Gastric cancer (GC) is the fifth most incident and the fourth deadliest cancer worldwide. GC is a heterogeneous disease from the histological and molecular standpoints. This malignancy is mostly diagnosed at advanced stages of the disease, where the available therapeutic interventions are not effective. The emergence of immunotherapy has transformed the landscape of cancer treatment, including GC, and currently immune checkpoint inhibitors have been approved for the treatment of patients with recurrent/metastatic GC. This review summarizes the main clinical trials evaluating the use of immune checkpoint inhibitors in GC. It also highlights the potential of biomarkers for patient selection for GC immune checkpoint inhibition therapy, including programmed cell death ligand 1 expression and tumor mutational burden, and characteristics of the GC molecular classification, such as microsatellite instability status and Epstein-Barr virus infection, as predictors of response to blockade of the programmed cell death 1/programmed cell death ligand 1 axis. It is the fifth most incident malignant disease worldwide, with a geographically heterogeneous incidence. The highest rates are observed mainly in Eastern Asia , Europe , and South America, and the lowest incidence rates in Africa and Northern America.4 Targeted therapy has been also introduced for a particular subset of GCs that overexpress human epidermal growth factor receptor 2 (HER2).9 More recently, a great deal of attention is being given to immunotherapy, which may be used in various types of cancers, including GC.10There are various approaches to GC therapy, including perioperative, adjuvant, and palliative chemotherapy, and tumor endoscopic/surgical resection, none of which is fully effective.11 In addition, mesenchymal tumors and B-cell lymphomas can also be found, although to a much lesser extent.13 Herein GC will be used as synonym of gastric adenocarcinoma.GC comprises various types of tumors. The great majority arise in glandular structures and are classified as adenocarcinomas.14 The intestinal subtype is usually diagnosed in older patients, most frequently in men, and appears in the distal part of the stomach, with a frequently exophytic growth pattern. The main histological characteristic of these tumors is the formation of glands and the synthesis of extracellular mucins. The diffuse subtype affects younger patients of both sexes equally, and arises mainly, although not exclusively, in the gastric body, frequently with linitis plastica growth pattern. Histologically, diffuse GC is characterized by the loss of cellular cohesion and the presence of isolated cells that contain high quantities of intracytoplasmic mucins (“signet ring cells”).14 An additional and rarer GC subtype, comprises tumors that present characteristics of both the intestinal and diffuse subtypes, and is denominated mixed type GC. According to the World Health Organization, GC comprises 5 main histological subtypes: papillary, tubular, poorly cohesive (characterized by “signet ring cells”), mucinous (when mucinous pools exceeds 50% of the tumor), and mixed adenocarcinomas. Other less common variations exist, such as the squamous cell, adenosquamous, hepatoid, and medullary carcinomas.15 Despite the heterogeneity referred above, histological subtypes have not provided significant contribution to therapeutic decisions.16GC is a heterogeneous disease from both histological and molecular standpoints. Histologically, and according to the Laurén's classification, there are 2 main GC subtypes.17 The proliferative GC subtype that corresponds to the intestinal type, presenting high TP53 mutations, high copy number alterations, and oncogenic activation. The mesenchymal GC subtype that corresponds to the diffuse type, having low copy number alterations, low number of TP53 mutations, and a high activity of the epithelial-mesenchymal transition (EMT) pathway, similarly to stem cells. Finally, the metabolic GC subtype is characterized by low number of TP53 mutations, high activity of the spasmolytic polypeptide-expressing metaplasia pathway, and high activity of metabolic pathways.18From the molecular standpoint, GC is also heterogeneous. The so-called “Singapore-Duke” classification considers 3 GC subtypes.19 The Epstein-Barr virus (EBV)-infected (EBV+) subtype constitutes 9% of all GCs and is more frequent in men and in younger patients. These tumors appear mainly in the upper part of the stomach, specifically, in the gastric fundus. Histologically, EBV+ GC is moderate to poorly differentiated, usually with dense lymphocytic infiltration.20 Molecular characteristics of EBV+ GC include extreme hypermethylation, CDKN2A methylation, but without MLH1 methylation, and PIK3CA and ARID1A mutations. The EBV+ GC subtype is also characterized by amplification of JAK2, ERBB2, and programmed cell death ligand ½ (PD-L1/2), the latter with an important role as targets of immunotherapy in the treatment of GC. An additional characteristic of this tumor subtype is the enhancement in immune cell signaling pathways.21The Cancer Genome Atlas (TCGA) classification considers 4 GC subtypes.ARID1A, PIK3CA, PTEN, ERBB2, ERBB3, EGFFR, KRAS, RNF43, and MHC1. TP53 mutations are frequent and MLH1 is hypermethylated. MSI tumors are enriched in mitotic and DNA damage pathways.21Microsatellite unstable (MSI) tumors make up to 15% to 30% of all GCs, and are more frequent in women and older patients. They arise mainly in the lower part of the stomach, particularly in the gastric antrum. The histology of MSI GC is similar to that of the intestinal subtype. MSI tumors are diploid and hypermutated, with mutations in CDH1, ARID1A, and RHOA, by CLDN18-ARHGAP fusion, and by high cell adhesion and angiogenesis pathways expression. In this subtype, TP53 mutations are not common.21Genomically stable (GS) tumors constitute 20% of all GCs. GS tumors affect both sexes equally and have an early onset diagnosis compared to other subtypes of GC. They are mainly located in the antrum and comprise histologically diffuse tumors. GS tumors are characterized by mutations in 20 CIN tumors originate in the gastric fundus and esophagogastric junction. Histologically, CIN tumors can be of the intestinal subtype, when associated with gains in copy number of 8q, 17q, and 20q, and of the diffuse subtype, when associated with gains of 12q and 13q.20 CIN tumors are aneuploid and harbor genomic amplifications in: RTKs and KRAS, which are mutually exclusive; transcription factors, including KLF5, GATA4, GATA6, and OCT1; cell cycle mediators, including CCNE1, CCND1, and CDK6. Mutations in HER2, BRAF, EGFR, MET, FGFR2, and RAS have also been identified. Unlike the other subtypes, CIN GC shows a high frequency of TP53 mutations.21Chromosomally unstable (CIN) GC comprises 50% of the cases.23 EMT/ microsatellite stable (EMT/MSS) comprises 15% of all GCs, it is located in the gastric body, and is histologically similar to the diffuse subtype. EMT/MSS presents EMT gene expression signature and loss of CDH1 expression. This subtype corresponds to the worst prognostic. The MSI subtype represents 23% of tumors, present in the antrum, and is closer to histological intestinal subtype. An MSI gene expression signature, together with loss of MLH1 expression and hypermutation is observed in this GC subtype that is associated to a better prognosis. The MSS/ TP53+ and the MSS/TP53—subtypes constitute respectively 26% and 36% of GCs. These subtypes are distinguished by the activation of TP53, in which MSS/TP53+ has an intact TP53 gene. Overall, the characteristics associated to specific GC subtypes can potentially provide novel therapeutic targets as well as new means for patient stratification.21The Asian Cancer Research Group classification also considers 4 GC subtypes, which unlike the TCGA classification, may predict disease progression or disease prognostic.24 Malignant tumors frequently use mechanisms of immune suppression and tolerance to prevent immune destruction. The idea underlying immune checkpoint blockade therapy is the removal of signals that inhibit T-cell activation and effector functions, which in turn allows the establishment of an efficient anti-tumor response.24Immunotherapy has revolutionized cancer treatment. Among the different forms of immunotherapy, immune checkpoint inhibitors are the best studied and the most used therapeutic agents.25 Following this breakthrough, other molecules for immune checkpoint inhibition followed, including nivolumab, pembrolizumab, and avelumab, which target the programmed cell death 1 (PD-1)/PD-L1 axis.28 In the context of GC, some of these immune checkpoint inhibitors were approved as a third-line therapy in advanced or recurrent GC, whereas their use as first-/second-line options has been and still is under evaluation. Results of major clinical trials are summarized in Table 33The pioneer immune checkpoint inhibitor was ipilimumab, an anti-cytotoxic T-lymphocyte–associated antigen-4 monoclonal antibody, originally approved for the treatment of unresectable/ metastatic melanoma.34 The randomized, double-blind, placebo-controlled, phase III ATTRACTION-2 trial assessed the efficacy and safety of nivolumab in patients with unresectable advanced/recurrent gastric or gastro-esophageal junction (GEJ) cancer, who had been treated with 2 or more chemotherapeutic regimens.27 The trial enrolled 493 Asian patients from Japan, South Korea, and Taiwan, who were randomly assigned to receive nivolumab or placebo. The results showed that nivolumab improved the overall survival (OS) in patients with GC refractory to standard chemotherapy; the median OS was 5.26 months in the nivolumab group, contrasting with 4.14 months in the placebo group. In this study, the 1-year OS rate was 26.2% in patients receiving nivolumab, in comparison with 10.9% in patients receiving placebo. The updates of the trial showed that the 2- and the 3-year OS rates were, respectively, 10.6% and 5.6% for nivolumab, and 3.2% and 1.9% for placebo, and confirmed the long-term efficacy of nivolumab.36 Following the first results of the ATTRACTION-2 trial, nivolumab was also approved in various Asian countries as a third-line or later option in patients with unresectable advanced or recurrent gastric or GEJ cancer.35 One of the limitations of the trial was that the patient population consisted only of Asian patients.38Nivolumab is a human monoclonal IgG4 kappa antibody that binds to the PD-1 receptor and blocks its interaction with PD-L1 and PD-L2. Nivolumab was initially approved in Japan for the treatment of several different types of cancers.26 The trial involved a cohort of 259 patients from 16 international locations with previously treated advanced GC or GEJ cancer.26 The objective response rate (ORR) associated with pembrolizumab treatment was 11.6%, 2.3% of the patients had complete response, and the median OS was 5.6 months. These results favored further developments of the use of pembrolizumab monotherapy in patients with advanced gastric or GEJ cancer with ≥2 previous lines of treatment. Based on the outcomes of this trial, the Food and Drug Administration (FDA)-approved pembrolizumab for treatment of recurrent and locally advanced or metastatic GC.39The KEYNOTE-59, open-label, phase II trial, evaluated the safety and efficacy of monotherapy with pembrolizumab, a humanized monoclonal IgG4 kappa antibody that binds to the PD-1 receptor, blocking its interaction with PD-L1 and PD-L2.28 Although the safety profile of avelumab was better than that of chemotherapy, the trial was not successful in meeting the primary or secondary end points; the median OS was 4.6 in the avelumab group and 5 months in the chemotherapy group; the progression-free survival (PFS) was 1.4 versus 2.7 months and the ORR was 2.2% versus 4.3% in the avelumab versus chemotherapy arms, respectively. Therefore, in the third-line setting, the use of avelumab as a single agent did not improve OS or PFS in comparison chemotherapy.An additional immune checkpoint inhibitor is avelumab, a human IgG1 lambda monoclonal antibody that by binding PD-L1, blocks its interactions with PD-1 and B7.1 receptors. The use of avelumab as third-line therapy was investigated in the JAVELIN Gastric 300 phase III trial, which enrolled 371 patients with recurrent locally advanced or metastatic gastric or GEJ cancer that were randomized to receive avelumab or the physician's choice of third-line chemotherapy.31 Patients were treated with nivolumab, or with combinations of nivolumab plus ipilimumab. The ORR, median PFS, and OS were, respectively, 12%, 1.4 months, and 6.2 months in the patients receiving nivolumab, 24%, 1.4 months, and 6.9 months in those receiving nivolumab 1 mg/kg and ipilimumab 3 mg/kg, and 8%, 1.6 months, and 4.8 months with the combination of nivolumab 3 mg/kg and ipilimumab 1 mg/kg. Although the combination of nivolumab 1 mg/kg plus ipilimumab 3 mg/kg had a numerically higher ORR than that of patients treated with nivolumab monotherapy, the median OS was similar in these patient groups. Nevertheless, the study demonstrated a reasonable safety profile and long-lasting responses. In addition, the clinical benefits of nivolumab monotherapy were similar to those of the ATTRACTIONS-2, suggesting consistent therapeutic benefit across patients from Asian and Western countries.The CheckMate-032 phase I/II trial tested the efficacy of nivolumab and ipilimumab in patients with chemotherapy-refractory metastatic gastric or GEJ cancer, in 160 patients from centers in the United States and Europe.29 Patients were randomized to receive pembrolizumab or paclitaxel. Pembrolizumab did not significantly improve the OS compared with paclitaxel (9.1 vs 8.3 months) or PFS (1.5 vs 4.1 months), although it had a better safety profile.29Although now established as a third-line therapy, immune checkpoint inhibitors have not been so successful in earlier lines of therapy for GC. The open-label, phase III KEYNOTE-061 trial enrolled 592 patients with advanced gastric or GEJ cancer that progressed on first-line chemotherapy, with a PD-L1 combined positive score (CPS) ≥1.30 Patients were randomized to receive pembrolizumab, pembrolizumab plus chemotherapy, or chemotherapy plus placebo. The OS of patients with PD-L1 CPS ≥1 treated with pembrolizumab was noninferior to that of patients treated with chemotherapy (10.6 vs 11.1 months). Interestingly, in patients with PD-L1 CPS ≥10 pembrolizumab extended OS versus chemotherapy (17.4 vs 10.8), but without statistical significance. Pembrolizumab plus chemotherapy was not superior to chemotherapy for OS in patients with CPS ≥1 (12.5 vs 11.1 months) or with CPS ≥10 (12.3 vs 10.8 months), or for PFS in patients with CPS ≥1 (6.9 vs 6.4 months).30The following randomized, controlled, phase III KEYNOTE-062 trial enrolled 763 patients with untreated, locally advanced, unresectable, or metastatic gastric or GEJ cancer, with a PD-L1 CPS ≥1.33 The results of the double-blind phase III part demonstrated a statistically significant improvement in PFS in patients receiving nivolumab plus chemotherapy in comparison with the other study arm (10.5 vs 8.3 months), reaching one of the primary endpoints of the trial. However, no differences in OS, which was the other primary study endpoint, were observed between the 2 groups (17.5 vs 17.2 months). Similarly, the randomized, open-label, phase III CheckMate-649 trial compared nivolumab plus chemotherapy with chemotherapy alone as a first-line treatment for patients with advanced or metastatic gastric or GEJ cancer. The first results of the trial that enrolled 1581 patients from geographic locations worldwide demonstrated that in patients with tumors with PD-L1 CPS ≥1 there was statistically significant benefit in OS those treated with nivolumab plus chemotherapy versus those treated with chemotherapy alone (14.0 vs 11.3 months). In particular, in patients with tumors expressing PD-L1 with CPS ≥5, nivolumab plus chemotherapy showed statistically significant improvements in both OS and PFS in comparison with chemotherapy alone.32 Based on the results of the CHECKMATE-649 trial, the European Medicines Agency validated40 and the US FDA accepted for priority review41 the application of nivolumab combined with chemotherapy as first-line treatment in metastatic GC, GEJ cancer, and esophageal adenocarcinoma.The ongoing randomized, phase II/III ATTRACTION-4 evaluates nivolumab plus chemotherapy versus placebo plus chemotherapy as a first-line treatment in HER2-negative, advanced, or recurrent gastric or GEJ cancer in Asian patients.The use of biomarkers for patient selection for immune checkpoint inhibition therapy aims to increase its efficacy, while reducing useless therapeutic exposure and health-related costs. In this section, we will summarize knowledge on biomarkers that are presently being tested in clinical trials addressing immune checkpoint blockade in GC.44 In these studies, higher expression levels of PD-L1, as evaluated by immunohistochemistry, were predictive of response to therapy with PD-1 and/or PD-L1 inhibitors. However, for other cancer types, including renal cell carcinoma and hepato-cellular carcinoma, PD-L1 expression did not show to be a good biomarker.46PD-L1 expression is the most widely studied biomarker for patient selection for PD-1/PD-L1 therapy. The usefulness of PD-L1 expression as a biomarker has been reported in various large clinical trials that assessed PD-1 and/or PD-L1 inhibitors in melanoma, non–small cell lung cancer, and urothelial carcino-ma.PDL1 gene amplification, structural variations in the 3’UTR of PDL1, polymorphisms in PDL1 promoter, activation of oncogenic PI3K signaling, and cytokine- and chemokine-mediated regulation.48 PD-L1 expression in GC has been associated with high density of tumor-infiltrating lymphocytes, with MSI, and with EBV infection.50In GC, between 25% and 65% of tumors express PD-L1, and multiple mechanisms have been associated with PD-L1 upregu-lation, including 51 Several meta-analyses have been now been performed to examine the clinicopathological and prognostic significance of PD-L1 expression. A meta-analysis that included 10 studies and 1901 patients with GC from Asia indicated that PD-L1 expression was associated with a shorter OS.47 The expression of PD-L1 was also associated with tumor size, and lymph node metastasis, but not with age or sex, tumor differentiation, invasion depth, or tumor stage. A more recent meta-analysis including 15 studies and 3218 patients from China, South Korea, Japan, and Germany, showed that PD-L1 expression was associated with a decrease in the 3-and 5-year survival rates.52 In the subgroup analyses of ethnicity, PD-L1 expression in Asian patients was also associated with a decrease in the 3- and 5-year survival rates. PD-L1 expression was associated with lymph node metastasis but not with tumor staging. These results point to the possibility of using PD-L1 expression as GC biomarker for PD-1/PD-L1-targeted therapy.The relationship between PD-L1 expression and prognosis in GC is controversial, and although some studies reported increased PD-L1 expression associated with adverse prognosis, others have shown a relationship with better patient outcome, or report that PD-L1 expression is not a prognostic factor.It is important, however, to mention that major problems exist regarding the comparisons between studies, namely the use of different antibodies, assays, or devices for PD-L1 immunohis-tochemistry, as well as differences in scoring criteria.36 In CHECKMATE-032, the benefits of nivolumab or the combinations of nivolumab with ipilimumab were observed, no matter the immunohistochemical status of PD-L1 independently.31 Finally, in the JAVELIN Gastric 300 that used the PD-L1 IHC 73-10 pharmDx assay, no differences were identified in the outcomes of avelumab treatment between patients with PD-L1-positive or -negative tumors. In these trials, however, the scoring of PD-L1 was performed using a tumor proportion score of ≥1%, which considers expression of PD-L1 in 1% or more of tumor cells.53In the 3-year update of the ATTRACTION-2 trial, PD-L1 was retrospectively analyzed using the PD-L1 IHC 28-8 pharmDx assay. In the 192 patients who had available tumor tissue, no differences were found regarding the efficacy of nivolumab compared to that of placebo in patients’ OS.53 In KEYNOTE-59, the ORR and the median response duration of patients with PD-L1-positive tumors were 22.7% and 8.1months, whereas the responses were significantly lower, 8.6% and 6.9months, respectively, in patients with tumors that were PD-L1 negative.26 In KEYNOTE-61, the ORR for patients treated with pembrolizumab versus paclitaxel was 16% versus 14% in patients with CPS ≥1 tumors, but in subgroup analysis, ORR was 25% versus 9% in the PD-L1 CPS ≥10 subgroup, and 2% versus 10% in the PD-L1 CPS <1 subgroup.29 In KEYNOTE-062, the OS for patients treated with pembrolizumab was 10.6 months in those with PD-L1 CPS ≥1 tumors, and a prolonged, though not statistically tested, OS of 17.4 months was observed in patients with PD-L1 CPS ≥10 tumors.30The trials that assessed pembrolizumab in GC showed efficacy of this immune checkpoint inhibitor in PD-L1-positive tumors, using the FDA-approved PD-L1 IHC 22C3 pharmDx assay, which scores PD-L1 expression in tumor cells, lymphocytes, and macrophages (CPS).54 comprehensively studied in post-hoc analyses the efficacy of pembrolizumab in patients with CPS ≥10 in the 3 trials mentioned above. In KEYNOTE-059 (median follow-up 6 months), median OS was 8 months, ORR was 17%, and median duration of response (DOR) was 21 months. In KEYNOTE-061 (median follow-up 9 months), median OS (pembrolizumab vs chemotherapy) was 10 versus 8 months, median PFS was 3 versus 3 months, ORR was 25% versus 9%, and median DOR was not reached versus 7 months. In KEYNOTE-062 (median follow-up 11 months), median OS (pembrolizumab vs chemotherapy) was 17 versus 11 months, median PFS was 3 versus 6 months, ORR was 25% versus 38%, and median DOR was 19 versus 7 months. This shows that more favorable clinical outcomes are consistently observed in first, second, and third lines of pembrolizumab therapy in patients with PD-L1 CPS ≥10 tumors, and suggest that PD-L1 expression could be used to identify patients who would benefit from PD-1/ PD-L1-targeted therapy.Very recently, and to better define CPS specificity as a predictor of clinical outcome, Wainberg et al56The accumulation of mutations in microsatellite regions of the genome, which are repeated sequences of nucleotides where DNA polymerase is more prone to replication errors, is known as microsatellite instability (MSI). MSI is generally caused by a deficiency in mismatch repair systems.58 Patients with MSI GC have better prognosis than those with MSS tumors.60 MSI tumors have distinct clinicopathologic features and are associated with older age, female sex, and Laurén's intestinal histology.58 A meta-analysis that included 48 studies and 18.612 patients, of which 9.2% had MSI tumors, confirmed the relationship between MSI tumors and these features, but also with the absence of lymph node metastases, and stages I–II (TNM classification). Patients with MSI tumors also had better OS than patients with MSS GC.60About 20% to 25% of gastric tumors have the MSI phenotype.62MSI and mismatch repair deficiency in tumor cells may lead to higher levels of mutations and the appearance of immunogenic neoantigens, leading to easier recognition by immune cells. This may facilitate the action of immune checkpoint inhibitors, as these types of tumors exhibit a high density of immune cells. Accordingly, in comparison with MSS GC, MSI gastric tumors have higher numbers of PD-L1-positive tumor and immune cells, and increased number of tumor-infiltrating lymphocytes.26 In CHECKMATE-32, and in all study arms, there were significantly better responses in patients with MSI-high GC compared to non–MSI-high patients.31 The ORRs for MSI-high versus non–MSI-high were for nivolumab: 29% versus 11%; for nivolumab 1 mg/kg plus ipilimumab 3 mg/kg: 50% versus 19%; and for nivolumab 3 mg/kg plus ipilimumab 1 mg/kg: 50% versus 5%. The OS for MSI-high versus non–MSI-high were for nivolumab: 57% versus 33%; for nivolumab 1 mg/kg plus ipilimumab 3 mg/kg: 50% versus 32%; and for nivolumab 3 mg/kg plus ipilimumab 1 mg/kg: 50% versus 23%.In KEYNOTE-59, 67% of the enrolled patients were assessed for GC MSI, of which 4% had MSI-high tumors. In patients with MSI-high GC, the ORR to pembrolizumab treatment was 57.1%, contrasting with an ORR of 9% for patients with non– MSI-high GC.29 In CHECKMATE 62, OS was enhanced in patients with MSI tumors with PD-L1 CPS ≥1, and overall outcomes proved more efficient in the MSI-high population. The predictive value of PD-L1 CPS ≥10 remained constant, regardless of the MSI status, which demonstrates the independent value of both biomarkers.In post-hoc analysis of the patients enrolled in KEYNOTE-61, those with MSI tumors had superior responses to pembrolizumab , compared to 17% in the paclitaxel group.63 The study included data from KEYNOTE-061, CHECKMATE-649, JAVELIN Gastric 100, and KEYNOTE-062,64 and provided evidence of improved survival and response in advanced patients with GC with MSI-high tumors who received anti-PD-1 blockade, with significantly greater OS compared with patients with MSS tumors.A recent meta-analysis of randomized clinical trials evaluated the role of MSI as a positive predictive factor for PD-1 immunotherapy as first- or second-line regimens in patients with advanced GC.65 Results from this study showed that pembrolizumab alone, or combined with chemotherapy, was associated with prolonged OS and PFS and with durable responses in comparison to chemotherapy alone, suggesting the MSI-high status as biomarker for patient selection, irrespectively of the line of therapy in which it is received.Also recently, a post-hoc analysis of 1614 patients, 84 of which had MSI-high gastric or GEJ cancer, and enrolled in the KEYNOTE-059, KEYNOTE-061, and KEYNOTE-062 trials, assessed the antitumor effects of pembrolizumab versus chemotherapy, irrespectively of the line of therapy.19 Among other features, these tumors are characterized by rich lymphocytic infiltrates, containing CD8-positive cytotoxic T cells and high number of mature dendritic cells, and are enriched in immune cell signaling pathways.66 Furthermore, about 15% of EBV-positive GCs have amplification of the PD-L1- and PD-L2-encoding genes, and have PD-L1 expression in both tumor cells and immune cells.67 These features suggest that EBV-positive GC may be more susceptible to PD-1/PD-L1 blockade.As discussed above, the TCGA identified EBV-positive tumors as a distinct GC subgroup.68 In a clinical trial that evaluated the impact of toripalimab (an anti-PD-1 antibody) on 55 advanced GC patients, of the 4 EBV-positive patients, 1 case had partial response, 2 cases had stable disease, and 1 case had progressive disease.22 In a prospective phase 2 clinical trial of 61 patients with metastatic GC that had been treated with pembrolizumab, whereas the general ORR was 24.6%, in the 6 patients with EBV-positive tumors the ORR was 100%, all responding to pembrolizumab.69 In this trial, there were also 7 patients with MSI-high GC and, in this group, the ORR was 85.7%. These finding suggest that EBV-positive GC patients may derive benefit from pembrolizumab therapy. Large prospective clinical trials are needed to evaluate EBV positivity as a biomarker for GC immune checkpoint therapy.A case report described response to avelumab treatment in 1 EBV-positive GC patient.70 Epitopes of these mutant proteins can be expressed at the cancer cell surface, thus rendering these cells recognizable as foreign by T cells. It has been shown that the tumor mutational burden (TMB), and consequently the neoantigen formation potential in a certain tumor, will determine the effectiveness of the response to immunotherapy, as highly mutated cells can be distinguished and, therefore, targeted more proficiently.71 Accordingly, melanoma and non–small cell lung cancer, the 2 tumor types with highest prevalence of somatic mutations,72 have excellent responses to immune checkpoint blockade.76 Interestingly, in a study that evaluated almost 9900 samples of 35 cancer types, no significant correlations between TMB and PD-L1 expression within most cancer subtypes were observed, suggesting that each may be used as a biomarker for predicting the response to immune checkpoint blockade.77During cancer initiation and progression, tumor cells acquire thousands of different mutations. Nonsynonymous mutations will cause tumors to express neoantigens, which are tumor cell specific and will distinguish them from normal cells.78 Objective responses were observed in 29% of patients with TMB-high status (≥10 mutations per megabase), in comparison to only 6% in patients with non–TMB-high. Noteworthy, TMB had predictive value, regardless of the tumor PD-L1 expression and of the MSI status.The relationship between TMB and response to therapy with pembrolizumab has been analyzed in a study that involved multiple cohorts of patients with different types of solid tumors.79 In another study of 63 South Korean patients with advanced GC treated with pembrolizumab or nivolumab, responders had significantly higher TMB than nonresponders with stable disease.38 In survival analysis, patients with high TMB had longer PFS. Although in univariate analysis, TMB, MSI, response to treatment, and ECOG performance status were all significantly associated with PFS, in multivariate analysis, both TMB-high and the ECOG ≤1 remained independent predictors of longer PFS.In a study of metastatic gastrointestinal cancer patients from China, including 57 patients with GC, treated with immune checkpoint inhibitors, patients with higher TMB had longer OS than those with lower TMB.80 Patients with TMB-high versus TMB-low also had enhanced ORR (33.3% vs 7.1%), and a numerically longer PFS, but without statistical significance. The analysis of the TMB in patients of KEYNOTE-061 showed that TMB ≥10 mut/Mb had a positive association with ORR, PFS, and OS in patients treated with pembrolizumab but not with paclitaxel.81 Interestingly, the OS benefits of pembrolizumab versus paclitaxel in TMB ≥10 mut/Mb remained, when patients with MSI-high tumors were excluded. Taken together, and although needing more consolidated data, the TMB appears to be a promising biomarker for GC immunotherapy.In a clinical trial that analyzed toripalimab therapy (a PD-1 antibody) in advanced GC, patients with TMB-high had significant higher OS (14.6 months) than those with TMB-low (4.0 months) with patients.The outcome of GC, in particular of advanced disease stages, remains poor. Immunotherapy based on immune checkpoint inhibition in advanced GC has shown promising benefits, in particular when patients who will derive most benefit from this type of therapy are selected. The heterogeneity of GC and the identification of GC subtypes with distinct molecular profiles has offered the opportunity to discover not only new GC therapeutic targets but also novel markers of response to immune checkpoint blockade. Nevertheless, further research is needed, as a lot of uncertainty still exists regarding immune checkpoint inhibitors and biomarker effectiveness. Future approaches should also consider additional biomarkers to identify patients who could better respond to the different inhibitors, thus contributing to improve the negative prognosis associated with advanced GC.Financial support and sponsorship: none.Conception and design: CF; Acquisition of data and interpretation: SR and CF; Drafting of manuscript: SR and CF; Revising of manuscript for important intellectual content: CF.The authors declare no conflicts of interest.


Specifically, intracellular Ca2+ homeostasis is mainly regulated by the endoplasmic reticulum and the mitochondria, whose Ca2+ exchange is mediated by appositions, termed endoplasmic reticulum–mitochondria-associated membranes (MAMs), formed by proteins resident in both compartments. These tethers are essential to manage the mitochondrial Ca2+ influx that regulates the mitochondrial function of bioenergetics, mitochondrial dynamics, cell death, and oxidative stress. However, alterations of these pathways lead to the development of multiple human diseases, including neurological disorders, such as amyotrophic lateral sclerosis, Friedreich’s ataxia, and Charcot–Marie–Tooth. A common hallmark in these disorders is mitochondrial dysfunction, associated with abnormal mitochondrial Ca2+ handling that contributes to neurodegeneration. In this work, we highlight the importance of Ca2+ signaling in mitochondria and how the mechanism of communication in MAMs is pivotal for mitochondrial maintenance and cell homeostasis. Lately, we outstand potential targets located in MAMs by addressing different therapeutic strategies focused on restoring mitochondrial Ca2+ uptake as an emergent approach for neurological diseases.Calcium (Ca Its versatility is achieved by its rich dynamics in concentration changes, which can be caused either by Ca2+ entry from the extracellular space or Ca2+ release from intracellular storage compartments [2+ out of the cell or to intracellular organelles. 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In ta cells .2+-dependent events occurring in cells, alteration of its signaling pathways contributes to the development of multiple human disorders. Therefore, the study of Ca2+ signaling is essential for understanding the pathophysiology of many diseases, including diabetes, carcinogenesis, cardio- and cerebrovascular diseases including endothelial dysfunction, as well as neurodegenerative disorders [Due to the huge amount of Caisorders ,14,15,162+ signaling in mitochondria and how the mechanism of communication between the ER and the mitochondria is pivotal to the mitochondria. Lately, we address different therapeutic strategies targeting mitochondrial Ca2+ uptake as an emergent therapeutic approach for neurological disorders.In this review, we describe the importance of CaMitochondria and the ER are structures that experience continuous remodeling to coordinate complex mechanisms of signal transduction and gene expression, generating physical interactions that facilitate a fast and efficient regulation of these processes . Termed The association between the ER and the mitochondria was described in the 1950s, when Copeland and Dalton observed a precise orientation of the ER with respect to the mitochondria . Besides2+ transfer [MAMs encompass an extensive variety of different proteins. The first independent proteomic studies identified 911 and 1212 proteins ,23 localtransfer .2+ exchange between the ER and the mitochondria requires the formation of a protein bridge composed by proteins of both compartments [2+, fundamental for mitochondrial function, dynamics, and the regulation of apoptosis [2+ upon the cation mobilization through the ER channel IP3R . Recently, the spatial relation between the ER and the mitochondria was described by the same group [2+ entry into the mitochondria in Hela cells [Caartments . In partpoptosis . In 1993me group . They obla cells .2+ in the cell (about 100–500 µM) compared to the cytosol (~100 nM). Ca2+ is released to the cytosolic space upon the input signals from the ER through the IP3R and through the RyR (ryanodine receptor) in the case of the SR [2+ signaling regulation. Sig-1R is enriched in MAMs and stabilizes activated IP3R, promoting Ca2+ influx into the mitochondria [The lumen of the ER is one of the main storages of free Caf the SR . Furtherchondria ,42.2+ must cross both mitochondrial membranes. The outer mitochondrial membrane (OMM) is Ca2+ permeable due to VDAC (voltage-dependent anion channel), which enables different metabolites  to cross from the cytosol to the mitochondria [2+ enters the mitochondrial matrix through the mitochondrial calcium uniporter (MCU) since this layer is ion-impermeable [For the mitochondria, Cachondria . In connermeable .3R and VDAC, maintaining the junction and ensuring an efficient transfer of Ca2+ to the mitochondria [In addition, another key protein that stabilizes the connections of both compartments is glucose-regulated protein 75 (GRP75), which chaperones IPchondria ,44. Alto3R-GRP75-VDAC-MCU is one of the complexes in MAMs that is not only essential for the regulation of Ca2+ homeostasis, but also for the control of mitochondrial function in the regulation of bioenergetics, mitochondrial dynamics, and cell death [IPll death ,46. Mitoll death . In partll death . Thus, d2+ levels. Enzymes of the tricarboxylic acid cycle and the electron transport chain require an increase in mitochondrial Ca2+ uptake to promote ATP synthesis [2+ increase in the mitochondrial matrix [2+ concentration. In this way, when energy demand increases, different neurotransmitter and hormone receptors increase mitochondrial Ca2+ through IP3R [The mechanism of ATP production depends on oxidative phosphorylation, and it is dynamically and promptly regulated by mitochondrial Caynthesis ,50. For l matrix ,52. Furtugh IP3R ,53.2+ uptake is also influenced by mitochondrial dynamics. Several properties of mitochondria, in terms of network, orientation, and shape, regulate the amounts of Ca2+ that reach the mitochondrial matrix and, thus, the subsequent functions regulated by these events [2+ more efficiently than disperse mitochondria. Mitochondrial fusion/fission events require elevated amounts of cytosolic Ca2+ to be transferred to the mitochondria [2+ can activate the cytosolic GTPase dynamin-related protein (Drp-1), which is recruited to form a ring around mitochondria to promote mitochondrial fission [2+ homeostasis [The first is distribution, because clustered mitochondria are able to buffer Cachondria ,55. In t fission . On the eostasis .2+ conductors, distributing the cation along the fused network. It has been described that, while fragmented mitochondria buffer Ca2+ from the ER in a heterogeneous manner, tubular mitochondria incorporate Ca2+ in an equilibrated and connected way [Connectivity, because elongated mitochondria are better Cacted way ,56.2+ buffering, since elevated concentrations of Ca2+ near mitochondria are required to promote mitochondrial Ca2+ uptake. In this context, Csordás et al. demonstrated the importance of spacing distance in MAMs for an efficient Ca2+ transfer, outlining that it is essential that the tethered bridge is properly assembled to ensure Ca2+ influx into the mitochondria [Vicinity is a dynamic property that also affects Cachondria ,57.2+ buffering, as forced mitochondrial expansion [2+ uptake capacity [Last but not least, the volume of mitochondria is also crucial for mitochondrial Caxpansion  and fragcapacity .Mitochondrial Cae events .The fir2+ accumulation in the mitochondria normally precedes cell death driven by necrosis and apoptosis [2+ handling can lead to mitochondrial Ca2+ overload, thus activating the opening mitochondrial permeability transition pore. This causes mitochondrial swelling, which leads to release of cytochrome c and caspase cofactors into the cytosol [Furthermore, abnormal Capoptosis ,60. Unde cytosol .2−), which are converted to the diffusible redox signaling molecule hydrogen peroxide (H2O2) by superoxide dismutase 2 (SOD2). There is mounting evidence about the role of H2O2 as a messenger located in redox nanodomains in MAMs [2O2, mainly generated in the mitochondria, can modulate the activity of IP3R and RYR channels, promoting the release of Ca2+ from the ER to the mitochondria. This event can, in turn, induce redox signaling through the activation of mitochondrial metabolism, further inducing the accumulation of additional H2O2. This positive feedback mechanism attenuates when Ca2+ returns from the mitochondrial matrix to the ER [2+ lead to high ROS levels, which may trigger cell death [2+ overload can inhibit H2O2 clearance, promoting its accumulation in the mitochondria [Mitochondria is considered the main source of reactive oxygen species (ROS) in cells with high metabolic rates. In circumstances of mitochondrial dysfunction, an uncontrolled production of ROS would lead to an imbalance in the cellular redox state which, in turn, might likely contribute to pathogenesis . Oxidati in MAMs . H2O2, mo the ER ,67. Thisll death . In addichondria .2+ exchange ER–mitochondria through proper contacts between the two compartments. Nonetheless, a growing knowledge and a better understanding about the translational and clinical role of Ca2+ homeostasis and oxidative stress in the physiopathology of neurological diseases are required to find novel therapeutic strategies.Hence, this outstands the necessity of an equilibrate Ca2+ exchange and regulate key mechanisms in cell homeostasis. These processes  are usually involved in the physiopathology of neurodegeneration. Mitochondrial dysfunction results in abnormal Ca2+ handling, leading to alterations in axonal transport, bioenergetics, redox status, contractility, and cell viability [As it has been discussed above, the regulation of MAMs is crucial to maintain a proper Caiability ,70,71. Iiability ,73,74,75iability ,77,78,792+ uptake, reduced cell viability, and dysfunction in the subsequent mechanisms regulated in this domain [The first proteomic description of MAMs detected proteins involved in mitochondrial dysfunction as well as in neuromuscular and degenerative diseases, including Huntington’s disease (HD), Parkinson’s disease (PD), and Alzheimer’s disease (AD) . Nowadays domain ,78,79.2+ accumulation in the cytosol, impairing the communication between the two compartments and displaying mitochondrial alterations. Several forms of amyotrophic lateral sclerosis (ALS) are characterized by mitochondrial disruption and abnormal mitochondrial Ca2+ handling. This includes ALS forms with mutations in superoxide dismutase 1 (SOD1), in which mouse models exhibit mitochondrial defects and deficits in mitochondrial Ca2+ uptake [2+ influx via IP3R, and lower ATP production. These defects lead to neuron vulnerability, directly associated with the physiopathology of the disease [Conversely, several neuropathies exhibit reduced ER–mitochondria connections and Ca+ uptake ,85. Homo+ uptake , showing disease .2+ handling defects, and oxidative stress that contribute to neurodegeneration [3R channel, which leads to the abnormal Ca2+ release to the cytoplasm, potentially inducing Ca2+ buffering defects in mitochondria [2+ accumulation, as well as impaired mitochondrial Ca2+ uptake and decreased inter-organelle interactions in MAMs. The protein involved in the disease, frataxin, has recently been found as a member of the protein network of MAMs, interacting with GRP75 and IP3R [GDAP1) and MFN2 [2+ and mitochondrial defects, coupled with altered interplay between ER–mitochondria and Ca2+ accumulation in the cytosol [3R, suggesting impaired ER–mitochondria communication [Accordingly, dominant cerebellar ataxias are multifactorial and progressive diseases with common mechanisms of mitochondrial dysregulation, Caneration . Specifichondria . Interesand IP3R ,91,92. Cand MFN2 , which e cytosol ,94,95. Hnication ,97,98.2+ modulation is fundamental to maintain the physiological mechanisms that regulate metabolism, mitochondrial dynamics, and cell death. Therefore, MAMs emerge as potential therapeutic targets of neurological disorders. In this review we will focus on different therapeutic approaches . Given eostasis ,123. Thi3R-dependent Ca2+ release and upregulation of key Ca2+-regulating genes [3R and Sig-1R with mitochondria in YAC128 neurons. Accordingly, this compound increased mitochondrial activity, elongation, and motility. In both HD human neural stem cells and YAC128 neurons, pridopidine increased mitochondrial respiration, rescued antioxidant response, and decreased mitochondrial ROS levels caused by Sig-1R knockdown. Additionally, apart from the improvement in motor coordination, YAC128 mice treated at early/pre-symptomatic age with pridopidine showed a reduction in mitochondrial ROS levels. Overall, these results highlighted the effects of pridopidine in mitochondrial function, contributing to neuroprotection mediated by Sig-1R [Pridopidine (ACR16) is a selective Sig-1R agonist and a dopamine stabilizer ,125. Greng genes . Furtherng genes . Interesy Sig-1R .Regarding clinical evaluation, pridopidine has demonstrated some improvements in motor symptoms. A phase-III randomized, double-blind, multicenter trial study  failed to provide evidence about the effects of pridopidine in the primary outcome (changes in modified Motor score). Despite this, some parameters related to motor scales improved significantly in the 90 mg/day group . AccordiThe efficacy of pridopidine has been tested in in vitro and in vivo models of ALS, due to the fact that different forms of ALS are caused by mutations in the Sig-1R gene ,129. AutRecently, pridopidine has demonstrated neuroprotective and neurorestorative effects in nigrostriatal dopamine neurons via Sig-1R in an animal model of PD . A phase2+ into the mitochondria. These effects attenuated neuronal apoptosis [2+ clearance in motoneurons and IP3R-mediated ER Ca2+ release in ALS mice [SA4503 or cutamesine is an orally available, potent, and selective Sig-1R agonist that exhibits antiarrhythmic and antidepressant effects . It has poptosis  and amelpoptosis . In addipoptosis . In ALS poptosis . In thisALS mice . In alphALS mice .In the clinical field, SA4503 has been evaluated in two trials. In 2008, the safety and efficacy of SA4503 was assessed in subjects with major depressive disorder (NCT00551109) . ResultsBlarcamesine (ANAVEX2-73) is a safe Sig-1R agonist and muscarinic receptor modulator with preliminary efficacy evidence in patients with AD and Rett syndrome . PreclinAfter demonstrating good safety, bioavailability, and tolerability in AD patients (NCT02244541) , blarcam2+ levels in preclinical studies using different disease models. For instance, PRE-084 exerted protective action against oxidation and improved viability in human retinal cells [PRE-084 is a selective Sig-1R agonist that has demonstrated promising effects against oxidative stress and modulating intracellular Caal cells . In a HDal cells .2+ influx into the mitochondria and ATP levels after incubating motor neurons of ALS with PRE-084. In all the experiments, this compound restored the function of IP3R impaired in the model, suggesting that Sig-1R activation by PRE-084 prevented the disruption of Sig-1R-IP3R interaction. In addition, intraperitoneal administration of PRE-084 in pre-symptomatic ALS mice successfully restored co-localization of Sig-1R and IP3R analyzed in neurons of the lumbar spinal cord. These data indicate that Sig-1R activation is crucial to prevent MAMs disruption and regulate the Ca2+ exchange between the ER and the mitochondria via IP3R [2+ levels [2+ homeostasis.Furthermore, in a model of AD, treatment with PRE-084 restored mitochondrial respiratory dysfunction in mouse hippocampus and prevented increases in lipid peroxidation levels and apoptosis markers . In thisvia IP3R . PRE-084via IP3R . Neverth+ levels . These f2+ uptake and ATP production in hypertrophic cardiomyocytes [Fluvoxamine is a selective serotonin reuptake inhibitor with high affinity for Sig-1R that has been widely used in clinical practice as an antidepressant. Fluvoxamine has shown to increase Sig-1R expression, inducing neuroprotection and protecting cells from ER-stress . This comyocytes  and protmyocytes . Furthermyocytes ,156. Formyocytes . Accordimyocytes . Specialmyocytes ). At the2+ transporting complexes [2+ and controlling the mitochondrial Ca2+ influx. In fact, it has been determined that MCU and IP3R must have optimal distance to ensure mitochondrial Ca2+ signaling in physiological conditions [2+-conducting subunit of the MCU complex is named Mcu, the regulatory subunit encompasses MICU1; MICU2; mitochondrial calcium uptake protein 1, 2, 3 (MICU3); essential MCU regulator (EMRE); mitochondrial calcium uniporter regulator 1 (MCUR1); and mitochondrial calcium uniporter regulatory subunit (MCUb) [The mitochondrial calcium uniporter (MCU) is one of the most important and highly selective Caomplexes . MCU is nditions . Locatedt (MCUb) ,163.2+ influx by interacting with Mcu, so when binding to Ca2+, its conformation changes from hexamers to oligomers and activates MCU [2+ uptake, preventing Ca2+ to cross the IMM unless it is above the threshold. In fact, elevated Ca2+ levels in the intermembrane space are required to reach the mitochondrial matrix through MCU due to its low Ca2+ affinity [2+ uptake, acting as a gatekeeper and preventing mitochondrial Ca2+ overload [2+ intake by the MCU [Specifically, MICU1 is an important gatekeeper located towards the mitochondrial matrix, next to the IMM. MICU1 controls the mitochondrial Caates MCU ,165. Knoates MCU . Besidesaffinity . MICU3 daffinity . EMRE isoverload ,170. MCUoverload ,172. MCU the MCU .2+ exchange, mainly in cancer [In the last decade, special interest has been taken in the modulation of MCU activity as a novel therapeutic target. Most current therapeutic approaches are focused on the inhibition of ER–mitochondria Can cancer ,175 and n cancer  (such asn cancer  and PD [n cancer ).2+ homeostasis, MAMs disruption, and defects in several functions regulated in this domain [2+ clearance by promoting mitochondrial Ca2+ buffering capability in cardiomyocytes with cardiac hypertrophy. In this cellular model, SR–mitochondria connections were decreased by the inhibition of Mfn2, leading to inhibition of ATP synthesis and contributing to pathogenesis [However, diseases such as type 2 diabetes point towards MCU activation to alleviate mitochondrial dysfunction associated with dysregulation in intracellular Cas domain ,180. Tarogenesis .2+ uptake [2+-induced oxidative stress and inhibition of mitochondrial Ca2+ uptake in SH-SY5Y human neuroblastoma cells [2+ import through MCU overexpression in glia recovered degenerative phenotypes and ATP production in a FRDA Drosophila melanogaster model [Besides, genetic modulation of MCU expression has proved to modulate mitochondrial Ca+ uptake . In thisma cells . The proer model .2+ import into the mitochondria and exhibiting neuro and cardioprotective properties [2+ entry through MCU activation. The most active compound was Kaempferol, which increased the rate of mitochondrial Ca2+ uptake by 85-fold [The most relevant MCU enhancer described until now is Kaempferol, which has demonstrated to enhance MCU, promoting Caoperties ,187,188. 85-fold . In addi 85-fold ,191. Spe 85-fold . A recen 85-fold .D. melanogaster model of PD, kaempferol delayed degenerative phenotype onset in a dose-dependent manner, accompanied by a reduction in oxidative stress markers [D. melanogaster model of AD [Accordingly, in cerebellar granule cells, Kaempferol prevented cells from apoptosis, exerting potent effects by blocking ROS production . In isch markers . Similarel of AD . Indeed,el of AD . Kaempfeel of AD .2+ uptake without a specific ER–mitochondria target.In addition to the aforementioned approaches, other compounds may be suitable to increase mitochondrial Ca2+ levels [2+ buffering in cerebellar granule cells. They demonstrated an active role in the regulatory mechanisms of Ca2+ homeostasis, suggesting an enhancement in mitochondrial function and regulation of intracellular Ca2+ [2+ uptake enhancement, though the specific receptor of taurine is not known.Taurine is a sulfur-containing amino acid present in abundance in many excitable tissues, including the brain, skeletal, and cardiac muscles. Physiological actions of taurine include membrane stabilization, neurotransmission, and modulation of cellular Ca+ levels . El Idrilar Ca2+ ,203. Bes2+ mobilization [2+ signaling, NGF has been demonstrated to increase cytosolic free Ca2+ concentration in C6-2B glioma cells and PC12 cells [2+ [Nerve growth factor (NGF) is a secreted neurotrophin involved in survival, maintenance, and regeneration of specific types of neurons in the central and peripheral nervous system . It has lization ,208. In 12 cells , as wellells [2+ . NGF wasells [2+ . For thi2+ uptake enhancers (MiCUps) are a group of compounds recently identified to increase mitochondrial Ca2+ import, especially in cardiomyocytes. Using molecular ligand–protein docking and mutational analysis, a recent study has determined Efsevin as a MiCUp that shifts the opening of the VDAC2 channel, promoting Ca2+ entry to the mitochondria [2+ uptake as a pharmacological target [Mitochondrial Cachondria . It has chondria ,212. Ezel target .2+ signaling to stabilize its redox state or directly target mitochondrial ROS [+2 flux and, thus, ensure a correct balance in the ROS–antioxidant system. In the context of the second strategy, treatment with antioxidants has been demonstrated to recover ER–mitochondria communication and Ca2+ exchange between the two compartments. Vitamin E restored mitochondrial Ca2+ uptake in a cardiomyocyte model of FRDA [2+ influx in the mitochondria.Targeting the mitochondrial redox state could be a suitable therapeutic strategy to recover ER–mitochondria communication. In this context, compounds may modulate mitochondrial Carial ROS . The fir of FRDA . Accordi of FRDA , suggestOn the other hand, when mitochondrial function is impaired, it is realistic to assume the involvement of different effectors and mechanisms. For this reason, some authors point towards the use of a combination of antioxidants also known as ‘mitochondrial cocktails’, due to its irrelevant toxicity, providing relevant benefits by increasing the spectrum of action ,217,218.SS-31 or elamipretide is an aromatic–cationic tetrapeptide that readily penetrates cell membranes and transiently localizes to the inner mitochondrial membrane. This mitochondrial-targeted agent was found to interact with cardiolipin, an anionic phospholipid located in the IMM and required for cristae formation. In addition, SS-31 accelerates ATP recovery and increases the enzymatic activities of Fe-S enzymes, including aconitase and complex II and III of the respiratory chain ,220. AnoMitoquinone or MitoQ is an orally active mitochondria-targeted antioxidant that mimics the role of the endogenous mitochondrial antioxidant coenzyme Q10 . In addiThese and other antioxidants targeting mitochondrial dysfunction in neurodegeneration have been extensively reviewed by several authors, including us ,237,238.2+ homeostasis and, therefore, in cell physiology. Since MAMs are the structures carrying out such communication, their disruption involves dramatic consequences that not only affect mitochondrial mechanisms, but also a plethora of intracellular signaling pathways. An example of such disarrangement is exerted in neurological disorders, as the proteins involved in these diseases are part of the protein network of MAMs [Since mitochondria are intracellular dynamic compartments involved in multiple mechanisms, the in-depth study of these mitochondrial-dependent pathways is crucial to understand the pathophysiology of neurological and neuromuscular disorders. Particularly, we highlight the importance of crosstalk communication between the ER and the mitochondria in intracellular Ca of MAMs ,90,239.On the other hand, proteins belonging to MAMs are a growing list, which contributes to the idea of the complexity and dynamism of these structures and, thus, the mechanisms involved in its regulation. For this reason, it is also important to determine the properties of MAMs in different cell types under distinct cellular conditions. The elucidation of these pathways will provide valuable information about the physiopathology of diseases that present impaired ER–mitochondria communication, opening new fields of research to identify adequate treatments for patients.2+ uptake can, therefore, exert antioxidant properties, making them more versatile in restoring the molecular defects involved in these diseases.We believe that restoration of MAMs communication may be a suitable strategy to reverse this impairment. In addition, some patients suffering from neuromuscular diseases usually undergo heart conditions ,241,242.2+ crosstalk and contribute to cardiac hypertrophy [In terms of future therapeutic approaches, special attention is being paid to miRNAs. These small molecules regulate genetic expression by binding to its target mRNA and can be detected in biological fluids. The identification of miRNA signatures could provide valuable information about diagnosis or prognosis, even in the early stages of the disease. Besides, this should address the nature of targets and the extent of the biological regulation of the occurring pathways. For instance, miR-20b has been found to mediate MFN2 signaling, which can impair ER–mitochondria Caertrophy . This opNeurological diseases, such as ALS, CMT, and FRDA, have no cure, so it is essential to find therapeutic approaches that can improve patient’s wellbeing by either slowing the onset of symptoms and/or delaying the progression of the disease, in addition to physical therapy, occupational therapy, and surgery. The repurposing of compounds outlined in this review could be a good strategy to test in vitro and in vivo models of disease. Furthermore, the use of new in silico approaches, such as pharmacophore modeling and molecular docking, are useful to ensure optimal molecular interactions with a specific biological target. This may contribute to identifying new targets, drug discovery, and optimization for specific treatments ,246,247.


ATM, BRAF, and SMAD4. The mutational co-occurrences of BRAF-HMCN and DNAH17-MDN1 as well as the methylation silence event of MLH-1 were only found in MSC-1. MSC-2 was linked to the carcinogenic process of age and tobacco chewing habit, exhibiting dominant SCNA such as MYC (8q24.21) and PTEN (10q23.31) deletion as well as CCND3 (6p21.1) and ERBB2 (17q12) amplification. MSC-1 displayed higher immunogenicity and immune infiltration. MSC-2 had better prognosis and significant stromal activation. Based on the two subtypes, we identified and validated the expression relationship of FAM83A and IDO1 as a robust biomarker for prognosis and distant metastasis of CRC in 15 independent cohorts and qRT-PCR data from 30 samples. These results advance precise treatment and clinical management in CRC.Genomic alterations constitute crucial elements of colorectal cancer (CRC). However, a comprehensive understanding of CRC genomic alterations from a global perspective is lacking. In this study, a total of 2,778 patients in 15 public datasets were enrolled. Tissues and clinical information of 30 patients were also collected. We successfully identified two distinct mutation signature clusters (MSC) featured by massive mutations and dominant somatic copy number alterations (SCNA), respectively. MSC-1 was associated with defective DNA mismatch repair, exhibiting more frequent mutations such as  Colorectal cancer (CRC) is the fourth most prevalent cancer and the second most lethal cancer globally . AlthougBRAF, TP53, and KRAS mutations, HNF4A amplification, and homozygous deletion of PTEN, but there is a wide range of genomic variations in CRC . The two subtypes displayed significant difference in the genomic variation, methylation profile, prognosis, immune landscape, and microsatellite instability (MSI) status. In addition, based on the two subtypes, we identified and validated the relationship between Mutation (derived from VarScan 2), somatic copy number alteration (SCNA), HumanMethylation450 array, and RNA-seq count data of CRC were obtained from The Cancer Genome Atlas (TCGA) portal. We also retrieved 14 expression microarrays datasets  from the Gene Expression Omnibus (GEO) database. For 11 microarray datasets derived from the Affymetrix Human Genome U133 Plus 2.0 Array, the “CEL” raw data were obtained and further processed via a robust multiarray averaging algorithm (RMA) implemented in the affy R package. RMA was used to perform background adjustment, quantile normalization, and final summarization of oligonucleotides per transcript using the median polish algorithm. For the three other microarray datasets, we directly retrieved the normalized matrix files. The corresponding clinical information was also downloaded, and the details are listed in De novo mutational signatures were then compared with curated signatures in COSMIC (https://cancer.sanger.ac.uk/cosmic/signatures_v2). The APOBEC enrichment analysis described by Roberts et al. , we then labeled it a subtype-specific MDG (ssMDG).We downloaded the HumanMethylation450 array data from the TCGA-CRC cohort and employed the IlluminaHumanMethylation450kanno.ilmn12.hg19 package for annotation. To identify the methylation-driven genes (MDGs) in CRC, we employed two methods to dissect the methylation profiling data. One method was MethylMix, which is based on the beta distribution and was designed to recognize gene expression that is significantly related to methylation events . The othWe performed gene set enrichment analysis (GSEA) between the two MSC subtypes, and the biological function terms with FDR < 0.05 were considered significant. Fifty hallmark pathways were also retrieved from the Molecular Signature Database (MSigDB v7.1). Based on the hallmark gene sets, we utilized the gene set variation analysis (GSVA) algorithm to transform the gene expression matrix into a pathway enrichment score matrix. The limma R package was applied to further reveal the discrepant pathways between the two MSC subtypes, with thresholds of FDR < 0.05 and |log2FC| > 0.2. The abundances of eight immune cell and two non-immune cell populations were assessed via the MCP-counter R package. In addition, we also calculated or retrieved data for 17 immunogenicity indicators from previous research: non-silent mutation rate, MSI score, single nucleotide variant (SNV) neoantigens, insertion and deletion (indel) neoantigens, cancer testis antigen (CTA) score, aneuploidy score (AS), intratumor heterogeneity (ITH) score, number or fraction of segments altered, homologous repair deficiency (HRD) score, loss of heterozygosity (LOH) score, B-cell receptor (BCR) or T-cell receptor (TCR) diversity score, and cytolytic activity (CYT) –30. The We aimed to identify the relationship between the mRNA expression of two genes with prognostic significance and utility for predicting distant metastasis to facilitate clinical management. To ensure the robustness and stability of our results, 11 independent CRC cohorts with complete prognostic information encompassing  were employed to identify promising prognostic markers, and seven independent CRC cohorts with distant metastasis information  were utilized to further explore the ability of the identified prognostic markers to predict metastasis . The pipThe use of the human cancer tissues in this study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University on December 19, 2019, and the TRN is 2019-KW-423. A total of 30 paired CRC tissues and matched adjacent non-tumor tissues were obtained from patients after receipt of surgical resection at The First Affiliated Hospital of Zhengzhou University. None of the patients received any preoperative chemotherapy or radiotherapy. Written informed consent was obtained from all patients. The inclusion criteria were as follows: no preoperative chemotherapy, radiotherapy, or targeted therapy; no other types of tumors; and no autoimmune diseases. The specimens obtained during surgery were immediately snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. Clinical staging of the specimens was based on the NCCN (2019) guidelines. For details on the patients, please refer to GAPDH. The sequences of the primers were as follows:Total RNA was isolated from CRC tissues and paired adjacent non-tumor tissues with RNAiso Plus reagent  according to the manufacturer's instructions. RNA quality was evaluated using a NanoDrop One C , and RNA integrity was assessed using agarose gel electrophoresis. An aliquot of 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) according to the manufacturer's protocol using a High-Capacity cDNA Reverse Transcription kit . Quantitative real-time PCR (qRT-PCR) was performed using SYBR Assay I Low ROX  and SYBR® Green PCR Master Mix  to detect expression. The data were normalized to the expression of GAPDH forward (5′- to 3′-): GGAGCGAGATCCCTCCAAAATGAPDH reverse (5′- to 3′-): GGCTGTTGTCATACTTCTCATGGFAM83A forward (5′- to 3′-): CAGATCTCTGACAGTCACCTCAAGFAM83A reverse (5′- to 3′-): CTGCCTGACTTGGCACAGTAIDO1 forward (5′- to 3′-): ATATGCCACCAGCTCACAGGIDO1 reverse (5′- to 3′-): AGCTTTCACACAGGCGTCAT.T-test. The Wilcoxon signed rank test was utilized to compare the gene expression differences between the paired CRC tissues and matched adjacent non-tumor tissues in the qRT-PCR assay. Kaplan–Meier and Cox regression analyses were performed with the survival R package. All P-values were two-sided, with p < 0.05 considered to indicate statistical significance. All data processing, statistical analysis, and plotting were conducted in R 3.6.4 software.Correlations between two continuous variables were assessed via Spearman's correlation coefficients. Fisher's exact test or Pearson's chi-squared test was applied to compare categorical variables. Continuous variables were compared between two groups through the Wilcoxon rank-sum test or APC had the highest number of deletion mutations (236), and TTN had the highest number of missense mutations (646)  . To gainns (646) . Subsequns (646) . Therefons (646) . Clocklip = 0.005) (p = 0.070) (p = 0.028) (n = 226) predominantly featured signatures 6, 15, 20, and 28. Signatures 6, 15, and 20 are linked to defective DNA MMR. MSC-2 (n = 309) was characterized by signatures 1 and 29, which are associated with age and tobacco chewing habits (APOBEC signature enrichment score was significantly higher (p = 0.003) in MSC-1, which indicated that MSC-1 had a higher occurrence of the C>T transition in TpCpW motifs than did MSC-2 (APOBEC family might contribute to a high tumor mutation burden (TMB)   . There w= 0.070) . We furt= 0.028) . Of noteg habits . We alsoid MSC-2 . A previen (TMB) . Therefo= 0.003) ; this fip < 0.001)   (The TMB in MSC-1 cells was significantly higher than that in MSC-2 cells (< 0.001) . A highe< 0.001) . We furt< 0.001) . Out of W7, etc. –39. In arable OS . In the rable OS . Consistgression , were dond USH2A . Interes< 0.001) . In addiable DFS  and commpoor DFS . Furtherpoor DFS . Hence, poor DFS , and the< 0.001) ; these rp < 0.05). Although there was no statistical significance in the focal level load between the two subtypes, slight trends for higher loads were shown in MSC-2 than in MSC-1 (MYC (8q24.21), CCND3 (6p21.1), ERBB2 (17q12), PTEN (10q23.31), SMAD4 (18q21.2), and APC (5q22.2)    . Althoug< 0.001) . These r< 0.001) . We repoonged OS , gain oforse DFS , and losable DFS . Moreove= 0.042) . ImportaTBX1, GREB1L, and CNNM1 was significantly associated with unfavorable OS   , which eTGF-β) signaling, and Wnt signaling  between the two MSC subtypes at the multiomics level . Obviousan MSC-2 . In addiin MSC-1 .HLA-B and ITGB2 induced significantly lower expression in only MSC-1, but a slightly increased expression was observed in MSC-2   .FAM83A|IDO1, FABP4|KLK12, and FABP4|GBP5   . ConversFAM83A|high and IDO1|high groups in all cohorts     . Due to = 1.000) , and mor= 0.278) . TherefoFAM83A was overexpressed in tumor tissues relative to adjacent non-tumor tissues, and the expression of IDO1 showed the opposite relationship (p < 0.001)  of 30 CRC patients are shown in FAM83A and IDO1. In line with the previous results, when the expression of FAM83A was higher than that of IDO1, patients had worse OS and DFS (log-rank p < 0.001) (p = 0.007) (qRT-PCR assays were performed in 30 paired CRC tissues and matched adjacent non-tumor tissues . We obse< 0.001) . The rol< 0.001) , as well= 0.007) .POLE somatic mutations (signature 10), and tobacco chewing habits (signature 29). Based on these signatures, we identified two heterogeneous subtypes, MSC-1 and MSC-2. The two subtypes exhibited tremendous differences in genomic alterations including mutation, SCNA, and DNA methylation. The distinct tumor microenvironment (TME) statuses and immune escape mechanisms of the two subtypes reinforced their molecular variability. We also observed significant clinical differences between the subtypes in terms of OS and DFS. In addition, to facilitate clinical application, we employed a gene pair pipeline to determine a prognostic and distant metastasis biomarker, FAM83A|IDO1, and further validated it in 15 independent datasets and qRT-PCR data from 30 samples.Elegant efforts have demonstrated that multifarious genomic alterations are critical to the prognosis and targeted therapy of CRC –48. We sATM, BRAF, and HMCN1, which play vital roles in the tumorigenesis and development of many cancers , SMAD4 (18q21.2), and PTEN (10q23.31) as well as gain of CCND3 (6p21.1) and ERBB2 (17q12). The oncogene ERBB2 has been shown to be amplified or overexpressed in multiple cancers, including colon cancers (ERBB2 amplification is an emerging therapeutic target and may also be a negative predictor of response to anti-EGFR therapy in CRC (SMAD4, a tumor suppressor that is the central node in TGF-β signaling (SMAD4 is associated with poor prognosis and predisposition to chemoresistance, such as resistance to 5-fluorouracil, leucovorin, and irinotecan (APC and KRAS, which occur early in the progression from colorectal adenoma to malignant carcinoma, were highest in MSC-2 (APC, SMAD4, TP53, KRAS, or PI3KCA, resulting in Wnt-β-catenin and TGF-β pathway activation; this process is referred to as the adenoma-to-carcinoma sequence. The above analysis suggests that the conventional adenoma-to-carcinoma sequence may be an important process of oncogenesis in MSC-2.MSC-2, a CNA-dominant subtype characterized by signatures 1 and 29, was related to spontaneous deamination of 5-methylcytosine and tobacco chewing habits. MSC-2 displayed loss of  cancers , 54. As y in CRC . Anotherignaling . Studiesinotecan . Of notein MSC-2 . As repoTGF-β signaling, and Wnt signaling. Combined with this weaker immunogenicity, the insufficient immune cell infiltration in MSC-2 contributes to immune escape, suggesting that patients with the MSC-2 subtype might exhibit an unfavorable response to immunotherapy. Therefore, comprehensive analysis of molecular and immune microenvironment variability might contribute to optimizing the treatment and clinical management of CRC patients.In the present study, we also assessed the differences in immune cell proportions, stromal cell infiltration, and immune escape mechanisms between the two subtypes. Consistent with the high mutation load in MSC-1, there was also infiltration of numerous innate and adaptive immune cells in the TME, which was linked to the immune inflammation status. The TME results were also supported by the finding of activation of immune-related pathways, including pathways related to the adaptive immune response, antigen processing and presentation of peptide antigens, and the response to interferon-gamma. In CRC, immune checkpoint inhibitors have been proven effective in heavily mutated tumors with MMR defects or high levels of MSI , which iEYS, as well as gain of MLST8 and MAP2K2, could prolong OS, while mutation of USH2, loss of DKK1, APC, MCC, and SMAD4, and methylation of TBX1 were linked to unfavorable prognosis. In addition, the prognostic value of some commutations was revealed for the first time. Commutation of APC-TP53 demonstrated favorable DFS, and commutation of APC-KRAS, KRAS-TP53, and KRAS-SYNE1 was significantly associated with poor DFS. Importantly, to facilitate clinical application, we identified three gene pairs with prognostic significance, FAM83A|IDO1, FABP4|KLK12, and FABP4|GBP5. FAM83A|IDO1 was best at predicting prognosis in 11 public datasets and our own cohort, and it was an independent prognostic factor for CRC. FAM83A|IDO1 also exhibited excellent performance in assessing the distant metastasis status in seven public datasets and our own cohort. Patients with FAM83A|high disease had a higher risk of metastasis than patients with IDO1|high disease. Traditionally, the batch effects of different platforms and the different cutoff values severely limit the clinical translation and application of biomarkers. In this study, we only focused on the mathematical relationship between the mRNA expression of two genes, which completely ignores the batch effects among different platforms and does not require definition of a cutoff value; it is just a binary relationship. Therefore, the relationship between FAM83A and IDO1 mRNA expression is a promising biomarker for predicting prognosis and metastasis in clinical applications.In addition, we comprehensively revealed many prognosis-relevant genomic events. In this study, we observed that mutation of FAM83A and IDO1 mRNA expression as a biomarker focused on the mathematical relationship between two genes, but the biological relationship was not studied.Our study also has a few limitations. First, it analyzed multidimensional data of genomic alterations in CRC but lacked microscopic experimental verification. Second, the identification of the relationship between We described a novel molecular classification method for categorizing CRC into two clusters, suggesting intertumoral molecular variability. The two subtypes displayed distinct genomic drivers, prognoses, functional statuses, immune microenvironments, and MSI statuses, and targeting these differences might advance precise treatment and clinical management in CRC. Promisingly, we also identified and validated a robust and promising biomarker for predicting the prognosis and metastasis of CRC patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the human cancer tissues used in this study were approved by Ethnics Committee of The First Affiliated Hospital of Zhengzhou University in December 19, 2019, and the TRN is 2019-KW-423. The patients/participants provided their written informed consent to participate in this study.ZLiu made the conceptualization. ZS, QD, and XH were involved in the methodology. ZLiu, DJ, ZLi, and XH provided the resources. ZLiu and QD analyzed the data. ZLiu, YZ, and KW prepared the original draft and reviewed and edited the manuscript. KW supervised the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Histone modifications (HMs) contribute to maintaining genomic stability, transcription, DNA repair, and modulating chromatin in cancer cells. Furthermore, HMs are dynamic and reversible processes that involve interactions between numerous enzymes and molecular components. Aberrant HMs are strongly associated with tumorigenesis and progression of breast cancer (BC), although the specific mechanisms are not completely understood. Moreover, there is no comprehensive overview of abnormal HMs in BC, and BC therapies that target HMs are still in their infancy. Therefore, this review summarizes the existing evidence regarding HMs that are involved in BC and the potential mechanisms that are related to aberrant HMs. Moreover, this review examines the currently available agents and approved drugs that have been tested in pre-clinical and clinical studies to evaluate their effects on HMs. Finally, this review covers the barriers to the clinical application of therapies that target HMs, and possible strategies that could help overcome these barriers and accelerate the use of these therapies to cure patients. Breast cancer (BC) has the highest incidence and mortality rate among cancer cases in women . The devWe summarize the existing evidence regarding HMs and their influence on BC progression. Furthermore, we review research done on enzymes involved in HMs and the potential regulation of these enzymes. Finally, we focus on existing and emerging drugs that target HMs and related enzymes, potential barriers to their clinical application, and possible strategies to help overcome those barriers.in situ (DCIS) and invasive ductal carcinoma (IDC) than in normal mammary epithelium  were evaluated in 880 BC specimens, and expressions of all seven markers were negatively correlated with tumor grade. Moreover, high levels of H4R3me2 and H3K9ac were detected at lower lymph node stages, and H4R3me2, H3K9ac, and H4K16ac are lower in large tumor sizes. More importantly, low or missing H4K16ac was detected in most specimens, implying that this change represented an early event in BC, which may be used for BC diagnosis. In addition, high levels of these markers in various BC subtypes were associated with a better prognosis, and were almost exclusively detected in luminal BC. In contrast, low levels of these markers were observed in triple-negative breast cancer (TNBC) and HER2-positive BC, which have a poorer prognosis . There iithelium . In BC, ithelium . Upon DNithelium . In BC, ithelium . In TNBCithelium , 2015. IHistone acetylation is characterized by addition of an acetyl group to the lysine residues of histone tails . This moBIRC3 , which increases H3K4me3 and histone acetylation, activates transcription, suppresses apoptosis, and potentiates the progression of BC  to the BRCA1 promoter, which is accompanied by loss of HDAC1, increased histone acetylation, and thus, increased BRCA1 transcription , which contributes to an aggressive biological behavior (The class III HDACs consist of sirtuins (SIRT1–7), with SIRT1 predominantly located in the nucleus, although it can shuttle between the nucleus and cytoplasm . In BC, d GATA-5 . In contr growth . The prer growth , 2018. Fbehavior . SIRT3-5WNT5A promoter and enhancer regions. Activation of WNT5A causes tumorigenicity, enhanced invasion, and CSC-like properties (BRCA1 and RAD51). Pharmacological inhibition of BRD4 impairs the ability of TNBC cells to manage DNA damage after exposure to platinum salts, which leads to massive cell death and synthetic lethality when the platinum salts are combined with poly ADP ribose polymerase (PARP) inhibitors (PARPi)  . Interes (PARPi) . The oncHistone methylation occurs on lysine and arginine residues, where it involves a more sophisticated set of modifications in contrast to acetylation. Lysine can be mono-, di-, or trimethylated, while arginine can be symmetrically or asymmetrically methylated . HistoneHistone methyltransferases or “writers” are generally divided in three groups. The first group of methyltransferases have the SET domain  and consists of lysine methyltransferases (KMTs) with the exception of DOT1L (KMT4) . The secThe KMTs include seven major families (KMT1–7) that alter HMs to activate or inactivate oncogenes and TSGs to either slow down or accelerate BC progression .TFF1 (an estrogen-dependent gene) via H3K4me1/2 at CpG islands of the enhancer region, which maintains a permissive chromatin structure for binding ERα and its pioneer factor (FOXA1). This process leads to chromatin relaxation to facilitate the binding of ERα and transcriptional activity in BC  . Increasty in BC . Moreovesistance . Finallysistance .HK2, G6PD, and TIGAR) and alters metabolism to enhance the pentose phosphate pathway. This ultimately promotes NADPH production and decreases the levels of reactive oxygen species (ROS)  , NSD1–3 , SETD2 (KMT3A), and SMYD2 (KMT3C) . NSD2 ises (ROS) . Moreovehibitor) . Other Hhibitor) .SNAIL, ZEB1, ZEB2) and the BCAT1, which enhances cell migration, sphere formation, and the EMT , whereas there are no reported enzymes that can induce H3K79 demethylation . In BC,  the EMT . Further the EMT .BRCA1-mutated BC, EHMT1 (KMT1D) is up-regulated, leading to H3K9 methylation and decrease in GCN5-mediated H3K9ac, which synergistically promote the inhibition of phosphatidylethanolamine N-methyltransferase to potentiate BC tumorigenesis , which generates H3K27me3 via its SET domain and contributes to transcriptional suppression. The PRC2 core complex consists of EED, SUZ12, NURF55, Rbap46/48, and two catalytic constituents: EZH2 (KMT6A) and EZH1 (KMT6B) . RelativParadoxically, EZH2 can be negatively correlated with H3K27me3 in BC, and high EZH2 with low H3K27me3 may predict poor outcomes even in ER-positive BC . This maIn humans, the PRDM family contains 17 members, which typically share a common structure that involves an N-terminal PR domain. This domain has similar structure and function to those of the SET domain, although its methyltransferase activity is restricted to lysine residues . ExistinThe PRMT family contains nine members that are divided into three types: type I , type II (PRMT5 and PRMT9), and type III (PRMT7). Type III PRMTs catalyze the formation of a mono-methylated intermediate, type I PRMTs catalyze the generation of asymmetric dimethylarginine, and type II PRMTs catalyze the formation of symmetric dimethylarginine. PRMTs regulate a myriad of biology process, including signal transduction, gene transcription, DNA repair, and mRNA splicing . Further2+/oxoglutarate-dependent enzymes that contain a JumonjiC (JmjC) domain  . However) domain . H3K4me1) domain . LSD1 is) domain . Neverth) domain . In TNBCblockade . Inhibitblockade . Moreoveblockade .Relative to LSD1, there are fewer studies on the role of LSD2 in BC, although LSD2 expression is significantly elevated in BC tissues . FurtherHistone H3K36 demethylation is catalyzed by KDM2 (H3K36me1/2), KDM4 (H3K36me2/3), and JMJD5 as the only member of the KDM8 family (H3K36me2/3) . In BC, MYC, PAX3, Cyclin D1, MMP-9, S100A4, and JUN , JMJD3 (KDM6B), and UTY (KDM6C). The UTX and JMJD3 members act on H3K27me2/3, whereas UTY has weak demethylase activity toward H3K27me3. These demethylation activities reduce the repression of oncogenes and TSGs . In BC, nd DICER . In addiand ZEB2 . Transacand ZEB2 .SNAIL1 transcription to facilitate the EMT  expression is induced by TGF-β, and epigenetically activates  the EMT . In thisreatment .HIC1, KISS1, and BRCA1 , and inhibit the Wnt/β-Catenin signaling pathway to inhibit invasion, metastasis and EMT  and involved in transcriptional inhibition, chromatin compaction, and DNA damage repair, while it is removed by BAP1 and USP16 deubiquitinase . BRCA1 pigration . In HER2igration . The deuigration . The PHD and EMT . Ubiquit and EMT .Histone citrullination/deimination is characterized as a conversion of peptidyl-arginine to peptidyl-citrulline mediated by peptidyl arginine deiminase (PAD) enzymes . AlthougHistone O-GlcNAcylation is dynamically regulated by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA) to add or remove O-GlcNAc to Ser or Thr. This modification is involved in mitosis, chromatin dynamics, and gene expression by crosstalk with other HMs . In BC, Sumoylation is characterized by the covalent attachment of a small ubiquitin-like modifier (SUMO) to lysine resides in histone tails which results in transcriptional silencing. It is catalyzed by four isoforms SUMO named SUMO-1, -2, -3, -4 and is reversed by sentrin-specific proteases . NeddylaHistone biotinylation is a rare modification of lysine residues that is catalyzed by biotinidase and holocarboxylase synthetase . BiotinyHistone can be mono- and/or poly-ADP-ribosylated by PARPs, which hydrolyze nicotinamide adenine dinucleotide and transfer ADP-ribose to lys/glu/asp residues . Histonecis and trans, which are regulated by prolyl cis-trans isomerization. This process is catalyzed by peptidyl-prolyl cis-trans isomerases. Proline isomerization has been extensively studied in non-histones, but rarely in histones. Proline isomerase Fpr4 is identified to catalyze isomerization of H3 proline P30 and P38 in yeast. The isomerization of H3P38 inhibits the SET2 methyltransferase activity of catalyzing H3K36me3, and contributes to enhanced transcription of target genes  prevent BC progression by restoring the aberrantly activated oncogenes or suppressed TSGs. Furthermore, epi-drugs help overcome drug resistance, enhance the effectiveness of other anti-cancer drugs, and improve radiotherapy effect . Many epin vitro experiments to in vivo experiments. The current inhibitors are also limited by their poor selectivity and low efficiency, although they may be useful starting points for developing new inhibitors.A few inhibitors targeting HATs have been studied but none have entered clinical trials. The currently available HATis include bi-substrate inhibitors, small molecule HATis , and library screened inhibitors . Bi-subsZEB1/2  is a cyclic peptide HDACi that is approved by the FDA. A combination of romidepsin and decitabine (a methyltransferase inhibitor) exerts synergistic inhibition of cell growth and induction of apoptosis . In inflSurvivin in a HDAC2-dependent manner  is the most widely studied aliphatic fatty acid HDACi, which can inhibit BC development by promoting cell cycle arrest and activating programmed cell death pathways . This drt manner . However unclear . Moreove unclear , epirubi unclear , cisplat unclear , camptot unclear , capecit unclear , and hyd unclear , which uEntinostat  is a synthetic benzamide derivative HDACi that has a strong immunomodulatory effect in BC . MoreoveSirtuin inhibitors can prevent BC progression via different targets, functions, and structures ; furtherin vivo and in vitro models (Inhibitors targeting the BRDs of histone acetylation readers have mainly been developed against the BET family of BRD-containing proteins. The currently available BET inhibitors (BETis), their use for treating BC, and efficacy data are shown in o models . Cells fo models . Anothero models , dBET6 , which include tazemetostat (EPZ-6438), CPI-0209, CPI-1205, GSK2816126, PF-06821497, and DS-3021. All of these inhibitors have been examined in clinical trials, although their roles in BC are not clear. In BC, EZH2is permit expression of TSGs, such as FOSB, FOXC1, RUNX3, CDKN1C, CHD1, and TET1 that had been silenced by EZH2-mediated H3K27me3 in the canonical role. Expression of these genes leads to suppressed proliferation, less invasion, and greater response to adriamycin-based treatment , DZNep and TSA can further enhance the level of CDKN1C which may be due to AZA-induced reduction of H3K9me2 in a DNA methylation independent manner  as the methyl donor. In 2007, Tan et al. first discovered that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) caused the degradation of PRC2 components: SUZ12, EZH2, and EED which was accompanied by a decreased in H3K27me3. Moreover, they showed the anti-cancer function of DZNep in inducing cell death in BC cells by re-expressing PRC2-repressed genes, such as reatment . In addiand MMP7 . Therefoand MMP7 . Relativto DZNep . Moreovet tumors . Howevert manner . More imt manner . In HER2t manner . Furthert manner . Numerout manner . Howeverin vivo experiments and clinical studies are needed to determine whether these drugs have clinical values.Binding modes can be used to divide G9a inhibitors into three groups. The first group contains substrate competitive inhibitors . The second group includes SAM cofactor competitive inhibitors . The third group includes inhibitors with unknown mechanism of action . As a substrate competitive inhibitor, BIX-01249 induces autophagy and increases intracellular ROS concentrations, which sensitizes BC cells to TRAIL by upregulating the expression of the ATF4/CHOP-dependent death receptor (DR5) , 2018. Ain vitro. The allosteric inhibitors may be more promising, which inactivate PRMTs by altering their conformation and show better selectivity. Moreover, different allosteric inhibitors can be designed for the same enzyme based on diverse binding sites. Therefore, their roles in BC should be further studied.Protein arginine methyltransferase inhibitors (PRMTis) include arginine competitive inhibitors , SAM cofactor competitive inhibitors , bi-substrate competitive inhibitors (SKI-72 and SGC8158), and allosteric inhibitors  . In 2004E-cadherin and other epithelial marker via disruption of the interaction between LSD1 and Slug (The most widely studied lysine demethylase inhibitors (KDMis) are LSD1is, which can be classified as irreversible or reversible inhibitors . Irreverand Slug . In TNBCand Slug . Compareand Slug . Many naand Slug , cryptotand Slug , flavoneand Slug ,b, resveand Slug , baicaliand Slug , and gerand Slug . IsoquerAnother group of KDMis contains JmjC inhibitors, which are mainly 2-oxoglutarate competitors and include hydroxamic acid derivatives, hydroxyquinoline derivatives, metal-chelating inhibitors, conjugated arylalkenes, cyclic peptides, metal-containing inhibitors, and inhibitors targeting JmjC-KDM non-catalytic domains. In BC, these inhibitors can kill cancer cells and exert synergistic effects with other anti-cancer drugs, such as trastuzumab , lapatinNANOG, SOX2, and KLF4. However, pharmacological inhibition of KDM6A by GSKJ4, a H3K27me3 demethylase inhibitor toward JMJD3 and UTX, overcomes the paclitaxel-induced BC stem cell enrichment  display synergetic action with AIs tamoxifen or letrozole in ER-positive tamoxifen-sensitive BC cells and overcome resistance in tamoxifen-resistant cells. Importantly, VPA can antagonize the drawback of tamoxifen in promoting proliferation of uterine endometrial cells . Howevernografts . Moreovenografts . A phasenografts .In HER2-positive BC, trastuzumab alone or in combination with chemotherapy are effective treatments; however, resistance is common. Treatment using Ent can overcome both trastuzumab and lapatinib resistance, and a clinical trial revealed that polytherapy using Ent, trastuzumab, and lapatinib was safe and effective . Both VPBRCA-mutated or PTEN loss in TNBC cells. However, the antagonism mediated by DNA repair-associated enzymes leads to resistance to PARPis, which calls for new strategies. Combination of SAHA or BETis  with PARPi (olaparib) shows synergistic effects. Interestingly, PTEN turned out to be a favorable factor to the combination of SAHA with olaparib in TNBC which promoted both apoptosis and autophagy to inhibit proliferation and accumulated DNA damage, while downregulation of PTEN was unfavorable  have been recognized as novel tools for BC treatment. However, the response to ICIs is poor because tumors recruit myeloid-derived suppressor cells to inhibit activation and infiltration of T-cells. Addition of Ent to anti-PD-1 or anti-CTLA-4 reversed the resistance by improving the infiltration and function of immune cells. This combination significantly reduced the inhibition functions of myeloid-derived suppressor cells and increased CD8 T cells .LIFR and activation of downstream JAK1/STAT3 pathway  pre-treatment and ionizing radiation induced increased γH2AX expression in MCF7 BC cells, while caused reduction in MCF10A cells . FurtherDue to the complexity of crosstalk among HMs and the synergistic anti-cancer effect of available drugs, more potent dual-target inhibitors have been developed. For example, MC3324  up-regulates H3K4me2 and H3K27me3, which caused growth arrest and apoptosis in both hormone-responsive and insensitive BC cells by inhibiting ERα signaling . A dual-https://www.clinicaltrials.gov/) in The promising preclinical results provide a solid foundation for translating epi-drugs into clinical trials for BC treatment. We have summarized the clinical trials of epi-drugs in treating BC  and dose-limiting toxicities (DLTs). AEs refer to side effects that occur during treatment and can be classified as dose-related toxicities and other problems. In an early phase II trial of SAHA monotherapy in BC, colorectal, and non-small cell lung cancer, drug-related AEs are similar to those of traditional chemotherapy, including anorexia, fatigue, nausea, diarrhea, vomiting, thrombocytopenia, weight loss, asthenia, anemia, constipation, stomatitis, cancer pain, dry mouth, dyspepsia, upper abdominal pain, and vertigo. Grade 3/4 AEs included thrombocytopenia, anemia, asthenia, and nausea . RelativSubstantial proteomic and genomic analyses have revealed that BC involves numerous molecular alterations. Although specific genetic alterations are known to drive BC, epigenetic pathways also play important roles in its oncogenesis. Furthermore, alterations in HMs may be used as biomarkers for predicting BC aggressiveness and the effectiveness of therapies that target epigenetic mechanisms. Additional research is needed to confirm whether correlations between early epigenetic changes and clinical characteristics can guide strategies to achieve better clinical outcomes.Unfortunately, there are numerous barriers to clarifying the roles and mechanisms of HMs in BC. First, HMs are dynamic processes that occur within and between nucleosomes . Second,Reducing the AEs of epi-drugs is a challenging issue in the clinical setting. Treatment-related AEs and DLTs are generally due to various absorptivity, penetrability, half-life, properties, and the complex interactions between different drugs in the human metabolism. Carefully designed combinations of epi-drugs and traditional anti-cancer drugs or synthesized multi-target drugs may produce better therapeutic effects, with greater ability to overcome drug resistance and fewer AEs . RelativIn conclusion, there is still substantial work needed to clarify the mechanisms of HMs and exploit these mechanisms to develop appropriate drugs and treatment regimens. Epi-drugs have achieved remarkable effects in terms of precise and effective personalized treatment for BC. Thus, it is reasonable to hope that more precise, specific, and effective drugs will be developed to target HMs as a strategy for treating BC.DP and SX conceived the idea and designed the study. WL was the first author, retrieved and analyzed the data and drafted the manuscript. HW, SS, and QW revised and polished the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.