LSD1 in drug discovery: From biological function to clinical application.

Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD) dependent monoamine oxidase (MAO) that erases the mono-, and dimethylation of histone 3 lysine 4 (H3K4), resulting in the suppression of target gene transcriptions. Besides, it can also demethylate some nonhistone substrates to regulate their biological functions. As reported, LSD1 is widely upregulated and plays a key role in several kinds of cancers, pharmacological or genetic ablation of LSD1 in cancer cells suppresses cell aggressiveness by several distinct mechanisms. Therefore, numerous LSD1 inhibitors, including covalent and noncovalent, have been developed and several of them have entered clinical trials. Herein, we systemically reviewed and discussed the biological function of LSD1 in tumors, lymphocytes as well as LSD1-targeting inhibitors in clinical trials, hoping to benefit the field of LSD1 and its inhibitors.

LSD1: an emerging face in altering the tumor microenvironment and enhancing immune checkpoint therapy.

Dysregulation of various cells in the tumor microenvironment (TME) causes immunosuppressive functions and aggressive tumor growth. In combination with immune checkpoint blockade (ICB), epigenetic modification-targeted drugs are emerging as attractive cancer treatments. Lysine-specific demethylase 1 (LSD1) is a protein that modifies histone and non-histone proteins and is known to influence a wide variety of physiological processes. The dysfunction of LSD1 contributes to poor prognosis, poor patient survival, drug resistance, immunosuppression, etc., making it a potential epigenetic target for cancer therapy. This review examines how LSD1 modulates different cell behavior in TME and emphasizes the potential use of LSD1 inhibitors in combination with ICB therapy for future cancer research studies.

Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness.

Several studies have examined the functions of nucleic acids in small extracellular vesicles (sEVs). However, much less is known about the protein cargos of sEVs and their functions in recipient cells. This study demonstrates the presence of lysine-specific demethylase 1 (LSD1), which is the first identified histone demethylase, in the culture medium of gastric cancer cells. We show that sEVs derived from gastric cancer cells and the plasma of patients with gastric cancer harbor LSD1. The shuttling of LSD1-containing sEVs from donor cells to recipient gastric cancer cells promotes cancer cell stemness by positively regulating the expression of Nanog, OCT4, SOX2, and CD44. Additionally, sEV-delivered LSD1 suppresses oxaliplatin response of recipient cells in vitro and in vivo, whereas LSD1-depleted sEVs do not. Taken together, we demonstrate that LSD1-loaded sEVs can promote stemness and chemoresistance to oxaliplatin. These findings suggest that the LSD1 content of sEV could serve as a biomarker to predict oxaliplatin response in gastric cancer patients.

Generation, secretion and degradation of cancer immunotherapy target PD-L1.

Cancer immunotherapy is a rapidly developing and effective method for the treatment of a variety of malignancies in recent years. As a significant immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) and its receptor programmed cell death protein 1 (PD-1) play the most significant role in cancer immune escape and cancer immunotherapy. Though PD-L1 have become an important target for drug development and there have been various approved drugs and clinic trials targeting it, and various clinical response rate and adverse reactions prevent many patients from benefiting from it. In recent years, combination trials have become the main direction of PD-1/PD-L1 antibodies development. Here, we summarized PD-L1 biofunctions and key roles in various cancers along with the development of PD-L1 inhibitors. The regulators that are involved in controlling PD-L1 expression including post-translational modification, mRNA level regulation as well as degradation and exosome secretory pathway of PD-L1 were focused. This systematic summary may provide comprehensive understanding of different regulations on PD-L1 as well as a broad prospect for the search of the important regulator of PD-L1. The regulatory factors of PD-L1 can be potential targets for immunotherapy and increase strategies of immunotherapy in combination.

LSD1 deletion decreases exosomal PD-L1 and restores T-cell response in gastric cancer.

BACKGROUND: Histone lysine-specific demethylase 1 (LSD1) expression has been shown to be significantly elevated in gastric cancer (GC) and may be associated with the proliferation and metastasis of GC. It has been reported that LSD1 repressed tumor immunity through programmed cell death 1 ligand 1 (PD-L1) in melanoma and breast cancer. The role of LSD1 in the immune microenvironment of GC is unknown. METHODS: Expression LSD1 and PD-L1 in GC patients was analyzed by immunohistochemical (IHC) and Western blotting. Exosomes were isolated from the culture medium of GC cells using an ultracentrifugation method and characterized by transmission electronic microscopy (TEM), nanoparticle tracking analysis (NTA), sucrose gradient centrifugation, and Western blotting. The role of exosomal PD-L1 in T-cell dysfunction was assessed by flow cytometry, T-cell killing and enzyme-linked immunosorbent assay (ELISA). RESULTS: Through in vivo exploration, mouse forestomach carcinoma (MFC) cells with LSD1 knockout (KO) showed significantly slow growth in 615 mice than T-cell-deficient BALB/c nude mice. Meanwhile, in GC specimens, expression of LSD1 was negatively correlated with that of CD8 and positively correlated with that of PD-L1. Further study showed that LSD1 inhibited the response of T cells in the microenvironment of GC by inducing the accumulation of PD-L1 in exosomes, while the membrane PD-L1 stayed constant in GC cells. Using exosomes as vehicles, LSD1 also obstructed T-cell response of other cancer cells while LSD1 deletion rescued T-cell function. It was found that while relying on the existence of LSD1 in donor cells, exosomes can regulate MFC cells proliferation with distinct roles depending on exosomal PD-L1-mediated T-cell immunity in vivo. CONCLUSION: LSD1 deletion decreases exosomal PD-L1 and restores T-cell response in GC; this finding indicates a new mechanism with which LSD1 may regulate cancer immunity in GC and provides a new target for immunotherapy against GC.

The design, synthesis and cellular imaging of a tumor-anchored, potent and cell-permeable BRD4-targeted fluorescent ligands.

Bromodomain 4 (BRD4) proteins play an important role in histone post-translational modifications and facilitate several important physiological and pathological processes, including cancers. The inhibition of BRD4 by small molecule inhibitors shows promise as a therapeutic strategy for cancer treatment. However, their clinical applications were limited, which is largely hampered by off-target effects-induced toxicity. We herein report the design, synthesis, and cellular imaging of a set of tumor-anchored and BRD4-targeted fluorescent ligands by introducing selective and potent BRD4 inhibitor into different fluorophores via variable linkers. One of the fluorescent conjugates (compound 6) was demonstrated to be cell-permeable and low cytotoxic, preferentially accumulated in cancer cells, and display pronounced fluorescent signal. More importantly, 6 was identified to show specific BRD4 engagement in the cellular content. Collectively, this study provides a pathway for developing labeled BRD4 ligands and highlights that compound 6 may represent a valuable tool for explorative learning and target delivery study of BRD4.

Recent advances on the intervention sites targeting USP7-MDM2-p53 in cancer therapy.

The ubiquitin-specific protease 7 (USP7)-murine double minute 2 (MDM2)-p53 network plays an important role in the regulation of p53, a tumor suppressor which plays critical roles in regulating cell growth, proliferation, cell cycle progression, apoptosis and immune response. The overexpression of USP7 and MDM2 in human cancers contributes to cancer initiation and progression, and their inhibition reactivates p53 signalings and causes cell cycle arrest and apoptosis. Herein, the current state of pharmacological characterization, potential applications in cancer treatment and mechanism of action of small molecules used to target and inhibit MDM2 and USP7 proteins are highlighted, along with the outcomes in clinical and preclinical settings. Moreover, challenges and advantages of these strategies, as well as perspectives in USP7-MDM2-p53 field are analyzed in detail. The investigation and application of MDM2 and USP7 inhibitors will deepen our understanding of the function of USP7-MDM2-p53 network, and feed in the development of effective and safe cancer therapies where USP7-MDM2-p53 network is implicated.

Skp2 in the ubiquitin-proteasome system: A comprehensive review.

The ubiquitin-proteasome system (UPS) is a complex process that regulates protein stability and activity by the sequential actions of E1, E2 and E3 enzymes to influence diverse aspects of eukaryotic cells. However, due to the diversity of proteins in cells, substrate selection is a highly critical part of the process. As a key player in UPS, E3 ubiquitin ligases recruit substrates for ubiquitination specifically. Among them, RING E3 ubiquitin ligases which are the most abundant E3 ubiquitin ligases contribute to diverse cellular processes. The multisubunit cullin-RING ligases (CRLs) are the largest family of RING E3 ubiquitin ligases with tremendous plasticity in substrate specificity and regulate a vast array of cellular functions. The F-box protein Skp2 is a component of CRL1 (the prototype of CRLs) which is expressed in many tissues and participates in multiple cellular functions such as cell proliferation, metabolism, and tumorigenesis by contributing to the ubiquitination and subsequent degradation of several specific tumor suppressors. Most importantly, Skp2 plays a pivotal role in a plethora of cancer-associated signaling pathways. It enhances cell growth, accelerates cell cycle progression, promotes migration and invasion, and inhibits cell apoptosis among others. Hence, targeting Skp2 may represent a novel and attractive strategy for the treatment of different human cancers overexpressing this oncogene. In this review article, we summarized the known roles of Skp2 both in health and disease states in relation to the UPS.

LSD1 deletion represses gastric cancer migration by upregulating a novel miR-142-5p target protein CD9.

LSD1 (histone lysine specific demethylase 1) takes part in the physiological process of cell differentiation, EMT (epithelial-mesenchymal transition) and immune response. In this study, we found LSD1 expression in metastatic gastric cancer tissues was significantly higher than that in normal tissues. Furthermore, LSD1 deletion was found to suppress gastric cancer migration by decreasing intracellular miR-142-5p, which further led to the upregulation of migration suppressor CD9, a newly identified target of miR-142-5p. While LSD1 was reported as a demethylase of H3K4me1/2, H3K9me1/2 and several non-histone proteins, this is a new evidence for LSD1 as a functional regulator of miRNA. On the other hand, our data suggested that promoting the secretion of miR-142-5p using small extracellular vesicles as vehicles is a new mechanism for LSD1 abrogation to down-regulate intracellular miR-142-5p. Taken together, this study uncovered a new mechanism for LSD1 that can contribute to gastric cancer migration by facilitating miR-142-5p to target CD9.

Natural protoberberine alkaloids, identified as potent selective LSD1 inhibitors, induce AML cell differentiation.

Natural protoberberine alkaloids were first identified and characterized as potent, selective and cellular active lysine specific demethylase 1 (LSD1) inhibitors. Due to our study, isoquinoline-based tetracyclic scaffold was identified as the key structural element for their anti-LSD1 activity, subtle changes of substituents attached to the core structure led to dramatic changes of the activity. Among these protoberberine alkaloids, epiberberine potently inhibited LSD1 (IC50 = 0.14 ± 0.01 µM) and was highly selective to LSD1 over MAO-A/B. Furthermore, epiberberine could induce the expression of CD86, CD11b and CD14 in THP-1 and HL-60 cells, confirming its cellular activity of inducing acute myeloid leukemia (AML) cells differentiation. Moreover, epiberberine prolonged the survival of THP-1 cells bearing mice and inhibited the growth of AML cells in vivo without obvious global toxicity. These findings give the potential application of epiberberine in AML treatment, and the isoquinoline-based tetracyclic scaffold could be used for further development of LSD1 inhibitors.

USP28 contributes to the proliferation and metastasis of gastric cancer.

USP28, a member of the deubiquitinating enzymes family, plays a vital role in the physiological process of cell proliferation, differentiation and apoptosis, DNA repair, immune response, and stress response. USP28 has been reported to be overexpressed in bladder cancer, colon cancer, breast carcinomas, and so on. Nevertheless, the role of USP28 in gastric cancer has not yet been investigated. In our study, we examined the USP28 expression in 87 paired samples of gastric cancer and normal gastric tissues. We found that USP28 was overexpressed in gastric cancer compared with normal gastric tissues (P < 0.01), and its overexpression was related to the degree of differentiation and metastases. Inhibiting USP28 expression in vitro suppressed the proliferation and invasion of gastric cancer cells by downregulating lysine specific demethylase 1. On the basis of our data, it can be concluded that USP28 may be a novel therapeutic target for gastric cancer.

Identification of osimertinib (AZD9291) as a lysine specific demethylase 1 inhibitor.

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In this study, osimertinib was characterized as a LSD1 inhibitor for the first time with an IC50 of 3.98 ±â€¯0.3 µM and showed LSD1 inhibitory effect at cellular level. These findings provide new molecular skeleton for dual inhibitor for LSD1 and EGFR. Osimertinib could serve as a lead compound for further development for anti-NSCLC drug discovery with dual targeting.

Sanggenon O induced apoptosis of A549 cells is counterbalanced by protective autophagy.

Sanggenon O (SO) is a Diels-Alder type adduct extracted fromMorus alba, which has been used for its anti-inflammatory action in the Oriental medicine. However, whether it has regulatory effect on human cancer cell proliferation and what the underlying mechanism remains unknown. Here, we found that SO could significantly inhibit the growth and proliferation of A549 cells and induce its pro-apoptotic action through a caspase-dependent pathway. It could also impair the mitochondria which can be reflected by mitochondrial membrane permeabilization. Besides, SQSTM1 up-regulation and autophagic flux measurement demonstrated that exposure to SO led to autophagosome accumulation, which plays a protective role in SO-treated cells. In addition, knocking down of LC3B increased SO triggered apoptotic cell rates. These results indicated that SO has great potential as a promising candidate combined with autophagy inhibitor for the treatment of NSCLC. In conclusion, our results identified a novel mechanism by which SO exerts potent anticancer activity.

Lysine-specific demethylase 1 activation by vitamin B2 attenuates efficacy of apatinib for proliferation and migration of gastric cancer cell MGC-803.

B vitamins play an essential role in the biosynthesis of nucleotides, replication of DNA, supply of methyl-groups, growth and repair of cells, aberrancies of which have all been implicated in carcinogenesis. Although the potential role of vitamin B in relation to the risk of cancer, including breast, and colorectal cancer, has been investigated in several observational studies, the mechanism of action is still unclear. In this study, vitamin B2 exhibited efficient activation of LSD1 by occupying the active sites where FAD stands. Interestingly, vitamin B2 significantly downregulated expression of CD86, a sensitive surrogate biomarker of LSD1 inhibition, and showed marked activation of gastric cancer cell migration and invasion. Meanwhile, vitamin B2 induced activation of LSD1 may attenuate the proliferation inhibition, and anti-migration effects of apatinib in gastric cancer cells. These findings suggested that vitamin B supplementation may interfere with the efficacy of apatinib in patients with gastric cancer.

LPE-1, an orally active pyrimidine derivative, inhibits growth and mobility of human esophageal cancers by targeting LSD1.

Histone lysine specific demethylase 1 (LSD1) plays an important role in epigenetic modifications, and aberrant expression of LSD1 predicts tumor progression and poor prognosis in human esophageal cancers. In this study, a series of LSD1 inhibitors were synthesized and proved to be highly potent against human esophageal squamous cell carcinoma (ESCC). Our data showed that these LSD1 inhibitors selectively suppressed the viability of esophageal cancer cell line (EC-109) bearing overexpressed LSD1. Among these, compound LPE-1 (LSD1 IC50=0.336±0.003µM) significantly suppressed proliferation, induced apoptosis, arrested cell cycle of EC109 cells at G2/M phase, and caused changes of the associated protein markers correspondingly. We also found that compound LPE-1 potently inhibited the migration and invasion of EC-109 cells. Docking studies showed that the cyano group formed hydrogen bonds with Val811 and Thr810. Additionally, the thiophene moiety formed arene-H interaction with Trp761 residue. In vivo studies showed that compound LPE-1 inhibited tumor growth of xenograft models bearing EC-109 without obvious toxicity. Collectively, our findings indicate that LSD1 may be a potential therapeutic target in ESCC, and compound LPE-1 could serve as a lead compound for further development for anti-ESCC drug discovery.

Discovery of tranylcypromine analogs with an acylhydrazone substituent as LSD1 inactivators: Design, synthesis and their biological evaluation.

Lysine specific demethylase 1 (LSD1), the first identified histone demethylase, plays an important role in epigenetic regulation of gene activation and repression, has been reported to be up-regulated and involved in numbers of solid malignant tumors. In this study, we identified a series of phenylalanyl hydrazones based LSD1 inhibitors, and the most potent one, compound 4q, can inactivate LSD1 with IC50 = 91.83 nM. In cellular level, compound 4q can induce the accumulation of CD86 as well as H3K4me2, and inhibit gastric cancer cell proliferation by inactivating LSD1. Our findings indicated that compound 4q may serve as a potential leading compound to target LSD1 overexpressed gastric cancer.

1,2,3-Triazole-Dithiocarbamate Hybrids, a Group of Novel Cell Active SIRT1 Inhibitors.

BACKGROUND/AIMS: Human SIRT1 is reported to be involved in tumorgenesis, mainly due to its modulating effect on p53 by deacetylation on lysine382. A large quantity of SIRT1 inhibitors was applied in chemotherapeutic study, but few of them were applied into clinical trials. METHODS AND RESULTS: In the current study, a novel series of compounds with 1,4-bispiperazinecarbodithioic acid methyl esters scaffold were characterized to have inhibitory potency to SIRT1 by molecular docking and biochemical evaluation. Further cell level study revealed that one of the most potent SIRT1 inhibitors, compound 3a, is cell active. It can upregulate the amount of p53 by accumulating the K382 acetylation of p53, which lead to the stabilization of p53 in human gastric cancer cell line MGC-803 cells. Meanwhile, we also found compound 3a can inactivate SIRT2 in cells, which suggests the compound as a non-selective SIRT inhibitor. CONCLUSION: All these findings indicate that compound 3a is a potent, reversible and cell active SIRT1 inhibitor and deserves further investigation as an anticancer agent or a biological tool.

Baicalin, a natural LSD1 inhibitor.

Baicalin is one of the active ingredients in the skullcap, with a variety of pharmacological effects, such as blood pressure reduction, sedation, liver-protection, gallbladder-protection, anti-bacteria, and anti-inflammation. In our study, baicalin was first characterized as a LSD1 inhibitor with an IC50 of 3.01µM and showed strong LSD1 inhibitory effect in cells. Baicalin may serve as a template for designing flavone-based LSD1 inhibitors.

A Systematic Review of Histone Lysine-Specific Demethylase 1 and Its Inhibitors.

Histone lysine-specific demethylase 1 (LSD1) is the first discovered and reported histone demethylase by Dr. Shi Yang's group in 2004. It is classified as a member of amine oxidase superfamily, the common feature of which is using the flavin adenine dinucleotide (FAD) as its cofactor. Since it is located in cell nucleus and acts as a histone methylation eraser, LSD1 specifically removes mono- or dimethylated histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) through formaldehyde-generating oxidation. It has been indicated that LSD1 and its downstream targets are involved in a wide range of biological courses, including embryonic development and tumor-cell growth and metastasis. LSD1 has been reported to be overexpressed in variety of tumors. Inactivating LSD1 or downregulating its expression inhibits cancer-cell development. LSD1 targeting inhibitors may represent a new insight in anticancer drug discovery. This review summarizes recent studies about LSD1 and mainly focuses on the basic physiological function of LSD1 and its involved mechanisms in pathophysiologic conditions, as well as the development of LSD1 inhibitors as potential anticancer therapeutic agents.

Design and synthesis of novel 1,2,3-triazole-pyrimidine-urea hybrids as potential anticancer agents.

A series of novel 1,2,3-triazole-pyrimidine-urea hybrids were designed, synthesized and evaluated for anticancer activity against four selected cancer cell lines (MGC-803, EC-109, MCF-7 and B16-F10). Majority of the synthesized compounds exhibited moderate to potent activity against all the cancer cell lines assayed. Particularly, compounds 26, 30 and 38 exhibited excellent growth inhibition against B16-F10 with IC50 values of 32nM, 35nM and 42nM, respectively. Flow cytometry analysis demonstrated that compound 26 induced the cellular apoptosis in a concentration-dependent manner.