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Dual-inhibition of NAMPT and PAK4 induces anti-tumor effects in 3D-spheroids model of platinum-resistant ovarian cancer – Cancer Gene Therapy

KPT-9274 is a potent and selective NAMPT inhibitor

To investigate the relevance of NAMPT and PAK4 in ovarian cancers, we first examined TCGA datasets. High expression of NAMPT was correlated with a significant reduction in overall survival in human ovarian cancer, suggesting that high NAMPT expression may be a prognostic factor in ovarian cancer. Similar results were observed in cervical and endometrial cancers, but there was no significant difference in breast cancer. High PAK4 expression lacked significant negative prognosis in these cancers, although a trend towards worse outcomes existed in endometrial cancer. (Fig. 1C and Supplementary Fig. 1B).

To assess preclinical effectiveness of KPT-9274 in gynecological cancers, we tested the effect on cell viability of 3D-spheroids from 11 cell lines of different histologic subtypes. The cell lines we used in this study had varying degrees of sensitivity to KPT-9274, and differed in their ability to be rescued by NMN or NA addition (Table 1). Based on manufacturer’s recommended concentrations and previous reports, KPT-9274 was tested up to 1000 nM as the highest concentration [24,25,26,27, 31]. The efficacy of KPT-9274 was demonstrated against A2780, 1A9CP80, CP80, IGROV1 and OVCAR8 in ovarian cancer, ACI-98 in endometrial cancer and T47D in breast cancer with IC50 25–83 nM. In contrast, KPT-9274 did not inhibit the viability of SKOV3, EFE-184, KLE and MCF-7 at the highest dose, indicating NAD+ synthesis independent from NAMPT in these cells (Supplementary Fig. 1C). As expected, addition of NMN (downstream of NAMPT) rescued KPT-9274 impact across all cell lines (NMN rescue). To further test whether the cell lines produced NAD+ from NA by other pathways, rescue experiments were performed. We observed that NA, but not NMN, rescued the cytotoxic effect of KPT-9274 in OVCAR8 (NA rescue) (Fig. 1D and Supplementary Fig. 1D). Notably, the NAD+ production pathway differed across cell lines, suggesting biomarker analysis might be necessary to clarify the pathway involved before clinical application of KPT-9274 (Supplementary Fig. 1E).

Table 1 A list of cell lines used in this study and a summary of the results for each cell line.

Next, to examine KPT-9274’s potential in platinum-resistant ovarian cancers, we tested using different cell lines, including platinum-sensitive (A2780) and platinum-resistant sub-lines (1A9CP80 and CP80). Based on clinical studies that reported the blood concentration of cisplatin [32, 33], the maximum concentration of cisplatin in this experiment was set at 20 μM. KPT-9274 demonstrated similar anti-tumor effects to cisplatin on A2780 (Fig. 1E). Notably, we observed KPT-9274 was significantly more effective than cisplatin in 1A9CP80 and CP80. Therefore, KPT-9274 could be a promising treatment for ovarian cancer that has developed resistance to platinum-based therapies.

KPT-9274 suppresses the production of NAD+, NADPH, and ATP

To assess KPT-9274 impact on NAMPT-dependent cell lines, we first measured NAD+ and NADPH production at various concentrations. Using 3D-cultured CP80, ACI-98, and IGROV1, KPT-9274 inhibited NAD+ and NADPH production in a dose-dependent manner (Fig. 2A, B).

Fig. 2: KPT-9274 suppressed the production of NAD+, NADPH, and ATP.
figure 2

A Change in total NAD levels in 3D-cultured CP80, ACI-98, and IGROV1 after treatment with KPT-9274 for 48 h at indicated doses relative to Control. (n = 4 independent experiments). B Change in total NADP levels in 3D-cultured CP80, ACI-98, and IGROV1 after treatment with KPT-9274 for 48 h at indicated doses relative to Control. (n = 4 independent experiments). C Change in total ATP levels in 3D-cultured CP80, ACI-98, and IGROV1 after treatment with KPT-9274 for 48 h at indicated doses relative to Control. (n = 4 independent experiments). Graph data were presented as mean ± SEM with n = 4 per group.

To further investigate the mechanism, we next tested the effect of KTP-9274 on ATP production, as NAD+ is essential for ATP generation through glycolysis and the TCA cycle [34]. Consistent with the effect on NAD+ and NADPH production, KPT-9274 treatment significantly reduced ATP levels (Fig. 2C). Together, KPT-9274 is a selective NAMPT inhibitor that causes a multifaceted anti-tumor effect against NAMPT-dependent cell lines. It inhibits NAD+, NADPH, and ATP production, suggesting a comprehensive disruption of vital cellular processes.

KPT-9274 causes the suppression of mitochondrial function

Based on the inhibitory effect on NAD+, NADPH and ATP, we next hypothesized that KPT-9274 affects mitochondrial functions. Using the Mito Stress Test with XFe96, we assessed KPT-9274 impact on mitochondria function in 3D-cultured CP80 and ACI-98. As anticipated, KPT-9274 reduced oxygen consumption rate (OCR), an established measure of mitochondrial function [35], in CP80 and ACI-98 cells in 3D-spheroids, affecting both basal and maximal respiration (Fig. 3A,B). Interestingly, KPT-9274 significantly suppressed not only OCR, but also maximal extracellular acidification rate (ECAR), reflecting glycolysis (Fig. 3C). NAD+ is a co-enzyme in the reaction catalyzed by Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is an enzyme essential for the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglyceric acid in glycolysis [36]. Hence, we hypothesized KPT-9274 inhibits GAPDH. As anticipated, KPT-9274 inhibited the GAPDH-mediated reaction, and adding NMN to the medium reversed the inhibition (Fig. 3D). These findings suggest KPT-9274 suppresses not only mitochondrial ATP production, but also glycolysis.

Fig. 3: KPT-9274 causes the suppression of mitochondrial function.
figure 3

A Representative OCR pattern in 3D-cultured CP80 and ACI-98 as a function of time (min), normalized with spheroid size. The spheroids were treated with KPT-9274 for 48 h at indicated doses. Oligomycin (Oligo), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A (R/A) were added to measure Basal OCR, ATP content, maximal OCR, and Non-mitochondrial OCR. (n = 8 independent experiments) Basal: Basal respiration, ATP-Linked: ATP-Linked Production, Maximal: Maximal respiration, Non Mt: Nonmitochondrial respiration. B Maximal respiratory capacity in OCR (n = 8 independent experiments). C Maximal glycolytic capacity in ECAR (n = 8 independent experiments). D Change in GAPDH-mediated reaction normalized by protein concentration in 3D-cultured CP80 and ACI-98 after treatment with KPT-9274 for 48 h at indicated doses relative to Control. NMN were added into media at indicated doses for confirming NMN rescue. (n = 3 independent experiments). E Change in TMRM intensity normalized with cell number in 3D-cultured CP80 and ACI-98 after treatment with KPT-9274 for 48 h at indicated doses relative to Control. (n = 6 independent experiments). F Change in MitoSOXTM Red intensity normalized with cell viability in 3D-cultured CP80 and ACI-98 after treatment with KPT-9274 for 48 h at indicated doses relative to Control. (n = 6 independent experiments). G Left: Fluorescence analysis of CP80 spheroids after treatment with KPT-9274 at indicated doses. The spheroids were treated 3days after seeding cells. Time-dependent bright field and fluorescent overlay images of Cleaved caspase-3/7 for the spheroids. Right: Quantification of Green Mean Intensity as a function of time (days) using IncuCyteTM S3. (n = 4 independent experiments). H Change in GF-AFC substrate intensity (left), bis-AAF-R110 substrate intensity normalized with viability (middle), and cleaved caspase-3/7 normalized with viability (right) in 3D-cultured CP80 after treatment with KPT-9274 for 96 h at indicated doses relative to Control. (n = 5 independent experiments). I Changes in treatment with Z-VAD-FMK 20 μM for 1 h before the same treatment as (H). (n = 5 independent experiments). J Comparison in cleaved caspase-3/7 normalized with viability in the absence and presence of prior Z-VAD-FMK. (n = 5 independent experiments). Graph data were presented as mean ± SEM with n = 3 or 4 or 5 or 6 or 8 per group.

Furthermore, we investigated the impact of KPT-9274 on mitochondrial membrane potential, using TMRM, a fluorescent dye dye which accumulates in active mitochondria with intact potentials, whch emits a bright signal in healthy cells. KPT-9274 significantly suppressed TMRM in CP80 and ACI-98 cells in 3D-spheroids (Fig. 3E). Conversely, we observed up-regulated MitoSOXTM Red which reflects reactive oxygen species (ROS) generated in mitochondria of live cells (Fig. 3F). Moreover, cleaved caspase-3/7 signal was monitored over time using IncuCyteTM Caspase-3/7 Green Dye. KPT-9274 treatment significantly up-regulated green fluorescence intensity per area of spheroid, indicating caspase 3/7 activity was induced by KPT-9274 (Fig. 3G and Supplementary Fig. 2A). To evaluate whether KPT-9274 induces cell death, we quantified viability, cytotoxicity, and apoptosis induction using ApoTox-Glo™ Triplex Assay Kit, with or without a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) [37], after 96 h of KPT-9274 treatment at varying doses. As anticipated, KPT-9274 suppressed cell viability and induced cytotoxicity as well as cleaved caspase-3/7 activity (Fig. 3H, J). Pre-treatment with Z-VAD-FMK inhibited only cleaved caspase-3/7 secretion, while having no considerable effect on cytotoxicity (Fig. 3I,J). These results suggest that caspase-3/7 activity is a part of anti-tumor effects of KPT-9274, but not entirely attributed to the cytotoxicity.

NAMPT correlates with inflammatory gene expression and PAK4 is associated with DNA repair genes in ovarian cancer patients

To further characterize the impact of NAMPT and PAK4 in ovarian cancer, we evaluated the ovarian cancer RNA sequencing data from TCGA. We first compared patients with high and low NAMPT expression (Lower percentile = 25% (n = 73), Upper percentile = 25% (n = 73)) and developed a heatmap and volcano plot to detect DEGs (Fig. 4A,B). Top 20 DEGs between NAMPT high and low expression patients included NAMPT, NAMPTP1, ARMC10, CAPZA2, CXCL8, CCDC71L, NCOA7, PMAIP1, SYPL1, PNPLA8, CXCL2, CEBPD, CCL20, ZBED6, FAM66A, PNMA8B, PYCR2, PSMC2, SOD2, and STEAP1 (Supplementary Fig. 3A and Supplementary Table. 1). GSEA revealed that patients with high NAMPT expression exhibited enriched gene sets related to inflammation in hallmark gene sets and KEGG pathway database. The top five up-regulated gene sets in NAMPT-high patients were TNF-α signaling via NFκB, Interferon-γ response, Interferon-α response, and Apoptosis. Moreover, using KEGG pathway, the top five up-regulated gene sets were Cytokine-cytokine-receptor interaction, Chemokine signaling pathway, JAK-STAT signaling pathway, Nicotinate and nicotinamide metabolism, and Apoptosis (Fig. 4C and Supplementary Fig. 3B). Notably, the findings highlight a connection between high NAMPT expression and increased inflammation, suggesting the increased inflammation may contribute to a poorer prognosis in ovarian cancer patients.

Fig. 4: NAMPT correlates with inflammatory gene expression and PAK4 is associated with DNA repair genes in ovarian cancer patients.
figure 4

A Heat map shows the comparison of transcripts from the ovarian cancer tumors of NAMPT high patients and NAMPT low patients in different samples from TCGA. (Cutoff used: p < 1e-6). B Volcano plot showing distinct transcriptomic signatures in the NAMPT high and NAMPT low tumors. Volcano plot was generated to identify genes that were differentially enriched. (Cutoff used: |Difference (Log2 Fold Change) of group means | > 1, and -Log10 (p-value) > 1). C Normalized enrichment score of various gene sets in NAMPT high group using Hallmark gene sets in MSigDB and KEGG pathway DB are shown in bar plots. D Heat map shows the comparison with transcripts with the ovarian cancer tumors of PAK4 high patients and NAMPT low patients in different samples from TCGA. (Cutoff used: p < 1e-11). E Volcano plot showing distinct transcriptomic signatures in the PAK4 high and PAK4 low tumors. Volcano plot was generated to identify genes that were differentially enriched. (Cutoff used: |Difference (Log2 Fold Change) of group means | > 1, and -Log10 (p-value) > 1). F Normalized enrichment score of various gene sets in PAK4 high group using Hallmark gene sets in MSigDB and KEGG pathway DB are shown in bar plots.

Similarly, we next identified DEGs in patients with high and low PAK4 expression (n = 73, respectively). Results were visualized with a heatmap and volcano plot, highlighting 20 significant DEGs between PAK4 high and low expression patients including PAK4, POLR2I, ECH1, CAPNS1, PTOV1, RBPJ, ZNF628, YIF1B, KPNA5, RPL26P6, NDUFS7, ZNF865, ZC3H3, FRA10AC1, ZNF574, LY96, FRG1, MRPL2, C19orf47, and PPDPF (Supplementary Fig. 3C and Supplementary Table. 2). All 23 DEGs were highly expressed in high PAK4 patients (p < 1e-11) (Fig. 4D, E). GSEA revealed that the top five up-regulated gene sets were G2M checkpoint, DNA repair, mTORC1 signaling, Wnt/β-Catenin signaling, and PI3K-AKT-MTOR signaling in Hallmark gene sets. Moreover, top five up-regulated gene sets were Cell cycle, DNA replication, Mismatch repair, Base excision repair, and Homologous recombination in the KEGG pathway (Fig. 4F and Supplementary Fig. 3D). Collectively, these findings suggest elevated gene repair and cell proliferation functions in high PAK4 patients, potentially contributing to tumor cell survival and replication.

KPT-9274 triggers suppression of inflammatory signaling

We hypothesized that the anti-tumor effects of KPT-9274 arose from inhibition of gene expression related to inflammation, gene repair, and cell proliferation signaling. To validate this hypothesis, we performed RNA-seq analysis on 3D-cultured CP80 cells treated with DMSO (Control) or KPT-9274 for 24 h. First, principal component analysis (PCA) demonstrated that technical replicates in each group clustered together, indicating low variation between the replicate samples (Fig. 5A). Next, we conducted hierarchical clustering analysis to detect the DEGs based on RNA-seq data and constructed a heatmap and volcano plot to visualize the impact of KPT-9274 treatment. The top 20 DEGs between Control and KPT-9274 treatment were CA14, NLGN3, SCARA5, HDGF, NQO1, HMGA2, ERP27, HSD17B7, PPP2R5B, MYOF, PYM1, CDC42EP4, ACTA2, NQO2, YIPF6, ATXN2, PTMA, SLC30A8, SCN9A, and ZBTB2 (Supplementary Table 3). Interestingly, SNHG25, known for promoting ovarian cancer progression [38], and TMEM52B, associated with EGFR and E-cadherin modulation and tumor/metastasis suppression [39], significantly decreased with KPT-9274 treatment (Fig. 5B, C). Next, GSEA revealed the top five up-regulated gene sets in the Control compared to KPT-9274 treatment: Myc-targets-V1, Hedgehog signaling, Epithelial mesenchymal transition, Allograft rejection, and Interferon-γ in Hallmark gene sets. The up-regulated gene sets in KEGG pathway included DNA replication, Proteasome, Mismatch repair, O-glycan biosynthesis, and Pentose phosphate pathway (Fig. 5D). Our findings suggest that KPT-9274 regulates cell proliferation by suppressing the expression of these tumor growth-associated genes and pathways.

Fig. 5: KPT-9274 triggers suppression of inflammatory signaling.
figure 5

A PCA showing gene profiles of 3D-cultured CP80 after treatment with KPT-9274 1000 nM for 24 h relative to Control. (Results shown are from four independent experiments). B Heatmap representing DEGs in treated 3D-cultured CP80 as described above. (Cutoff used: p < 1e-5). C Volcano plot generated to identify DEGs in 3D-cultured CP80 after KPT-9274 treatment relative to Control. (Cutoff used: |Difference (Log2 Fold Change) of group means | >1, and -Log10 (p-value) >1). D Left: Normalized enrichment score of various gene sets in Control group relative to KPT-9274 treatment are shown in bar plots. Right: GSEA in Control group relative to KPT-9274 treatment. (Top: Hallmark gene sets in MsigDB, bottom: KEGG pathway DB). E Top: Pathways affected by KPT-9274 treatment as identified by Ingenuity pathway analysis (IPA). Bottom: Normalized gene expression levels associated with Interferon Signaling in Control and KPT-9274 treatment. (n = 4 independent experiments). F Top: Immunoblotting for assessing the expression of IFNGR1, IFIT1, and IFITM2/3 in 3D-cultured CP80 cell lysates with KPT-9274 treatment at indicated doses. GAPDH and LaminB1 were shown as controls. (Left: cytoplasm lysate, Right: nuclear lysate) Bottom: Cytoplasmic protein levels normalized by GAPDH in Control and KPT-9274 treatment. (n = 4 independent experiments). G Schematic showing that KPT-9274 inhibits Wnt/β-Catenin signaling by reducing the expression of inflammatory-related proteins, including IFNGR1 and IFIT1. Graph data were presented as mean ± SEM with n = 4 per group.

IPA revealed that KPT-9274 treatment suppressed the Interferon signaling pathway, Remodeling of epithelial adherens junctions, and ILK signaling. The genes linked to Interferon signaling, namely IFNGR1, IFIT1, IFITM1, IFITM2, and IFITM3, showed varying expression patterns upon treatment (Supplementary Fig. 4A, B). Specifically, IFNGR1, which encodes the IFN-γ receptor-1, was upregulated, while the others were downregulated (Fig. 5E). IFIT1 affects cancer cell behavior through Wnt/β-Catenin signaling [40], and IFITM1, IFITM2, and IFITM3 are related to antiviral functions [41]. To validate how these changes in transcriptomes affect protein expression, we tested expression of IFNGR1, IFIT1, IFITM1, IFITM2, and IFITM3 using Western blotting. IFITM1 was not detected (data not shown), and IFITM2/3 showed no significant differences between Control and KPT-9274 treatment. Interestingly, contrary to RNA-seq data, KPT-9274 significantly suppressed IFNGR1 expression, a membrane surface protein. Given that the protein is the functional component of IFNGR1, not the transcript, we concluded that the inhibition of IFNGR1 protein expression by KPT-9274 treatment observed in this experiment contributes to the suppression of cell proliferation. Moreover, IFIT1 cytoplasmic expression was significantly down-regulated by KPT-9274 (Fig. 5F), suggesting that KPT-9274 downregulates Wnt/β-Catenin pathway via a suppression of IFNGR1 and IFIT1, contributing to the anti-tumor effects (Fig. 5G).

KPT-9274 down-regulates multiple kinase activities in the cytoplasm through a localization change of PAK4

It has been shown that PAK4 regulates β-Catenin phosphorylation and mTOR complex function [19,20,21,22,23]. Hence, suppressing PAK4 leads to reduced kinase activity of various proteins, such as AKT, that are controlled by mTOR complexes. To validate the effect of KPT-9274 on kinase activity, we evaluated the expression of PAK4-affected proteins with Western blotting using cytoplasm and nuclear lysate. We also evaluated Poly (ADP-ribose) (PAR), which reflects the function of DNA repair [42], because RNA-seq results suggested KPT-9274 inhibited DNA repair. As expected, PAR expression was suppressed in both cytoplasm and nucleus following KPT-9274 treatment, suggesting impaired DNA repair by KPT-9274. Notably, PAK4, which was mostly localized in the cytoplasm before treatment, migrated into the nucleus after KPT-9274 treatment. In parallel to the shift of the localization of PAK4, cytoplasmic expression level of RAPTOR, Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), and Phospho-β-Catenin (Ser675) was decreased (Fig. 6A). RAPTOR and Phospho-S6 Ribosomal Protein (Ser235/236) reflect mTORC1 function [23]. Similar protein suppression was observed in whole cell lysates of 3D-cultured A2780, ACI-98, and CP80 cells (Supplementary Fig. 5A, B). Next, using FK-866, the first-in-class NAMPT inhibitor, and GNE-617, a specific NAMPT inhibitor [43], we conducted a similar validation. Despite successfully inhibiting NAD+ production, the subcellular distribution of PAK4 remained unaltered with specific inhibition of NAMPT alone, while and the impact on key proteins like RAPTOR, S6 Ribosomal Protein, AKT, and β-Catenin was inconsistent, displaying distinct patterns between NAMPT inhibitors (Supplementary Fig. 6A, B). These findings highlight that the alteration of PAK4 localization seems to be specific to KPT-9274.

Fig. 6: KPT-9274 inhibited cell proliferation by down-regulating kinase activity in the cytoplasm through a localization change of PAK4.
figure 6

A Immunoblotting for assessing the activity of DNA repair, Serine/threonine protein kinase, mTORC1, mTORC2, and Wnt/β-Catenin signaling in 3D-cultured CP80 cell lysates with KPT-9274 treatment at indicated doses. PAR for NAD+-mediated DNA repair, PAK4 for Serine/threonine protein kinase, RAPTOR and the phosphorylation of S6 (p-S6) at S235/236 for mTORC1, the phosphorylation of AKT (p-AKT) at S473 for mTORC2, and the phosphorylation of β-Catenin (p-β-Catenin) at S675 for Wnt/β-Catenin signaling. Total S6, AKT, β-Catenin, GAPDH, and LaminB1 were shown as controls. (Left: cytoplasm lysate, Right: nuclear lysate). B Representative images of CP80 spheroids after treatment with KPT-9274 1000 nM for 48 h relative to Control. (Results shown are from three independent experiments.) The spheroids were stained with phosphorylated S6 at S235/236 (orange), phosphorylated AKT at S473 (green), and NucBlueTM (blue). Scale bars, 100 μm (low magnification). C 3D-cultured CP80 stably expressing the IncucyteTM Kinase Akt Green/Red Indicator were treated with KPT-9274 1000 nM for 48 h. The image panel shows green fluorescence channel on the left, red fluorescence channels in the middle, and overlap channel on the right. Scale bars, 10 μm (high magnification). D Left: The kinetic graph shows cell proliferation in 2D-cultured CP80 with KPT-9274 treatment at indicated doses as a function of time (hours) using IncuCyteTM S3. (Results shown are from six independent experiments.) Right: The kinetic graph shows 2D-cultured CP80 change in the Nuclear Translocation Ratio, which reflects translocation of the green fluorescent sensor from the cytoplasm to the nucleus, with KPT-9274 treatment at indicated doses as a function of time (hours) using IncuCyteTM S3. (n = 6 independent experiments). E Schematic showing that PAK4 reduction in the cytoplasm by KPT-9274 treatment regulates phosphorylation of AKT, S6, and β-Catenin. Graph data were presented as mean ± SEM with n = 6 per group.

In support of these Western blotting findings, immunofluorescence confocal imaging of the spheroids also revealed the fluorescence intensity of Phospho-S6 Ribosomal Protein (Ser235/236) and Phospho-AKT (Ser473) in the 3D-spheroids was suppressed with KPT-9274 treatment (Fig. 6B). Phospho-β-Catenin (Ser675) was difficult to detect (data not shown). To assess kinase activity from different perspectives, IncuCyteTM Kinase AKT Assay was performed. AKT phosphorylation moves the green sensor from nucleus to cytoplasm. Conversely, AKT inhibition retains the sensor in the nucleus [44]. Interestingly, KPT-9274 treatment maintained the green sensor in the nucleus, indicating suppressed AKT kinase activity (Fig. 6C). Nuclear Translocation Ratio, reflecting sensor movement [44], was reduced by KPT-9274 in a concentration-dependent manner, linked to inhibited cell proliferation (Fig. 6D). Overall, these findings suggested that KPT-9274 hindered cell proliferation by lowering cytoplasmic kinase activity through altering PAK4 localization (Fig. 6E).

Suppression of PAK4-mediated kinase activity by KPT-9274 is NAD+ dependent

To uncover whether the ability of KPT-9274 to suppress multiple kinase activities is a NAD+-dependent mechanism, we silenced NAMPT expression using siRNA. NAMPT-silenced cells showed approximately 60% less NAD+ content and about 75% less GAPDH corrected NAMPT expression than control siRNA-treated cells (Fig. 7A,B). Adding NMN to the medium had no effect on NAMPT expression, while rescued total NAD to 80% of the control. Consistent with the NAD+ production, PAR was suppressed in NAMPT-silenced cells and was rescued by NMN addition. However, NAMPT silencing did not impact PAK4, Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), and Phospho-β-Catenin (Ser675) (Fig. 7A,B). These findings suggest that reducing NAD+ through NAMPT silencing alone does not strongly suppress kinase activity.

Fig. 7: The suppressed PAK4-mediated kinase activity by KPT-9274 treatment is NAD+-dependent.
figure 7

A Change in total NAD levels in 3D-cultured CP80 with Control siRNA, NAMPT siRNA, and NAMPT siRNA plus NMN 500 μM. Spheroids were harvested 72 h after transfection. NMN was administered at the same time the trypsinized cells were seeded. (n = 3 independent experiments). B Immunoblotting for assessing the activity of NAMPT, DNA repair, Serine/threonine protein kinase, mTORC1, mTORC2, and Wnt/β-Catenin signaling in 3D-cultured CP80 cell lysates with Control siRNA, NAMPT siRNA, NAMPT siRNA plus NMN 500 μM. PAR for NAD+-mediated DNA repair, and the phosphorylation of S6 (p-S6) at S235/236 for mTORC1, the phosphorylation of AKT (p-AKT) at S473 for mTORC2, and the phosphorylation of β-Catenin (p-β-Catenin) at S675 for Wnt/β-Catenin signaling. Total S6, AKT, β-Catenin, GAPDH, and LaminB1 were shown as controls. (Left: cytoplasm lysate, Right: nuclear lysate). C Change in total NAD levels in 3D-cultured CP80 with Control, KPT-9274 1000 nM for 48 h, and KPT-9274 1000 nM plus NMN 500 μM for 48 h. (n = 4 independent experiments). D Immunoblotting for assessing the activity of NAMPT, DNA repair, Serine/threonine protein kinase, mTORC1, mTORC2, and Wnt/β-Catenin signaling in 3D-cultured CP80 cell lysates with Control, KPT-9274 1000 nM for 48 h, and KPT-9274 1000 nM plus NMN 500 μM for 48 h. The evaluated proteins are the same as those described in B. Graph data were presented as mean ± SEM with n = 3 or 4 per group.

Next, we tested whether supplemental NMN could rescue the kinase activity reduction caused by KPT-9274 treatment. As expected, KPT-9274 decreased NAD+ production by approximately 90% (Fig. 7C), while GAPDH corrected NAMPT expression increased (Fig. 7D), suggesting NAMPT upregulation due to NAD+ reduction. Importantly, NMN addition largely restored the suppressed PAK4, Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), and Phospho-β-Catenin (Ser675), indicating that suppressed PAK4-mediated kinase activity by KPT-9274 is NAD+-dependent (Fig. 7C,D). In conclusion, KPT-9274 demonstrated a promising activity against NAMPT or PAK4-driven cancer growth, suggesting it is a potential novel treatment for platinum-resistant ovarian cancer.