Biological Functions and Therapeutic Potential of NAD+ Metabolism in Gynecological Cancers

NAD + is a key cofactor that plays a role in various metabolic and signaling pathways. Understanding its ability to regulate cellular homeostasis also sheds light on its role in tumorigenesis and disease progression in gynecologic cancers. By reviewing the enzymes that consume NAD + as well as the pathways and enzymes that synthesize NAD + , the vast influence NAD + has on the survival of cancer cells becomes clear. With that information, advances have been made in the development of unique inhibitors to target NAD + synthesizing and consuming enzymes.

NAD + is a biomolecule integral to numerous essential biological processes. It is a key driver of energy production and a crucial signaling molecule, highlighting its significance in health and disease [ 1 ]. Cancer cells can reprogram their metabolism based on genetic, epigenetic, or microenvironmental changes, allowing increased growth rates and survival under limited energy resources and adverse conditions, thereby providing an advantage during tumor progression [ 2 ]. Along with its role in metabolic regulation, NAD + acts as a key cofactor in various cellular signaling pathways through its ability to be consumed by enzymes such PARPs and sirtuins, giving it a key role in aging, the immune response, and various other pathways [ 3 ]. NAD + signaling influences various processes that are dysregulated in cancer, including DNA repair, cell proliferation, differentiation, redox regulation, and oxidative stress [ 4 ]. Tumor cells exhibit higher ratios of the oxidized forms of NAD + and NADP + compared to nontumor cells, highlighting the crucial role of NAD + in metabolic plasticity [ 5 ]. Tumor metabolism relies heavily on the pentose-phosphate pathway (PPP), serine biosynthesis, and fatty acid synthesis, all of which are dependent on NAD + and NADP + . Thus, NAD + biosynthesis becomes a key driver of cancer metabolism [ 6 , 7 , 8 ]. Furthermore, NAD + is a vital regulator of cell signaling and survival pathways due to its role as a cofactor for enzymes essential in cellular energy production and various metabolic processes such as glycolysis, fatty acid oxidation, and the citric acid cycle [ 1 ]. NAD + and NADH are crucial coenzymes in redox reactions, and an imbalance in their ratio can disrupt the flow of these pathways’ reactions, leading to a dysregulated cellular metabolism. Numerous studies have discovered that changes in NAD + levels significantly contribute to metabolic disorders, neurodegenerative diseases, and tumorigenesis [ 5 , 9 , 10 , 11 , 12 , 13 ]. Here, we discuss the importance of cellular NAD + metabolism in gynecologic cancer.

The Preiss–Handler pathway highlights the intricate process by which dietary niacin and nicotinic acid (NA) are utilized to produce NAD + ( Figure 1 B). The enzyme nicotinic acid phosphoribosyl transferase (NAPRT) is the rate-limiting step in this pathway, converting nicotinic acid to the intermediate nicotinic acid mononucleotide (NAMN). NAMN is then converted into nicotinic acid adenine dinucleotide (NAAD) by nicotinamide mononucleotide adenylyl transferases (NMNATs) [ 19 ]. Its final step then utilizes NAD + synthase (NADSYN1 or NADS)-to catalyze them replacement of the carboxylic group in NAAD with an amide group from glutamine to produce NAD + as its final product [ 20 ].

NAD + acts as a crucial cofactor in metabolic processes and is also required for several enzymatic reactions. Most notably, NAD + is reduced to NADH or NAD + to NADPH in several reactions during glycolysis and oxidative phosphorylation [ 31 ]. These reactions themselves are indicative of the pertinence of these redox couples in maintaining cellular metabolism and redox homeostasis. Yet, the NAD + levels in cells are often regulated by its consumption through enzymatic cleavage to produce NAM, which is subsequently shuttled into the NAD + salvage pathway to resynthesize NAD + [ 32 ]. The predominant consumers can be categorized into four classes: poly (ADP-ribose) polymerases (PARPs), Sirtuins (SIRTs), CD38 and CD157 (two cADP-ribose synthases (cADPRs)), and nicotinamide N-methyltransferase (NNMT) ( Figure 1 C and Figure 2 ) [ 13 ]. These enzymes utilize NAD + in order to maintain various cellular signaling pathways that include but are not limited to DNA repair, the inflammatory response, and post-translational modifications [ 20 ]. 2.2.1. Poly (ADP-Ribosyl) Polymerases (PARPs) The human PARP enzyme family is composed of 17 unique proteins that are responsible for cleaving NAD + to produce NAM and ADP-ribose [ 33 ]. The ADP-ribose is then used to build single or covalently linked polymers by the PARP enzymes, referred to as mono(ADP-ribosyl)ation or poly(ADP-ribosyl)ation, respectively [ 19 ]. Nuclear PARP enzymes are activated in response to single-stranded breaks in DNA, a process referred to as base excision repair ( Figure 2 A). PARP1, PARP2, and PARP3 are the members that are unique in their DNA-dependent activation, making them key regulators in DNA repair pathways [ 34 , 35 ]. The DNA-dependent PARP enzymes bind tightly to the DNA break and begin to build the poly(ADP-ribose) (PAR) chains to help build the scaffold that recruits DNA repair enzymes to repair the damaged DNA [ 36 ]. PARP1 in particular is a major consumer of NAD + , with it accounting for close to 90% of the total PARP activity [ 32 ]. However, as mentioned before, the other members of the PARP family are unique in their localizations, structure, and function [ 37 ]. Though PARP1 and PARP2 are known for their poly(ADP-ribosyl)ation (PARylation), most of the other PARP enzymes are monoenzymes responsible for catalyzing mono(ADP-ribosyl)ation (MARylation) reactions [ 38 ]. PARP7 (TiPARP) is one of the mono(ADP-ribosyl) transferases found in both the cytosol and nucleus. It was found to modify hundreds of different proteins, with targets identified through an orthogonal assay system using NAD + analogs [ 39 ]. Of its known functions, PARP7 has been shown to modify α-tubulin on microtubules in the cytoskeleton of cancer cells [ 37 ] and to act as a negative regulator of type 1 interferon (IFN) signaling in tumor microenvironments [ 40 ]. Another member with a cytosolic localization is PARP16: a tail-anchored endoplasmic reticulum transmembrane protein functioning as an ER stress sensor [ 41 ]. PARP16 is pertinent in the activation of endoplasmic reticulum (ER) stress sensors such as PERK and IRE1α through ADP-ribosylation during the unfolded protein response (UPR) [ 41 ]. With PARPs being responsible for the regulation of various cellular stress responses as well as DNA damage repair pathways, the necessity of sustained NAD + levels become pertinent for PARP function and ultimately cell survival in high-stress environments.

CD38 and CD157 are cell-surface anchored, dual-receptor enzymes that are genetically homologous and have multifunctionality, allowing for both glycohydrolase and ADP-ribosyl cyclase activities [ 47 ]. This allows for CD38 and CD157 to utilize their glycohydrolase activity to cleave NAD + into NAM and ADP-ribose and use their ADP-ribosyl cyclase activity to form cyclic ADP-ribose, a key messenger molecule in Ca 2+ signaling ( Figure 2 C) [ 48 ]. Though they belong to the ADP-ribosyl cyclase family and share similar enzymatic function as well as their ability to be antigens [ 49 ], they vary in their structure, localization, and overall role. CD38 is a transmembrane protein that is universally expressed, particularly during inflammation [ 50 ], and an adhesion receptor that interacts with CD31 to mediate immune cell trafficking [ 51 ]. CD157 is a glycophosphatidylinositol-anchored protein that was originally identified as an Mo5 myeloid differentiation marker but has since been understood to have a much more diverse function in immunity that is not limited to just the myeloid compartment [ 52 ]. However, CD157’s possible cell surface receptor activity and its overall role in human immunity have yet to be fully understood [ 53 ].

3.1. NAD + Biosynthesis Pathway in Gynecologic Cancers With their most distinctive feature being accelerated and unregulated growth, gynecologic cancer cells, like many other cancers, require altered metabolism to maintain this phenotype [ 60 ]. In cancer cells, the NAD + levels are much higher compared to normal cells, which is likely due to an upregulation of NAD + synthesis [ 5 ]. To meet the peak NAD + requirements of both redox and non-redox processes, NAD + is produced from dietary precursors such as tryptophan and forms of vitamin B3, as described previously ( Figure 1 ) [ 14 ]. In this section, we describe how enzymatic activity in the NAD + biosynthesis pathways shift during tumorigenesis and the influence they have over the tumor microenvironment and disease progression. 3.1.1. Preiss–Handler Pathway: Nicotinic Acid Phosphoribosyl Transferase (NAPRT) NAPRT has been shown to have heightened expression in several prevalent cancer types, including ovarian cancer [ 61 , 62 ]. Mutations in breast cancer gene 1 and 2 (BRCA1 and 2) increase the risk of developing ovarian cancer due to dysregulation of DNA damage repair. This is because BRCA genes play a role in in DNA double strand break repair, which is essential for the homologous recombination repair (HRR) pathway [ 63 , 64 , 65 , 66 ]. In epithelial ovarian cancer, it was found that high NAPRT expression was correlated with BRCA gene expression [ 67 ]. Consistent with these findings, NAPRT was found to have a role in DNA repair processes in the deficit of BRCA2 in response to chemotherapeutics [ 68 ]. It was then suggested that in cancers with defective HRR, including ovarian cancer, an increase in NAPRT expression confers a selective advantage by ensuring the maintenance of sufficient NAD + levels, allowing for PARP activity and cellular protection from oxidative stress and DNA damage ( Figure 3 A) [ 68 ]. Due to NAPRT overexpression potentially causing resistance to DNA-damaging agents, the enzyme could act as a possible therapeutic target for patients with BRCA-mutated ovarian cancer to prevent or reverse treatment resistance.

As the rate-limiting enzyme in the NAD + biosynthetic salvage pathway, NAMPT influences intracellular NAD + levels through its pivotal role in NAD + biosynthesis from nicotinamide [ 10 , 99 ]. In the context of cancer biology, NAMPT is often regarded as a potent oncogene ( Figure 3 A and Table 1 ) [ 100 ]. Several studies have highlighted the upregulated expression of NAMPT in various cancers, including colorectal, breast, ovarian, endometrial, prostate, and pancreatic cancers, with higher NAMPT expression often being correlated with poorer survival outcomes [ 100 , 101 , 102 , 103 , 104 ]. In ovarian cancer, increased NAMPT expression has been found to be linked to tumor lymph node metastases, invasion, advanced clinical stage, and poor survival outcomes [ 101 ]. BRCA1 knockdown was found to effectively increase NAMPT levels in ovarian cancer cells and primary non-BRCA1-mutated cells, suggesting a critical role for NAMPT in BRCA1-related NAD + synthesis in ovarian cancer [ 105 ]. Numerous studies have shown that changes in NAMPT expression occur with the development of drug resistance to targeted therapies [ 100 , 106 , 107 , 108 ]. Platinum resistance is a lasting issue for most ovarian cancer patients, with most platinum-based therapies operating by inducing cellular senescence and causing the induction of cancer-stem-like cells as a response [ 109 ]. This new stem-like phenotype ultimately causes chemoresistance, as ovarian cancer cells will adapt a quiescent phenotype allowing for an autophagic state [ 110 ]. When analyzing the NAMPT derived from patient ascites, the authors found that the NAMPT was inducing an increased migratory ability in ovarian cancer cells. This phenotype was observed to be caused by the activation of Rho-ROCK-mediated signaling that induced actin polymerization [ 111 ]. A study by Kudo et al. has provided evidence of a link between using KPT-9274—a dual NAMPT and PAK4 inhibitor—and the reduction of inflammation, decrease in cell growth, and downregulation of DNA-repair-related genes in platinum-resistant 3D-cultured ovarian cancers spheroids [ 112 ]. Another study by Narcelli et al. showed that treatment using the NAMPT inhibitor FK866 in combination with cisplatin treatment reduces tumor growth, delays tumor relapse, and improves the survival outcomes of cisplatin-treated epithelial ovarian cancer (EOC) in pre-clinical models [ 109 ]. In endometrial cancer, NAMPT expression is shown to be relatively higher in endometrial carcinoma patient tissues when compared to normal tissues and that high NAMPT levels lead to poor survival outcomes [ 113 ]. Additionally, eNAMPT levels were found to be elevated in the serum from endometrial cancer patients, and this was found to be correlated with NAMPT expression in the tumor tissue [ 113 ]. In pre-clinical models, NAMPT enhanced endometrial cancer progression by targeting the PI3K/AKT and MAPK/ERK signaling pathways, altering its progression through the G1/S phase of the cell cycle ( Table 1 ) [ 114 ]. In vivo xenograft models demonstrated that NAMPT-treated mice showed enhanced tumor growth in endometrial carcinoma, exhibiting a high Ki-67 proliferation index [ 114 ]. Though NAMPT is greatly responsible for the conversion of NAM to NMN, it is not the only source of NMN that cells have access to. Cells can uptake NMN directly from the extracellular environment, as well as use extracellular NR as previously mentioned. Ecto-5′-nucleotidase CD73 is a GPI-anchored glycoprotein on the extracellular side of the plasma membrane [ 115 , 116 ]. CD73 consumes NMN and converts it to NR [ 117 ], which then becomes internalized and converted back to NMN by nicotinamide riboside kinases (NRK1/2) ( Figure 1 C) [ 118 ]. The CD73 found in tumor immune environments is overexpressed in different cancers, including ovarian cancer [ 119 ]. In cancer cells, CD73 generates adenosine, an immunosuppressive metabolite, thereby affecting the anti-tumor T-cell response [ 120 ]. CD73 activity leads to an enhancement in DNA damage repair and causes chemotherapy resistance through its NR production [ 121 ]. High CD73 expression has been reported to be associated with worse clinical outcomes in ovarian cancer patients and may lead to the development of metastasis [ 119 ]. In fibroblasts, increased expression of CD73 enhances tumor immunosuppression and promotes tumor growth [ 119 ]. CD73-mediated adenosine production can also promote tumor cell migration and metastasis by activating adenosine’s A2A and A2B receptors [ 82 ]. The inhibition of the adenosine A2A and A2B receptors (A2AR/A2BR) was found to enhance the immune system’s ability to exert an anti-tumor response against cancer cells; conversely, stimulating the A2A and A2B receptors limits T-cell proliferation and cytotoxicity [ 120 , 122 , 123 ].

With NAD + levels being so closely linked to tumorigenesis and tumor progression, a better understanding of the changes to NAD + consumers in gynecologic cancers can give us insight into how cancer cells differentiate from their parent tissue cell type. In the following section, we will discuss the changes in NAD + consumers in gynecologic cancers and note their distinct roles in gynecologic cancer progression ( Table 1 ). 3.2.1. PARPs in Gynecologic Cancers PARP1, the most studied member of the PARP family, plays a crucial role in repairing single-strand breaks (SSBs) and is activated in response to DNA damage, thus ensuring the safeguarding of DNA integrity and maintaining genomic stability [ 124 ]. When activated in cells experiencing DNA damage or external stress, PARP1 consumes substantial amounts of NAD + during the DNA repair process, leading to NAD + depletion [ 125 ]. PARP1 induces apoptosis by activating p53 under normal conditions in response to severe DNA damage [ 84 ]. However, excessive activation of PARP1 following genotoxic exposure can deplete NAD + and ATP levels, disrupting sirtuin activity and cellular homeostasis and ultimately leading to cell death [ 85 , 126 ]. PARP1 can function as either a tumor promoter or suppressor depending on its degree of activation [ 127 , 128 ]. Still, many tumors upregulate PARP1 to aid in DNA damage repair, particularly under the influence of cellular stressors like radiotherapy and chemotherapy ( Table 1 ). Elevated PARP1 expression is commonly observed in various carcinomas such as breast, ovarian, and lung cancers and non-Hodgkin’s lymphoma [ 84 ]. When examining patient epithelial ovarian cancer tissue, it was found that 73.3% of a 60 patient cohort had overexpression of PARP1 when measured using immunohistochemistry [ 87 ]. Though the mechanism has yet to be explored, studies have shown that platinum-treatment-resistant epithelial ovarian cancer (EOC) tissue has even greater levels of PARP1 expression, leading to speculation of its potential as a marker for predicting poor prognosis and platinum resistance [ 87 , 129 ]. In addition to PARP1, other members of the PARP family play distinct roles in ovarian cancers. The cancer cells’ accelerated growth creates the necessity for the proper regulation of protein synthesis and other metabolic processes to ensure cell survival. Previous studies have also shown that to achieve proper cell growth and survival, many transformed cancer cells have altered levels of proteotoxic stress [ 86 ]. Many ovarian cancer cell lines have shown overexpression of NMNAT2, indicating an increase in cytosolic NAD + levels as well as cell growth [ 38 ]. Due to the direct endoplasmic reticulum localization of PARP16, the direct interaction between PARP16 and NMNAT2 influences protein synthesis levels. When investigating the compartmentalization of NAD + synthesis in ovarian cancer cells, Challa et al. identified a pathway where NMNAT2 and PARP16 together regulate ribosome function and protein homeostasis in ovarian cancer cell lines ( Figure 3 A) [ 38 ]. As mentioned in the previous section, the activity of PARP16 is directly influenced by the overexpression of NMNAT2, which increases the MARylation of ribosomal proteins by PARP16, leading to the inhibition of protein synthesis and preventing the protein aggregation that would lead to greater cellular stress that reduces growth and increases cell death [ 38 ]. Similar to PARP16, MARylation of key cytosolic proteins by PARP7 has been found to regulate the biology of ovarian cancer cells. The expression levels of PARP7 differs between normal and tumor tissues, with malignant ovarian cancer cells showing higher expression of PARP7 [ 130 , 131 ]. PARP7 was found to MARylate α-tubulin, causing the destabilization of microtubules in the cytoskeleton. This has led to the hypothesis that PARP7 is linked to greater motility and migration in ovarian cancer cells [ 37 ]. When NAD + homeostasis is dysregulated, there is a direct effect on PARP activity and DNA damage repair. PARPs have been targeted using inhibitors such as olaparib, niraparib, rucaparib, and veliparib to prevent the activation of PARPs in diseases like cancer where the DNA damage is high in cells [ 132 ]. The use of PARP inhibitors (PARPis), particularly PARP1 inhibitors, has been shown to suppress cell viability and motility in EOC cells and could potentially increase sensitivity to platinum treatments and other chemotherapeutics [ 133 ]. Current studies are also focused on developing inhibitors for the PARP monoenzymes that are key therapeutic targets for ovarian cancers such as PARP7 and PARP16 [ 134 , 135 ]. These topics will be discussed in further detail in the coming sections.

Zhu et al. conducted a recent study where the authors analyzed CD38 mRNA expression levels in normal ovaries and epithelial ovarian cancer (EOC) using online datasets like GEPIA, Oncomine, and TISIDB. It was found that CD38 expression in EOC is significantly higher in borderline ovarian tumors when compared to normal tissue [ 140 ]. Variations in CD38 expression across the immune subtypes of ovarian cancer were noted, with the C2 subtype (IFN-γ dominant) showing the highest levels [ 140 ]. This analysis emphasizes CD38’s strong association with immunological characteristics in the tumor microenvironment. Furthermore, high CD38 expression is correlated with better survival outcomes in EOC, particularly in stages III and IV and grades II and III [ 140 ]. Studies have shown that CD157 expression in ovarian cancer cells enhances tissue invasion and cell migration [ 141 ], thus playing a pivotal role in promoting ovarian cancer metastasis by regulating the interaction of ovarian cancer cells with the mesothelium and extracellular matrix proteins through its expression [ 142 ]. CD157-overexpressing ovarian cancer cells also exhibit mesenchymal traits, promoting tumor cell proliferation while reducing apoptosis [ 141 ]. Furthermore, high CD157 expression was seen to be correlated with increased malignancy and a higher risk of relapse in ovarian cancer [ 143 ]. When comparing serous ovarian cancer primary tissue cells to those obtained from the patient’s ascites, Ortolan et al. were able to note that nearly all the primary tumor cells expressed CD157, yet only 10% of the cells isolated from ascites expressed CD157. Furthermore, the authors found that cells were able to re-express CD157 after being cultured on plates, allowing for the theory that the expression is adhesions dependent. Thus, regarding the prospect of reversing the trend that was seen, the authors proposed targeting CD157 with monoclonal antibodies in order to reduce tumor cell adhesion to extracellular proteins like collagen, fibronectin, and laminin [ 143 ].

The upregulation of NAD + synthesis has been found to be associated with cancer development and progression [ 149 ]. Inhibiting NAD + synthesis and targeting NAD + consumers show promise in cancer treatment, likely due to the differing roles of these enzymes [ 150 ]. NAD + synthesis enzymes mainly influence tumor cell behaviors, while NAD + -consuming enzymes affect the immunosuppressive tumor microenvironment [ 149 ]. Enzymes such as Sirtuins, PARPs, CD38, and other consumers influence critical cellular functions and are closely linked to cancer progression, as described in the previous section [ 151 ]. Recent studies have revealed these enzymes’ roles in regulating cell metabolism, cytokine release, neoantigen expression, and modifying the tumor immune microenvironment, thereby exhibiting anticancer effects [ 83 , 152 ]. Drug development in targeting these enzymes is ongoing. Here, we summarize the recent advancements in inhibitors targeting the NAD + biosynthesis and consumption pathways in gynecologic cancers ( Table 2 ). 4.1. Targeting NAD + Biosynthesis via the Salvaage Pathway—Targeting NAMPT Nicotinamide phosphoribosyl transferase (NAMPT) regulates the intracellular NAD + levels crucial for cellular redox reactions and NAD + -dependent enzyme function, and it also exhibits cytokine-like activity [ 19 ]. NAMPT influences metabolic disorders and cancer by modulating oxidative stress, apoptosis, lipid metabolism, inflammation, and insulin resistance [ 10 ]. NAMPT is overexpressed in various cancers due to the need to meet increased energy demands, driving cellular metabolism and responses to NAD + depletion [ 150 ]. This overexpression, coupled with increased NAD + levels, enhances cancer cell survival by making cells resistant to anti-cancer treatments [ 171 ]. Additionally, NAMPT-specific inhibitors lower NAD + levels and the activity of metabolic pathways including glycolysis, the citric acid cycle, and OXPHOS, thereby helping to suppress cancer cell proliferation [ 172 ]. Several reports have also indicated a link between elevated NAMPT levels and negative clinical outcomes including reduced survival rates across various types of cancer [ 173 , 174 ]. Specifically, an analysis using TCGA datasets by Kudo et al. found that an elevated level in NAMPT expression was consistent with worse overall survival in patients with ovarian, endometrial, and cervical cancers [ 112 ]. As a result, NAMPT has been a target for the development of a number of inhibitors over the past few decades. NAMPT inhibitors have demonstrated significant anticancer activity in both in vitro and in vivo cancer models [ 172 ]. Targeting NAMPT for oncology has led to the development of compounds with improved inhibitory effects, such as FK866 (also known as (E)-Daporinad), CHS828, GEN617, OT-82, GMX1777, and other cytotoxic agents [ 175 , 176 , 177 ]. Among the identified NAMPT inhibitors, the prototypical compound FK866 and CHS-828, with its prodrug GMX1777, have been evaluated in early-phase clinical trials involving cancer patients. Ongoing research aims to develop NAMPT inhibitors with enhanced therapeutic efficacy and optimized dosing strategies [ 178 ]. In this section, we will highlight the antitumor effects of NAMPT inhibitors both in vitro and in vivo in gynecologic cancer models, as well as advancements in their clinical applications. 4.1.1. FK866 (E)-N-[4-(1-Benzoylpiperidin-4-yl)butyl]-3-(pyridin-3-yl)acrylamide (FK866) was the first NAMPT inhibitor reported by Hasmann et al. in 2003 [ 175 ]. FK866 has an IC50 of 1nM and binds to the same binding pocket as nicotinamide but with higher affinity, acting as a partially noncompetitive inhibitor to NAMPT. FK866 indirectly suppresses mitochondrial respiratory activity while selectively inhibiting NAMPT, thereby causing a gradual depletion of NAD + [ 175 ]. This conclusion was further supported by Gogola-Mruk et al. in ovarian cancer spheroids, where the authors were able to show that visfatin, another name for NAMPT, increased ATP levels and mitochondrial activity, while its inhibition with FK866 reversed these effects [ 153 ]. FK866 and PARP Inhibitors Poly(ADP-ribose) polymerase inhibitors (PARPis) are the standards of care for the treatment of advanced and recurrent epithelial ovarian cancer [ 179 ]. PARPis target the PARP1 protein that synthesizes chains of poly(ADP-ribose) (PAR) onto its target DNA [ 180 ]. PARP1 cleaves NAD + into nicotinamide (NAM) and ADP-ribose (ADPr), attaching the ADPR moieties to its target DNA damage. NAM is recycled to form NAD + through the salvage pathway via NAMPT and the NMNATs [ 181 ]. This process is critical for DNA damage repair, chromatin remodeling, and cell death [ 180 ]. Due to the long exposure to this drug treatment, resistance to PARPis has been observed as leading to recurrence; efforts have been made to study the mechanisms and ways to overcome resistance to PARPis [ 182 ]. FK866 was found to sensitize PARPi-resistant cell-based

In gynecologic cancers, FK866 treatment was shown by Nacarelli et al. to result in better survival outcomes in mice transplanted with the ovarian cancer cell line OVCAR3 when combined with cisplatin [ 109 ]. In this study, the authors demonstrated that inhibition of NAMPT resulted in the suppression of senescence-associated cancer-stem-like cells (CSCs), which are induced by platinum-based chemotherapy and can contribute to chemoresistance in ovarian cancer. The combination of FK866 and cisplatin also resulted in reduced tumor outgrowth and improved overall survival in mice bearing epithelial ovarian cancer [ 109 ].

P21 activated kinase 4 (PAK4) plays a critical role in various signaling pathways in cancer, including the Wnt/β-catenin, RAS-ERK, and androgen/estrogen receptor pathways [ 189 ]. PAK4 is overexpressed across different cancer types and has been proposed as a biomarker for cancer [ 10 , 190 ]. KPT-9274 is a dual inhibitor, targeting both NAMPT and PAK4 [ 112 ]. Using 3D spheroids with ovarian cancer cells resistant to platinum-based therapy that mimic the CSC-enriched tumor masses floating intraperitoneally, Kudo et al. demonstrated that KPT-9274 inhibited NAD + production via direct NAMPT inhibition and also reduced PAK4 activity, resulting in an NAD + -dependent decrease in the phosphorylation of S6 ribosomal protein, AKT, and β-Catenin [ 112 ]. In clinical trials, KPT-9274 was used in the PANAMA trial ( NCT02702492 ), where patients with advanced solid tumors were recruited for the study [ 160 ]. This study was terminated in 2021, and the results have not been reported; it is also unclear whether any of the participants had GYN cancer. Despite promising preclinical results, NAMPT inhibitors have shown limited tumor responses in phase I/II clinical trials, suggesting the need for biomarkers to identify patients who may benefit from these inhibitors.

4.3.1. PARP Inhibitors As a major NAD + consumer, PARP inhibitors are probably the most clinically utilized inhibitors of NAD + metabolic pathways in treating GYN cancers. Olaparib was the first PARP inhibitor approved in 2014 following Study 42, which demonstrated its effectiveness as a monotherapy in patients with germline BRCA1/2 mutations who had had previously multiple lines of chemotherapy [ 161 ]. Olaparib was then brought to the front line as maintenance therapy in patients with BRCA-mutated ovarian cancer after SOLO1, a phase III clinical trials in 2018 [ 162 ]. Rucaparib was the second PARP inhibitor approved, receiving FDA accelerated approval in 2016 based on the findings of the ARIEL2 trial for patients with BRCA-mutated, relapsed ovarian cancer and, subsequently, approval as a maintenance therapy for patients with platinum-sensitive ovarian cancer based on the results of the ARIEL3 trial in 2018 [ 163 ]. Niraparib was the most recent PARPi to receive approval as a treatment for ovarian cancer, with the first approval for maintenance therapy happening in 2017 in the NOVA trial and its approval for first-line maintenance therapy in newly diagnosed ovarian cancer in the PRIMA trial [ 164 ]. PARP inhibitors are, overall, quite well tolerated, with common side effects including fatigue, anemia, and nausea. In 2022, the FDA approval was withdrawn for olaparib, niraparib, and rucaparib for patients with multiple-line therapies based on the findings from the ARIEL4, SOLO3, and QUADRA trials [ 165 , 166 , 167 , 168 ]. However, PARP inhibitors remain an important frontline maintenance therapy for patients with BRCA-mutated, platinum-sensitive ovarian cancer Resistance to PARP inhibitors, both endogenous and acquired, has been identified to contribute to disease progression. A review by Guidice et al. discussed HR function restoration, DNA replication fork stabilization, BRCA reversion mutations, dissociation of PARP1 and PARG, and epigenetic modifications as some of the well-studied mechanisms of PARP inhibitor resistance [ 193 ]. Current studies are working to identify strategies to overcome resistance, including various combinations of PARP inhibitors and other pathway inhibitors [ 193 ].

NNMT has been found to have elevated expression in peritoneal stroma and omental metastases in ovarian cancer, and its expression correlates with the aggressive behavior of ovarian cancers with poor prognosis [ 93 , 194 ]. NNMT is a potential target for cancer therapy development; there are currently three main types of NNMT inhibitors: competitive, bi-substrate, and covalent [ 195 ]. Among these existing inhibitors, 5-amino-1-methylquinolinium (5-amino-1MQ) was recently used in a study by Eckert et al. to demonstrate the critical role of NNMT in the tumor microenvironment in facilitating ovarian cancer growth and metastasis [ 93 ]. When CAFs were treated with 5-amino-1MQ, histone methylation increased, supporting the authors’ hypothesis that ovarian cancer progression via NNMT was driven by epigenetic changes in the stromal cells. Mice treated with 5-amino-1MQ after being injected with ovarian cancer HeyA8 cells were found to have decreased tumor burden by about threefold and increased stromal H3K27 methylation [ 93 ]. 5-Amino-1MQ was also used in another study on ovarian cancer cells by Huang et al. [ 170 ]. In this study, the authors found that expression of fat mass and obesity associated protein (FTO) increased the sensitivity of ovarian cancer cells to platinum both in vitro and in vivo. NNMT was found to be targeted by FTO, which was upregulated and found to demethylate NNMT transcripts in FTO-overexpressing ovarian cancer cells. When NNMT was knocked down or inhibited using 5-amino-1MQ, FTO-induced sensitivity to platinum was rescued, suggesting a novel function of FTO-dependent RNA modifications in regulating the platinum response by targeting the enzyme NNMT [ 170 ]. More experimental studies are still needed to evaluate the therapeutic efficacy of these inhibitors, especially in humans.