effect of Dual Inhibition of NAMPT and PAK4 in a 3D Spheroid Model of Platinum-Resistant Ovarian Cancer

Platinum-resistant ovarian cancer is a malignant gynecological tumor that is difficult to treat and has a short patient survival period. In recent years, researchers have sought new treatment strategies, targeting cancer stem cells (CSCs) has become an important direction. CSCs are enriched in ovarian cancer tumor bulk within the abdominal cavity, not only possessing high tumorigenic potential but also being resistant to conventional platinum-based drugs. This study investigates the antitumor effect of an innovative dual-target inhibitor KPT-9274 in a 3D spheroid model, specifically targeting platinum-resistant ovarian cancer.

Research Background

Platinum-based drugs are currently the mainstay treatment for ovarian cancer, but over 80% of high-grade cases will relapse due to tumor resistance to platinum drugs. Although VEGF inhibitors, PARP inhibitors, and immune checkpoint inhibitors have shown some efficacy in certain cases, most patients eventually relapse. Therefore, new treatment strategies are urgently needed.

Researchers have found that targeting CSCs is a potential direction because these cells have growth mechanisms different from ordinary cancer cells. NAD+ is an essential coenzyme for cell survival and growth, and NAMPT (Nicotinamide Phosphoribosyltransferase) is a key rate-limiting enzyme in the NAD+ regeneration cycle. In various cancers, NAMPT inhibitors such as FK-866 and GNE-617 have shown significant antitumor effects. KPT-9274 is a novel oral NAMPT inhibitor and also a PAK4 (P21-Activated Kinase 4) inhibitor, but its effect on ovarian cancer has not been verified.

Study Information

This paper is written by Kei Kudo et al. The authors are from organizations including the National Cancer Institute (NCI) in the United States and Tohoku University School of Medicine in Japan. The paper is published in the journal “Cancer Gene Therapy.”

Research Process

Samples and Cell Culture

This study tested 11 different cancer cell lines, including ovarian cancer, endometrial cancer, and breast cancer. To simulate the actual tumor environment, a 3D spheroid model was used in this experiment. These spheroids are composed of stem cells cultured on ultra-low attachment plates, which can more accurately simulate the morphology and drug response of in vivo tumors.

Cell Viability and Measurements of NAD+, NADPH, and ATP Levels

The XTT method was used to measure cell viability, and the concentrations of NAD+, NADPH, and ATP were measured using colorimetric and fluorometric methods, respectively. The results showed that KPT-9274 significantly inhibited the production of NAD+, NADPH, and ATP in NAMPT-dependent cell lines, indicating that it obstructed cell energy metabolism by inhibiting NAMPT function.

Mitochondrial Function Testing

Mitochondrial function was assessed using the Seahorse XF analyzer. The results showed that in CP80 and Aci-98 cells, KPT-9274 significantly reduced the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), indicating that it inhibited mitochondrial oxidative metabolism and glycolysis. Additionally, KPT-9274 induced a decrease in mitochondrial membrane potential and an increase in reactive oxygen species (ROS), along with significant Caspase ³⁄₇ activity, indicating that it exerts antitumor effects through mitochondrial dysfunction and pro-apoptotic mechanisms.

RNA Sequencing and Bioinformatics Analysis

RNA sequencing technology was used to analyze CP80 cells. The results showed that after KPT-9274 treatment, genes involved in inflammatory responses and cell proliferation were significantly downregulated, while genes related to DNA repair were inhibited. Specifically, the expression levels of genes such as IFNGR1 and IFIT1 were significantly decreased, indicating that KPT-9274 achieves its antitumor effects through the inhibition of multiple signaling pathways.

Protein Expression and Kinase Activity Assay

These findings were further validated by Western blotting and immunofluorescence staining. After KPT-9274 treatment, PAK4 mainly transferred from the cytoplasm to the nucleus, accompanied by a decrease in the activity of key proteins such as Raptor, Akt, and β-catenin. Akt kinase activity analysis showed that KPT-9274 significantly inhibited Akt activity in a concentration-dependent manner.

Research Results

KPT-9274 exhibited significant multiple antitumor effects in NAMPT-dependent cell lines:

1. Inhibition of the production of NAD+, NADPH, and ATP, thereby hindering cell energy metabolism.
2. Inhibition of mitochondrial function, leading to reduced oxygen consumption and glycolysis, while inducing apoptosis.
3. Inhibition of multiple signaling pathways involved in inflammation, cell proliferation, and DNA repair.
4. Alteration of PAK4 localization within the cell, thereby inhibiting its cytoplasmic kinase activity.

Conclusion and Significance

This study confirms the potential therapeutic effect of KPT-9274 on platinum-resistant ovarian cancer, indicating the importance of dual-target inhibitors of NAMPT and PAK4 in precision medicine. Additionally, it suggests that before clinical application, further clarification of the NAMPT dependency of cancer cells is needed to improve the therapeutic effect and predictive accuracy of the drug.

Research Highlights

1. KPT-9274 demonstrated significant antitumor effects in a 3D spheroid model, which more accurately simulates in vivo tumors.
2. Multiple experimental techniques validated the impact of KPT-9274 on cell energy metabolism, mitochondrial function, and apoptosis, proposing new mechanisms.
3. Bioinformatics analysis revealed multiple antitumor pathways of KPT-9274, providing extensive gene expression and signaling pathway data.

This study provides a new treatment strategy for platinum-resistant ovarian cancer, while also highlighting the importance of identifying the NAMPT dependency of tumors in clinical applications through precision medicine.