Inhibition of NAMPT targets DNA damage response to sensitize alkylating chemotherapy in TP53 mutant mantle cell lymphoma

Key Points

• •NAMPT is a TP53-stratified therapeutic target in MCL.
• •NAMPT inhibitors synergize with DDR agents to overcome TP53 mutant chemoresistance without added toxicity.

Visual Abstract

Abstract

Patients with TP53 mutant mantle cell lymphoma (MCL) face poor chemotherapy response and early progression, requiring novel therapies. Nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting nicotinamide adenine dinucleotide salvage enzyme overexpressed in MCL cell lines and patient tissues, emerges as a therapeutic target. The NAMPT inhibitor KPT-9274 reduced viability and induced apoptosis in MCL cells irrespective of TP53 status. Mechanistic studies reveal a striking dichotomy: in TP53 mutant cells, NAMPT inhibition triggers synthetic lethality through catastrophic DNA damage response (DDR) pathway disruption, whereas in TP53 wild-type cells, it selectively suppresses B-cell receptor (BCR) signaling and immune checkpoint activation. This biological divergence translates to clinically actionable synergies: TP53 mutant cells exhibit marked sensitization to alkylating agents and DDR-targeting therapies, whereas TP53 wild-type models show potential for overcoming BTK inhibitor resistance. In vivo studies confirm that NAMPT-based combinations achieve profound tumor regression in TP53 mutant xenografts without exacerbating toxicity. Our findings establish NAMPT as a dual-context therapeutic node, providing a precision medicine framework to circumvent chemoresistance in high-risk MCL. These results advocate for the clinical evaluation of TP53 status–guided NAMPT inhibitor combinations to address this unmet oncologic challenge.

Introduction

Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin lymphoma characterized by overexpression of CCND1 and the presence of t(11;14)(q13;q32).1, 2, 3 Although chemoimmunotherapy is effective for patients with MCL, there is significant heterogeneity in disease biology and outcome. Certain high-risk subgroups of patients experience poor disease control and overall survival (OS) with standard approaches.^2,4 Karolová et al⁵ observed enrichment of genetic aberrations of the DNA damage response (DDR) pathway in MCL. The most common copy number variations at diagnosis and relapse included TP53 and CDKN2A/B deletions and PIK3CA amplifications.⁵ Detection of TP53 mutations has been shown to be the most reliable predictor of poor response to chemoimmunotherapy, early disease progression, and death.^2,6 The median OS duration was 1.8 years in patients with TP53 aberrations vs 12.7 years in patients without TP53 aberrations.⁷ There is an urgent need to develop novel drugs to overcome resistance to chemotherapy caused by TP53 mutations.

Nicotinamide (NAM) adenine dinucleotide (NAD⁺) is an abundant metabolite that plays an essential role in maintaining cellular homeostasis. Higher NAD⁺/nicotinamide adenine dinucleotide hydrogen ratios are found in cancer cells to support their high proliferation rate and enable tumor progression, development, and survival, as well as regulate the expression of DNA damage repair genes and stress responses.⁸ Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for the biosynthesis of NAD⁺ in the salvage pathway, which is overexpressed in various cancers, associated with a poor prognosis and tumor progression.9, 10, 11, 12 Evidence from other studies suggests that NAMPT may be a potential therapeutic target for hematologic tumors, including B-cell and T-cell lymphomas.^9,13, 14, 15, 16, 17 While NAMPT dysregulation has been reported across various hematologic malignancies, its specific role and therapeutic potential in MCL remain unexplored, representing a critical knowledge gap given MCL’s unique biological characteristics and clinical challenges. A previous study by our group provided evidence of the significant impact of the NAMPT inhibitor KPT-9274 on the growth and viability of Waldenström macroglobulinemia cells, representing a potential new therapeutic intervention for this malignancy. We first identified the DNA damage and repair pathway that is significantly affected by NAMPT inhibition via KPT-9274, leading to synergistic activity in combination with DNA-damaging agents in vitro and in vivo.¹⁴

In this work, we describe the role of NAMPT in MCL cell viability and identify the DNA damage and repair pathway that is significantly affected by the NAMPT inhibitor KPT-9274. This leads to synergistic activity in TP53-deficient MCL when combined with DNA-damaging agents.

Materials and methods

Cells and reagents

The MCL cell lines (Mino, Jeko-1, Z138, and Granta-519) were cultured in RPMI medium containing 10% fetal bovine serum (VivaCell, C04001), 10 000 units per mL of penicillin, and 10 000 μg/mL of streptomycin (HyClone, SV30010). The cells were maintained in a humidified incubator at 37°C with 5% carbon dioxide. Mino and Jeko-1 were selected as TP53 mutant-type cells, whereas Granta-519 and Z138 were selected as TP53 wild-type cells. Human MCL Granta-519 cells and Mino cells were provided by the Tianjin Blood Research Institute. Jeko-1 and Z138 cells were purchased from Shenzhen Huatuo Biotechnology Co, Ltd KPT-9274 (S844), bendamustine (S1212), and AZD7762 (S1532) were purchased from Selleck Chemicals. AZD1775 (HY-10993) and olaparib (HY-10162) were purchased from MedChemExpress.

Cell viability and apoptosis assay

Cell viability was determined using the Cell Counting Kit-8 assay. The MCL cells were seeded in a 96-well plate and subjected to their respective treatments. Subsequently, 10 μL of the Cell Counting Kit-8 solution (Bimake) was added to each well, and the cells were incubated at 37°C for 3 hours. The absorbance was measured at 450 nm using a microplate reader. For apoptosis determination, the MCL cells were stained with annexin V–fluorescein isothiocyanate and propidium iodide (Beyotime Biotechnology, China) and analyzed via flow cytometry according to the manufacturer’s instructions.

NAMPT activity

NAMPT activity was analyzed via the NAD/nicotinamide adenine dinucleotide hydrogen Assay Kit (Beyotime Biotechnology, S0175) according to the manufacturer’s instructions.

Immunohistochemistry

Lymph node samples were obtained from patients with MCL diagnosed at the Second Hospital of Dalian Medical University. Biopsy specimens were processed into formalin-fixed, paraffin-embedded sections; affixed to adhesive-coated slides; and baked at 60°C for 1 hour. Subsequently, the sections underwent sequential treatments including dewaxing, hydration, antigen retrieval, endogenous peroxidase blocking, and protein blocking. Sections were incubated overnight at 4°C with primary antibodies against NAMPT (Servicebio, GB113478-10) and p53 (Servicebio, GB15627-100), followed by a secondary antibody, horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (Servicebio, GB23303). After 3,3′-diaminobenzidine chromogenic development and hematoxylin counterstaining, samples were dehydrated, cover-slipped, and imaged using a Nikon E100 light microscope. Images were then imported into the 3DHISTECH CaseViewer 2.4 software (Hungary) for quantitative analysis, and a series of high-resolution .tif files were exported for further processing.

Immunoblotting

Western blotting (WB) was performed to evaluate the expression levels of total protein and phospho-specific isoforms using the following antibodies: FANCD2 (Santa Cruz Biotechnology, sc-20022), RAD51 (Santa Cruz Biotechnology, sc-398587), PBEF (Santa Cruz Biotechnology, sc-166946), cleaved Caspase3 (Cell Signaling Technology, 9664s), PARP (Cell Signaling Technology, 9532s), cleaved PARP (Cell Signaling Technology, 5625s), γ-H2AX (Ser139; Cell Signaling Technology, 9718s), p-CHK1 (Ser345; Cell Signaling Technology, 2341s), p53 (Cell Signaling Technology, 2527s), p-ATR (Ser428; Cell Signaling Technology, 2853s), p-ATM (Ser1981; Cell Signaling Technology, 4526s), and p-CHK2 (Thr68; Cell Signaling Technology, 2197s). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, 2118) and α-tubulin (Santa Cruz Biotechnology, sc8035) were used as loading controls.

Murine xenograft model of human MCL

NOG mice were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd. A total of 20 mice were randomly divided into 4 groups. Mice were subcutaneously inoculated in the right flank with 1 × 10⁷ Mino cells in 0.1 × 10³/μL of phosphate-buffered saline. After detection of the tumor (∼1 week after the injection), mice were treated as follows: intraperitoneally with bendamustine (25 mg/kg of body weight) at 1 dose per week for 2 consecutive weeks, orally gavaged with KPT-9274 (100 mg/kg) daily for 5 consecutive days per week for 3 weeks, or with a combination of the 2 agents using the same dosing regimens as for the individual agents. The control group received the vehicle at the same schedule as the combination group. Tumor volume was evaluated in 2 dimensions via caliper measurements performed approximately twice a week using the following formula: V = 0.5a × b², where “a” and “b” are the long and short diameter of the tumor, respectively.

Statistical analysis

All values are expressed as mean plus or minus the standard deviation. The statistical significance of differences between treatments was analyzed using the t test with the GraphPad Prism analysis software. Differences were considered significant when P was ≤.05. Drug interactions were assessed using the SynergyFinder software, and the mean synergy score was reported using the highest single agent method. The intensity of the red color indicates the synergy score for each dose combination, whereas green indicates antagonism. A combination index (CI) of 1 indicates an additive effect, CI <1 indicates synergism, and CI >1 indicates antagonism.

All animal studies were conducted in accordance with protocols approved by the experimental animal ethics committee of Dalian Medical University (ethical approval AEE22096).

Results

NAMPT is highly expressed in MCL, and its targeting by KPT-9274 significantly affects MCL viability

According to our analysis of the Gene Expression Profiling Interactive Analysis and Human Protein Atlas databases, patients with lymphoma had higher levels of NAMPT protein expression, which was associated with a poor prognosis (supplemental Figure 1A-B). We further performed a bioinformatic analysis of 7 public Gene Expression Omnibus data sets to compare NAMPT messenger RNA levels across lymphoma subtypes: diffuse large B-cell lymphoma showed significant upregulation; peripheral T-cell lymphoma was significantly downregulated in 1 data set; and T-cell lymphoblastic lymphoma and MCL displayed non–statistically significant downregulation and upregulation trends, respectively (the latter likely due to small MCL sample sizes; supplemental Table 1). In addition, the expression of NAMPT and p53 proteins was examined in pathological tissue samples from 28 patients with MCL from the Department of Oncology of the Second Hospital of Dalian Medical University (ethical approval 2023-XWLW number 14) between 2013 and 2023. The results further supported the database findings by showing that NAMPT protein was highly expressed in 27 cases and negative in only 1 case (supplemental Figure 2A; Figure 1A shows representative examples). Patients showed differential expression of p53 protein, indicating a positive correlation between high NAMPT expression and poor p53 expression. In addition, it was shown that higher levels of NAMPT expression were associated with later clinical stages, a higher percentage of bone marrow invasion, and more B symptoms. These results suggest that individuals with higher levels of NAMPT expression had worse clinicopathological features (supplemental Table 2).

Figure 1.

NAMPT is expressed and functional in MCL cells. (A) Representative images of NAMPT and p53 immunocytochemistry stain in 8 different patients with MCL. Positive staining appears in a brown color (scale bars, 100 and 5 μm). Positive immunostaining for NAMPT is characterized by brown staining or brown cytoplasmic granules localized in the cytoplasm of positive cells. The immunostaining score of NAMPT is determined by 2 parameters: staining intensity and percentage of positive-stained cells. Regarding staining intensity scoring, a score of 0 indicates no staining, a score of 1 indicates weak staining, a score of 2 indicates moderate staining, and a score of 3 indicates strong staining. Regarding positive cell percentage scoring, a score of 0 indicates that ≤25% of the cells are positive, a score of 1 indicates that 26% to 50% of the cells are positive, a score of 2 indicates that 51% to 75% of the cells are positive, and a score of 3 indicates that ≥76% of the cells are positive. The total score is the sum of the staining intensity score and the positive cell percentage score. This total score defines the expression level of NAMPT protein in each case: a total score of ≥3 indicates high NAMPT expression, and a total score of 1 to 2 indicates low NAMPT expression. The immunostaining score of p53 is determined based on the percentage of positive-stained cells, with the following criteria: negative (˗) indicates that <25% of the cells are positive; weakly positive (+) indicates that 25% to 50% of the cells are positive; moderately positive (++) indicates that 50% to 75% of the cells are positive, with a small subset of strongly positive cells; and strongly positive (+++) indicates that >75% of the cells are positive. (B) Protein lysates from 4 MCL cell lines were analyzed for NAMPT and p53 expression via WB. GAPDH was used as the loading control. (C) Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay on 4 MCL cell lines treated with KPT-9274 in culture for 24, 48, and 72 hours. (D) Protein lysates from 4 MCL cell lines treated with KPT-9274 in culture for 48 hours were analyzed for NAMPT using WB. GAPDH was used as the loading control. (E) Cellular NAD levels in Mino cells treated with KPT-9274 were measured using an enzyme cyclic assay and normalized to total cell number. (F) Mino cells were treated with KPT-9274 (0, 0.05, 0.1, and 0.25 μmol/L) in the absence or presence of NAM (10 and 100 μmol/L) for 48 hours. Cell viability was evaluated using the CCK-8 assay. (G) Apoptotic cell death was assessed via flow cytometric analysis after annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining in cells treated with KPT-9274 (0-1 μmol/L). (H) Whole-cell lysates from Z138 and Mino cells treated with control or KPT-9274 (1 μmol/L) for 48 hours were subjected to WB analysis and probed with antibodies against indicated proteins, with GAPDH as the loading control. mut, mutant; wt, wild-type.

We next evaluated the expression of NAMPT and p53 in 4 MCL cell lines, including Mino and Jeko-1 as TP53 mutant cell lines and Granta-519 and Z138 as TP53 wild-type cell lines. NAMPT was expressed in all 4 cell lines as shown through WB, and p53 was differentially expressed (Figure 1B), indicating that there was no significant correlation between p53 and NAMPT expression. Subsequently, we investigated the effect of pharmacological inhibition of NAMPT using KPT-9274. We observed a significant dose- and time-dependent decrease of MCL cell viability that had no significant correlation with the mutation status of TP53 (Figure 1C). We confirmed that KPT-9274 treatment significantly reduced the activity of NAMPT in MCL cells without affecting NAMPT protein levels (Figure 1D-E). Meanwhile, repletion of NAD⁺ through biosynthesis from NAM can rescue the effect of KPT-9274 treatment on NAMPT inhibition; consistently, we found that exogenous NAM rescued KPT-9274–induced MCL cell death in a dose-dependent manner (Figure 1F), demonstrating even more how crucial NAD⁺ depletion is to KPT-9274’s antitumor effect on MCL cells.

Based on the flow cytometry assay, we discovered that the percentage of apoptosis in MCL cells treated with KPT-9274 increased as the drug concentration gradient increased (Figure 1G), and WB verified that proteins linked to the apoptosis pathway were activated (Figure 1H).

Inhibition of NAMPT affects DDR pathways and the B-cell receptor pathway in TP53 mutant and TP53 wild-type MCL cells, respectively

To further elucidate the mechanism of action of NAMPT in MCL and explore novel combination protocols, we strategically selected TP53 mutant Mino and TP53 wild-type Z138 cells for conducting quantitative proteomics experiments. Our findings revealed distinct genetic backgrounds between the 2 MCL cell lines with different TP53 mutation statuses (Figure 2A; supplemental Figure 3A). Furthermore, our gene ontology protein function enrichment analysis unveiled significant disparities in monocarboxylic acid metabolism, NAM nucleotide metabolism, and pyruvate metabolism processes, particularly highlighting noteworthy differences in the NAD metabolism process between the 2 cell lines (Figure 2B-C; supplemental Figure 3B).

Figure 2.

KPT-9274 affects DDR pathways and the B-cell receptor pathway in TP53 mutant and TP53 wt MCL cells, respectively. (A) The relative expression levels of multiple differential proteins in Z138 (TP53 wt) and Mino (TP53 mutant) treated with KPT-9274 in culture for 48 hours were detected via tandem mass tag labeling quantitative proteomics. The clustering relationship of the relative expression of these differential proteins is presented. Each row represents 1 differential protein, and each column represents 1 sample. Red indicates high expression, blue indicates low expression, and gray indicates nonquantifiable values in the corresponding sample. (B-C) A specific function was executed in order, called the biological process. Gene ontology (GO) annotation was conducted to analyze the identified proteins using the EggNOG-mapper software (v2.0). The software is based on the EggNOG database. The latest version is the fifth edition, covering 5090 organisms (477 eukaryotes, 4445 representative bacteria, and 168 archaebacteria) and 2502 virus genome-wide coding protein sequences. The GO identification was extracted from the results of each protein note, and the protein was then classified according to cellular component, molecular function, and biological process. (D) All differentially expressed protein database accessions or sequences were searched against the STRING database version 11.5 for protein-protein interactions. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence; we fetched all interactions that had a confidence score of ≥0.7 (high confidence). The interaction network from STRING was visualized using the R package “networkD3.” (E-F) Whole-cell lysates from 2 TP53 mutant MCL cell lines treated with control or KPT-9274 for 48 hours were subjected to WB analysis and probed with antibodies against indicated proteins, with GAPDH as the loading control. (G) Mino cells were treated with KPT-9274 (1 μmol/L) in the absence or presence of NAM (10 and 100 μmol/L) for 48 hours. Whole-cell lysates from Mino cells treated with control or KPT-9274 for 48 hours were subjected to WB analysis and probed with antibodies against indicated proteins, with α-tubulin as the loading control. CNT, control; dUMP, deoxyuridine monophosphate; mut, mutant; tRNA, transfer RNA, wt, wild-type.

To account for these baseline variations, proteomics overlap analysis (Venn diagram) identified KPT-9274–altered shared proteins in both TP53 wild-type and mutated cells (supplemental Figure 3C). Enriched in NAD⁺ metabolism, cellular redox homeostasis, and mitochondrial respiratory chain pathways, these proteins confirm that KPT-9274 effectively targeted NAMPT and induced NAD⁺ depletion, serving as a critical internal positive control for a common initial trigger (supplemental Figure 3D).

Consequently, the signaling pathways and biological processes in the 2 cell lines illustrated significant divergence after treatment with KPT-9274. Notably, quantitative proteomics results showed significant enrichment of B-cell proliferative activity, lymphocyte-mediated immunomodulation, regulation of adaptive immune response, acute inflammatory response, and other biological processes in Z138 cells (TP53 wild type) after treatment with KPT-9274 (Figure 2B). In contrast to TP53 wild-type cells, our analysis of quantitative protein results from KPT-9274–treated Mino cells (TP53 mutant) showed significant enrichment of relevant signaling pathways related to DNA damage repair compared to untreated cells (Figure 2C). These pathways included DNA-dependent DNA replication, RNA priming synthesis, mitotic DNA damage checkpoint signaling, cell cycle checkpoint, and other biological processes.

Protein-protein interaction network analysis was carried out on TP53 mutant Mino cells treated with KPT-9274 and a control group. Analysis showed that CHK1 and RAD51, 2 proteins essential for homologous recombination repair, were significantly downregulated after KPT-9274 treatment (Figure 2D).

Furthermore, we performed a WB analysis to confirm the proteomics findings. The results indicated that, in comparison to control cells, Mino cells (TP53 mutant) treated with KPT-9274 accumulated DNA damage (increased γ-H2AX expression) and downregulated the expression of proteins related to the homologous recombination pathway, such as p-ATR, p-CHK1, FANCD2, RAD51, and other proteins (Figure 2E-F). NAM complementation was also found to rescue the function of KPT-9274 on DNA damage and repair, which is in line with these findings (Figure 2G).

KPT-9274 inhibits the growth of MCL in vivo

We examined the anti-MCL effect of KPT-9274 in vivo in a murine xenograft model of human MCL using Mino cells (TP53 mutant) in NOG mice. After tumors were found, mice were given either 100 mg/kg of KPT-9274 or a vehicle orally 5 days a week for 3 weeks, as illustrated in Figure 3A. Treatment with KPT-9274 markedly reduced the growth of MCL cell tumors when compared to the vehicle alone. During the course of treatment, the mice’s daily activity and overall body weight changes revealed no associated toxicity (data not shown). WB analysis of protein lysates from retrieved tumor cells confirmed significant induction of γ-H2AX and cleaved PARP as well as decreased FANCD2 and RAD51 expression (Figure 3B).

Figure 3.

Inhibition of NAMPT function via KPT-9274 inhibits tumor growth in vivo. (A) NOG mice were injected subcutaneously with Mino cells. After detection of the tumor, the mice were randomized and treated orally with either KPT-9274 or the vehicle for 5 consecutive days per week for 3 weeks. Tumor volume was evaluated via caliper measurement. Differences between the 2 groups were evaluated using the standard t test. (B) Tumor cells collected from mice were lysed in radio-immunoprecipitation assay buffer, and the whole-cell lysate was subjected to WB analysis and probed with antibodies against Cl-PARP, FANCD2, RAD51, and γ-H2AX. GAPDH was used as the loading control. ns > .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001.

NAMPT inhibition potentiates sensitivity to bendamustine via synergistic modulation of DDR markers in TP53 mutant MCL cells

As we have shown in previous studies, alkylating agents can become more sensitive when the NAMPT enzyme is inhibited.¹⁴ This is accomplished by downregulating proteins linked to the homologous recombination repair pathway and inducing DNA damage. To explore a novel targeted chemotherapy combination regimen, we chose to combine KPT-9274 with bendamustine, a chemotherapy agent frequently used in the treatment of MCL. When KPT-9274 and bendamustine were administered to MCL cells at varying concentrations at the same time, it was found that the combination dramatically reduced MCL cell proliferation and caused apoptosis (Figure 4A-B). Moreover, equivalent line graphs and CI analyses revealed robust synergistic effects of the combination compared to single agents, with highest single agent values of >10 at all doses tested and a CI of <1.0 (supplemental Figure 4A-B).

Figure 4.

KPT-9274 induces synergistic growth-inhibitory effects in combination with bendamustine in MCL cells in vitro. (A) MCL cell lines were simultaneously treated with KPT-9274 and/or bendamustine for 48 hours. Cell viability was tested using the Cell Counting Kit-8 (CCK) assay and expressed as a percentage of the cell viability of untreated cells. Data represent the mean ± standard deviation (SD) of 3 experiments performed in triplicate. (B) Apoptotic cell death was assessed via flow cytometric analysis after annexin V–FITC and PI staining in cells treated with single agents or their combination. One representative experiment is shown in the left panel, whereas the percentage of annexin V⁺ cells in different MCL cell lines after treatment is shown on the right. Data represent the mean ± SD of 3 experiments. (C-D) Whole-cell lysates from Mino (left) and Jeko-1 (right) cell lines treated with the control, single agents, or their combination were subjected to WB analysis and probed with the indicated antibodies. GAPDH was used as the loading control. ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. BEN, bendamustine; CNT, control; COM, combination; mut, mutant.

The proteins FANCD2 and RAD51, which are involved in homologous recombination repair and the DNA damage repair pathway, were found to be downregulated in the combined group according to our findings. This implies that tumor cells were unable to finish repairing DNA damage, which resulted in the accumulation of DNA damage (increased expression of γ-H2AX) and, ultimately, cell death (Figure 4C). Furthermore, downregulation of p-ATR and p-CHK1 was also observed in the combined group of TP53 mutant cells, resulting in the inability to activate the ATR-CHK1 pathway for DNA damage repair (Figure 4D). Taken together, inhibition of NAMPT function can lead to defective homologous recombination repair function, induce DNA accumulation, lead to cell death, and increase sensitivity to alkylating drugs.

KPT-9274 and bendamustine act synergistically to suppress the growth of human MCL cells in vivo

In vitro studies have shown that KPT-9274 and bendamustine work in concert to induce apoptosis in MCL cells. The effectiveness of this combination was then evaluated in vivo by creating a human xenograft mouse model by subcutaneously injecting Mino cells into NOG mice. After tumorigenesis (approximately a few weeks after cell injection), mice were treated with the vector, KPT-9274 (100 mg/kg given orally once daily, 5 days a week), and bendamustine (25 mg/kg given intraperitoneally once a week for 2 weeks) either separately or in combination (Figure 5A). The findings showed that, in comparison to mice given the mediator or either agent alone, the combination of bendamustine and KPT-9274 significantly reduced the growth of tumors (Figure 5B-C). It is noteworthy that there was no increase in toxicity as a result of the combination (data not shown). The combination’s effect on the DDR pathway was further validated through WB analysis of cell lysates from recovered tumors (Figure 5D).

Figure 5.

KPT-9274 induces synergistic growth-inhibitory effects in combination with bendamustine in MCL cells in vivo. (A) Schematic illustration of the experimental design. NOG mice were engrafted with Mino cells and treated with vehicle, 100 mg/kg of KPT, 25 mg/kg of bendamustine, or KPT-9274/bendamustine in combination. (B) Average and SD of tumor volume (cubic millimeter) from groups of mice (n = 5 per group) vs time (days) when the tumor was measured. Mino cells (1 × 10⁷ in 0.1 × 10³/μL of phosphate-buffered saline) were implanted in the axilla of NOG mice. After tumor detection, mice were randomized to intraperitoneal treatment with vehicle, KPT-9274, bendamustine, or combination at the indicated doses for 3 weeks. A significant decrease in tumor growth was noted in combination-treated mice vs vehicle-treated mice. Data are mean tumor volume ± SD. (C) Comparison of tumor volume in control and treated mice 3 weeks after initial assessment of tumor appearance and start of treatment. (D) Whole-cell lysate from tumor cells retrieved from mice was subjected to WB analysis and probed with the indicated antibodies. GAPDH was used as the loading control. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. BEN, bendamustine; COMB, combination.

Small molecule inhibitors of the DDR pathway combined with KPT-9274 may be a new option for combination therapy in MCL

Based on the previous findings supporting the mapping of the DDR signaling pathway through NAMPT inhibition in MCL, we aimed to further validate the combined effects of KPT-9274, which inhibit the function of NAMPT, with the small molecule inhibitors of the DDR pathway in MCL cells. We performed cell proliferation assays to confirm that the combination of KPT-9274 with AZD2281 (PARPi), AZD7762 (CHK1i), and AZD1775 (WEE1i) showed significant synergistic effects (Figure 6A), indicating the feasibility of the combination regimen. Nevertheless, additional mechanistic studies and in vivo experimental validation are still required for further follow-up.

Figure 6.

KPT-9274 induces synergistic growth-inhibitory effects in combination with small molecule inhibitors of the DDR pathway in MCL cells in vitro. (A) Mino cells were selected, and cell survival was detected after 48 hours of PARPi, WEE1i, and CHK1i treatment with or without KPT-9274. Data represent the mean ± SD of 3 experiments (all P values were ≤.0001). (B) A schematic illustration shows the possible mechanisms through which MCL cells are sensitized to NAMPT inhibition through the suppression of DNA repair processes. CNT, control; COM, combination treatment; mut, mutant.

Discussion

Recent therapeutic advances have improved the prognosis of MCL; however, drug resistance and relapse remain major challenges, rendering MCL largely incurable.⁷ Mutations in DNA damage repair genes, particularly ATM and TP53, represent the most frequently identified secondary oncogenic events in MCL.18, 19, 20 TP53 mutations or deletions occur in ∼15% to 25% of MCL cases19, 20, 21 and constitute one of the most powerful prognostic markers, strongly predicting early disease progression and inferior survival in patients receiving conventional chemoimmunotherapy.^22,23 Eskelund et al reported a striking disparity in median OS between TP53-mutated and wild-type patients (1.8 years vs 12.7 years, respectively).²² These findings underscore the critical need for novel therapeutic agents and combination strategies specifically designed to overcome chemoresistance and improve outcomes in TP53-mutated MCL.

Our study identifies the pivotal role of NAMPT in MCL progression, positioning it as a compelling therapeutic target. As the rate-limiting enzyme in the NAD⁺ salvage pathway, NAMPT is frequently overexpressed in aggressive malignancies, a phenomenon observed in other B-cell lymphomas such as diffuse large B-cell lymphoma.24, 25, 26, 27, 28, 29 The high NAMPT expression we identified in MCL aligns with these reports, reflecting the elevated metabolic requirements of rapidly proliferating tumor cells.

The high expression of NAMPT was verified in this study in pathological samples from patients with MCL, as well as in the lymphoma database and MCL cell lines. A comprehensive analysis of tissue specimens from a total of 28 patients with MCL was conducted, revealing that NAMPT exhibited high levels of expression in 27 patients (96.4%). Of these patients, 85.2% were classified as stage IV, and 77.8% exhibited involvement of the bone marrow (supplemental Table 2). These findings suggest a potential correlation between high NAMPT expression and a poor prognosis for MCL. However, the limited number of patients represents a significant challenge in determining whether NAMPT expression is associated with the prognosis of patients with MCL with TP53 mutations. To address this gap in knowledge, further investigation is necessary, which will require an increase in the sample size.

A central finding of our study is that the therapeutic mechanism of NAMPT inhibition in MCL is fundamentally dictated by the cellular TP53 status. Our in vitro and in vivo data demonstrate that the NAMPT inhibitor KPT-9274 effectively reduces proliferation and induces apoptosis in MCL cells irrespective of their TP53 mutation status, robustly validating NAMPT as a promising therapeutic target in this malignancy. Crucially, we uncovered a striking mechanistic divergence: in TP53 mutant cells, the antilymphoma activity of KPT-9274 stems primarily from the dysregulation of the DDR pathway, leading to an accumulation of cytotoxic DNA damage. Conversely, in TP53 wild-type cells, the drug’s efficacy is mediated predominantly through the disruption of the B-cell receptor, NF-κB, and related immune signaling pathways. This discovery of distinct, nonoverlapping modes of action supports our proposition of NAMPT as a “dual-context therapeutic node” in MCL, a concept not previously elucidated in other lymphoma types. This mechanistic insight provides a clear rationale for designing personalized therapeutic strategies, suggesting that the selection of combination agents should be stratified by the tumor’s TP53 status.

It is well established that dysregulation of the DDR pathway drives resistance to numerous therapies, including alkylating agents and PARP inhibitors, with TP53 status being a critical determinant of therapeutic response.³⁰ Building on this, our finding that NAMPT inhibition downregulates homologous recombination proteins in TP53 mutant MCL cells aligns with previous reports in other B-cell malignancies. This logically led us to hypothesize that combining NAMPT inhibition with DDR-targeting agents would yield synergistic antitumor effects in a TP53 mutant context. Our results robustly confirmed this hypothesis, demonstrating that NAMPT inhibition significantly sensitizes MCL cells to the alkylating agent bendamustine both in vitro and in vivo. Mechanistically, this synergy is rooted in an impaired DNA repair capacity, which causes an accumulation of lethal DNA damage and subsequent apoptosis. A similar high degree of synergy was also observed with other DDR pathway inhibitors, including PARPi, CHK1i, and WEE1i. Furthermore, we noted that NAMPT inhibition downregulated mutant p53 protein expression in Mino cells, an intriguing preliminary finding that suggests a potential role in modulating TP53 dysfunction itself, warranting further investigation.

Beyond this study, KPT-9274’s role within the tumor microenvironment and its potential in combination with chimeric antigen receptor T cells and bispecific antibodies represents a critical area. Notably, immunotherapy has recently emerged as a significant focus in MCL research, with previous reports also highlighting NAMPT’s involvement in the immune microenvironment. Our current study, particularly in TP53 wild-type MCL cells, further demonstrates that NAMPT inhibition can influence changes in immune-related pathways, thus warranting further investigation into NAMPT’s role in MCL immunotherapy.

While our study provides significant insights into KPT-9274’s mechanism of action, certain limitations should be acknowledged. Our in vivo findings used xenograft models, which do not fully recapitulate the human tumor microenvironment; thus, future studies will use more physiologically relevant models such as patient-derived xenografts. Furthermore, the reliance on cell lines and a limited number of primary patient samples necessitates expansion to larger, more diverse cohorts for broader validation and biomarker identification.

Notwithstanding these limitations, our work herein demonstrates, to our knowledge, for the first time the potent tumor-suppressive activity of the NAMPT inhibitor KPT-9274 in MCL. Notably, dual targeting of NAMPT with alkylating agents or DDR inhibitors represents a novel therapeutic strategy for TP53 mutant MCL, effectively overcoming chemoresistance through synthetic lethality (Figure 6B). Equally significant, in TP53 wild-type MCL, NAMPT inhibition may reverse BTK inhibitor resistance and potentiate immunotherapy via immune pathway modulation, whereas its combination with BCL2 inhibitors can enhance apoptosis induction, thereby exerting synergistic antitumor effects and providing a novel therapeutic direction for relapsed/refractory MCL, although the clinical translation of this strategy still requires further verification. These findings collectively establish NAMPT as a pivotal therapeutic node in MCL and provide, to our knowledge, the first evidence supporting TP53 status–guided, subtype-specific precision therapy. Our work bridges mechanistic discovery with clinical translation, offering actionable insights for therapeutic development in this molecularly heterogeneous disease. While these preclinical insights are promising, further validation in more physiologically relevant models and diverse patient cohorts, particularly regarding synergy with emerging immunotherapies, will be crucial for realizing KPT-9274’s full clinical potential.

Conflict-of-interest disclosure: The authors declare no competing financial interests.