Abstract
Kynurenine 3-monooxygenase (KMO), a key enzyme within the kynurenine (KYN) pathway of tryptophan (TRY) metabolism, enables the excess production of toxic metabolites (such as 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid and quinolinic acid), and modulates the balance between these toxic molecules and the protective metabolite, kynurenic acid (KYNA). Despite its importance, KMO suppression as a treatment for cancer has not been fully explored. Instead, researchers have focused on prevention of KYN pathway activity by inhibition of enzymes indoleamine 2,3-dioxygenase (IDO1 and IDO2) or tryptophan 2,3-dioxygenase (TDO, also known as TDO2). However, studies using IDO/TDO inhibitors against cancer have not yet shown that this type of treatment can be successful. We argue that KMO suppression can be an effective strategy for treatment of cancer by 1) decreasing toxic metabolites within the KYN pathway and 2) increasing levels of KYNA, which has important protective and anticancer properties. This strategy may be beneficial in the treatment of aggressive breast cancer, particularly in patients with triple-negative breast cancer. A major challenge to this strategy, when searching for an effective treatment for tumors, especially tumors like breast carcinoma that often metastasize to the brain, is finding KMO inhibitors that adequately cross the blood-brain barrier.
Excess or unbalanced activity of the kynurenine (KYN) pathway has been demonstrated in numerous diseases, including Alzheimer’s (1), Parkinson’s (2), schizophrenia (3), post-traumatic stress disorder (4), myocardial infarction (5), and cancer. The KYN pathway is particularly important in cancer, causing it to be resistant to therapy by secreting the enzyme IDO and TDO. This increases the synthesis of toxic kynurenines, such as 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid, and quinolinic acid, resulting in T cell apoptosis and the creation of a highly immunosuppressive environment in the patient (6-8). Cancer cells also increase the activity of kynurenine 3-monooxygenase (KMO), a downstream enzyme in the KYN pathway (Figure 1).
Tryptophan metabolism along the kynurenine (KYN) pathway with key enzymes IDO-1, IDO-2, and TDO (circled) and KMO (circled with dashes). Metabolites surrounded by a rectangle and in red can have major destructive effects.
Although the majority of TRY metabolism occurs normally through the KYN pathway (and the KYN pathway has many important protective and immune regulatory functions), excess stimulation of this pathway by inflammation, injury or other factors can result in increased production of toxic metabolites. This increased activity of the KYN pathway may be stimulated by multiple factors (Table I), but it is primarily caused by increases in the levels of pro-inflammatory cytokines, which shift tryptophan metabolism away from the serotonin-melatonin pathway to the KYN pathway (9-23).
Factors associated with increased production of toxic kynurenines.
The most important cytokines involved in the stimulation of the KYN pathway are interferon γ (IFN-γ), TNF-α, IL-1β, and IL-6, although other cytokines also have a major role (Table II) (24-27). For instance, TGF-β is an anti-inflammatory cytokine, but also has pro-inflammatory functions. It causes T effector cell paralysis through suppression of IFN-γ and TGF-β receptor. TGF-β inhibits tumorigenesis in early stages, but promotes tumor growth in later stages (24, 25, 27).
Toxic Kynurenines
As noted, increased stimulation of the KYN pathway results in increased levels of toxic kynurenines in the main branch of the pathway, including KYN, 3-hydroxykynurenine (3HK), anthranilic acid (AA), xanthurenic acid (XA), 3-hydroxyanthranilic acid (3HAA), and quinolinic acid (QA). These metabolites are known to be toxic to the immune system, brain, heart, eye, as well as to other organs (28).
Current Methods of Suppressing Toxic Kynurenines
Much work has been done over the last decade in attempts to find ways to reduce production of these toxic kynurenines. Many attempts have been made to decrease the activity of the KYN pathway directly by suppressing enzymes, such as IDO and TDO. For example, commonly used agents, such as aspirin, indomethacin, statins, acyclovir, chloroquine, and anti-estrogens all have mild anti-IDO activity (28). Numerous natural products have now been shown in experimental models to suppress IDO, such as resveratrol, rosmarinic acid (rosemary extract), and brassinin.
However, none of these have proven to be effective in clinical studies. Likewise, despite promising pre-clinical results, treatments specifically designed as IDO inhibitors from NewLink, Incyte, and other companies, have failed in patient trials. The most dramatic example of this was the failure of Incyte’s epacadostat, which was given in combination with Merck’s pembrolizumab against malignant melanoma in the ECHO-301 trial (29, 30). It has been speculated that the failure in the trial may be due to epacadostat itself, rather than to the approach of using IDO inhibition, or due to the fact that epacadostat is a pure IDO-1 inhibitor that does not affect TDO or IDO-2 and that suppression of all three enzymes that produce toxic kynurenines may be necessary (31, 32).
Kynurenine 3-Monooxygenase (KMO)
Others have suggested that, rather than attempting to suppress excess activity of the KYN pathway with inhibition of enzymes IDO and TDO, a better approach might be to suppress the activity of KMO, a key enzyme within the KYN pathway. Inhibition of KMO decreases levels of toxic TRY metabolites in the main branch of the KYN pathway. KMO inhibition and the resulting decreases in toxic kynurenines have been linked to decreases in disease severity as well as significant increases in longevity in yeast, fruit fly, C. elegans, worm, and mouse models (33-35). These benefits are lost when 3HK levels are restored in these models (31). Additionally, after KMO suppression, increased levels of the neuroprotective metabolite KYNA are formed. There is much information on the role of KMO in multiple types of diseases including Huntington’s disease, Alzheimer’s disease, and cancer. Over-expression of KMO in cancer is linked to malignancy and poor prognosis.
KMO in Triple-negative Breast Cancer
It has been reported that KMO is elevated in all breast invasive carcinomas, as well as in the normal breast cells adjacent to the tumor (36). Triple-negative breast cancer (TNBC) is a breast cancer subtype characterized by estrogen receptor negative, progesterone receptor negative, and human epidermal growth factor receptors type 2 (HER2) negative. Incidence of TNBC is higher in African-American women (37). TNBC typically has a poor prognosis and is minimally responsive to conventional therapy. Liu et al. showed that KMO is amplified in TNBC and that this over-expression leads to an increase in cell growth and colony formation (38). Huang et al. also investigated the role of KMO in TNBC using KMO alteration and expression data from public databases, and found that KMO was amplified and associated with poorer survival (39). KMO promoted cancer aggressiveness by interacting with β-catenin, while KMO suppression caused a decrease in tumor growth and β-catenin expression. They also found that an increased level of KMO results in shorter overall survival and shorter relapse-free interval in patients with breast cancer. Girithar et al., in an update on recent clinical research, reported similar results (40).
In addition, it has been reported that more aggressive breast cancer subtypes, including triple-negative and HER2 type breast cancers, have an even greater level of KMO over-expression compared to other less aggressive breast cancer subtypes (41-44). Wu et al. investigated KMO in 103 patients with TNBC who had surgical resection and found that the patients with the highest KMO had most advanced disease progression and minimal survival, and that a median threefold higher KMO expression was seen in cancer versus non-tumor areas (44). These studies support our previous findings, where we explored tryptophan levels in breast cancer tissue samples from patients with different histologies using fluorescence spectroscopic techniques (45). We found that more aggressive, high grade breast cancer had higher levels of tryptophan content compared to low grade breast cancer (45). Large tumor size, in part due to its connection with high grade, was also linked to high tryptophan content in breast cancer tissue samples, whereas the number of lymph nodes did not correlate with tryptophan levels.
These findings support the idea that KMO plays a major role in decreasing survival for patients with TNBC. Lai et al. identified surface KMO as a potential target for the treatment of TNBC (42). Surface KMO is expressed in the mitochondria and on the cell surface in breast cancer, including in TNBC. In a commentary by Park et al., they argued that KMO inhibition may have clinical value in the treatment of patients with breast cancer, especially TNBC (43).
KMO in Other Cancers
KMO over-expression has been reported in numerous cancers including hepatocellular carcinoma (46), colorectal (47, 48), and glioblastoma (49-51). Jin et al. studied 120 paired hepatocellular (HCC) tissue and adjacent normal liver tissue samples, as well as 205 clinical HCC samples (46). They found that KMO was considerably higher in HCC compared to normal liver tissue samples. They also found that KMO is involved in overall survival and recurrence rates, and in regulating proliferation and invasion of HCC cells in vitro. Similarly, Liu et al. showed that KMO is up-regulated in colorectal cancer tissues in comparison to normal healthy tissues (47). A decrease in KMO resulted in a decrease in the expression of cancer stem cell markers and a reduction in the sphere-formation and invasiveness of colorectal cancer cells.
KMO expression is also important in brain tumors. Cervantes et al. studied KMO activity in cell lines A172, LN-18, U87, and U373, and in samples from patients with astrocytoma (48). They showed that KMO is expressed in astrocytoma and found that high KMO expression correlated with a highly immunosuppressive environment and a lower survival in patients with gliomas. The toxic kynurenine metabolite QUIN accumulates in gliomas. The neuroprotective metabolite, KYNA, which is normally increased when KMO is suppressed, is decreased in plasma samples from patients with GBM (49). The KYNA/KYN ratio was also reduced in human glioma cells. Interestingly, Vezzani et al. recently reported that KYNA levels were much lower in glioblastoma (GBM) (which is very high grade) than they are in lower grade astrocytoma (50). Walczak et al. confirmed that KYNA was present in human GBM T98G cells, and, even at low concentrations, inhibited GBM proliferation and migration, and had synergistic effects when given with temozolomide, a chemotherapy drug frequently used in the treatment of patients with GBM (51).
KMO Inhibitors
Initially, before important structural information of KMO was discovered, KMO inhibitors were designed as analogs of KYN. Nicotinylalanine was one of the first KMO inhibitors and, when tested in rat brains, was able to prevent the accumulation of 3-HK and QUIN, while increasing KYNA levels (52-54). However, nicotinylalanine was weak and non-specific, inhibiting both KMO and kynureninase with low potency. The first KMO specific inhibitor m-nitrobenzoylalamine (m-NBA) was the most potent. It decreased the levels of 3-HK, and increased the levels of KYNA in mice (55). Later KMO inhibitors include 3,4-dichlorobenzoyl alanine (a m-NBA derivative), which was even more potent and was able to inhibit quinolinic acid via blocking interferon gamma (INF-γ). Ultimately, however, this inhibitor was abandoned when it was discovered that it forms cytotoxic hydrogen peroxide by uncoupling NAD(P)H and O2 (56, 57). Other potent KMO inhibitors, based on KMO’s crystal structure, have been developed, including 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulfonamide (Ro-61-8048), UPF648, and Ianthellamide A (56, 58, 59) (Table III). However, these have not been shown to cross the blood brain barrier (BBB) adequately.
Classes of KMO inhibitors and corresponding key KMO structures.
It is, of course, not necessary for a KMO inhibitor to cross the BBB to be effective in the treatment of many types of cancer. However, to be an effective therapy for neurological diseases or for tumors metastatic to the brain, it is likely that a KMO inhibitor would have to be substantially brain-penetrating. Finding effective KMO inhibitors that cross the BBB significantly has thus become a major research goal; and, until recently, these studies have had little success. Chen et al. (60), Hughes et al. (56), Gotina et al. (61), and others have extensively discussed the challenges of finding a KMO inhibitor that is non-toxic, effective, and brain-penetrating. Zhang’s group has reported the discovery of and are continuing to investigate promising KMO inhibitors which do cause decreases in KYN and other toxic TRY metabolites, although the degree of brain penetration and the potency of these compounds are still unclear (62).
Future Directions
It has been suggested that the quantum computer could be an invaluable tool in drug discovery, especially when used in conjunction with machine learning (63-66). We are using the quantum computer to build a 4 billion molecule library of KMO inhibitors that successfully cross the BBB, and will then test the 20 most effective and non-toxic candidates.
Footnotes
Authors’ Contributions
The Authors contributed equally to all aspects of this manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received October 16, 2023.
- Revision received November 4, 2023.
- Accepted November 7, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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