Enhancer of zeste homolog 2 inhibitors (EZH2i) have garnered increased attention owing to their anticancer activity by targeting EZH2, a well-known cancer-promoting factor. However, some lymphomas are resistant to EZH2i, and EZH2i treatment alone is ineffective in case of EZH2-overexpressing solid tumors. The anti-cancer efficacy of EZH2i may be improved through safe and effective combinations of these drugs with other treatment modalities. Preclinical evidence indicates that combining EZH2i with other therapies, such as immunotherapy, chemotherapy, targeted therapy, and endocrine therapy, has complementary or synergistic antitumor effects. Therefore, elucidating the underlying mechanisms of the individual constituents of the combination therapies is fundamental for their clinical application. In this review, we have summarized notable clinical trials and preclinical studies using EZH2i, their progress, and combinations of EZH2i with different therapeutic modalities, aiming to provide new insights for tumor treatment.

EZH2 inhibitors (EZH2i), a class of small-molecule inhibitors that target EZH2 to exert anti-tumor functions, have just been approved by the US Food and Drug Administration (FDA) in treatment of adults and adolescents with locally advanced or metastatic epithelioid sarcoma. The application of EZH2i in several solid tumors is still in different stages of clinical trials and needs to be further validated. As a key epigenetic regulator, besides its role in controlling the proliferation of tumor cells, EZH2 has been implicated in the regulation of various immune cells including macrophages. But there are still controversial research results at present. Colorectal cancer (CRC) is a common malignant tumor that highly expresses EZH2, which has the third highest incidence and is the second leading cause of cancer-related death worldwide. Studies have shown that the numbers of M2-type tumor-associated macrophages (TAMs) are highly associated with the progression and metastasis of CRC. In the current study, we aim to investigate how EZH2 modulates the polarization of macrophages in the tumor microenvironment (TME) of CRC, and compare the role of two different EZH2 inhibitors, EPZ6438 and GSK126. We applied a 3D culture method to demonstrate that EZH2i did indeed suppress the proliferation of CRC cells in vitro. In vivo, we found that the percentage of CD206⁺ macrophages of the TME was decreased under the treatment of EPZ6438, but it increased upon GSK126 treatment. Besides, in the co-culture system of macrophages and CRC cells, EPZ6438 led to significant elevation of M1 markers and reduction of M2 markers. Furthermore, mechanistic studies validated by ChIP-qPCR demonstrated that EZH2i inhibit EZH2-mediated H3K27me3 levels on the promoters of STAT3, an essential transcription factor for M1 macrophage polarization. Therefore, our data suggested that EZH2i not only suppress CRC cell proliferation directly, but also regulate macrophage by skewing M2 into effector M1 macrophage to exert a tumor suppressive effect. Moreover, our study provided new insight for better understanding of the role of two kinds of EZH2i: EPZ6438 and GSK126, which may pave the way in treating CRC by targeting cancer cells and immune cells via this epigenetic approach in the future.

Sensitivity has been a key issue for Enhancer of zeste homolog 2 (EZH2) inhibitors in cancer therapy. The EZH2 inhibitor EPZ-6438 was first approved by the US Food and Drug Administration (FDA) in 2020. However, its inadequate anti-cancer activity in solid tumors limits its clinical application. In this study, we utilized the multiple cancer cell lines, which are less sensitive to the EZH2 inhibitor GSK126, combining animal model and clinical data to investigate the underlying mechanism. IncuCyte S3 was used to explore the difference in the responsiveness of hematological tumor cells and solid tumor cells to GSK126. Transcriptome and metabolome of B16F10 cells after GSK126 treatment were analyzed and the distinct changes in the metabolic profile were revealed. Real-time quantitative PCR and western blot experiments were used to further verify the multi-omics data. ChIP-qPCR was performed to detected H3K27me3 enrichment of target genes. Finally, the anti-tumor effects of combining GSK126 and lipid metabolism drugs were observed with IncuCyte S3 platform, CCK-8 and animal model respectively. We found that although the proliferative phenotype did not show strong difference upon treatment with GSK126, the transcriptome and metabolome changed profoundly. GSK126 treatment led to broad shifts in glucose, amino acid, and lipid metabolism. Lipid synthesis was strengthened manifested by the increasing abundance of unsaturated fatty acids. SCD1 and ELOVL2 were regulated by H3K27me3 at gene regulatory region, and upregulated by EZH2 knockdown and inhibitors. SCD1 knockdown increased cellular sensitivity to GSK126. Based on the findings above, the application of the combination with SCD1 inhibitor significantly attenuated the proliferation of cancer and increased the sensitivity to GSK126 by suppressing desaturation of fatty acids. Dysregulated lipid metabolism can blunt the sensitivity of cancer cells to GSK126. These characteristics shed light on the novel combination therapy strategies to combat tumor resistance. National Natural Science Foundation of China (No. 81672091, No.91749107 and No. 81972966).

Rationale: Stroke is a leading causes of human death worldwide. Ischemic damage induces the sterile neuroinflammation, which directly determines the recovery of patients. Lipids, a major component of the brain, significantly altered after stroke. Cholesterol sulfate, a naturally occurring analog of cholesterol, can directly regulate immune cell activation, indicating the possible involvement of cholesterol metabolites in neuroinflammation. Sulfotransferase family 2b member 1 (Sult2b1) is the key enzyme that catalyzes the synthesis of cholesterol sulfate. This study aimed to investigate the function of Sult2b1 and cholesterol sulfate in the neuroinflammation after ischemic stroke.

Rationale: Ischemic stroke poses a significant health burden with limited treatment options. Lymphocyte Cytosolic Protein 1 (LCP1) facilitates cell migration and immune responses by aiding in actin polymerization, cytoskeletal rearrangements, and phagocytosis. We have demonstrated that the long non-coding RNA (lncRNA) Maclpil silencing in monocyte-derived macrophages (MoDMs) led to LCP1 inhibition, reducing ischemic brain damage. However, the role of LCP1 of MoDMs in ischemic stroke remains unknown.

Stroke induces a robust inflammatory response. However, it still lacks a systematic view of the various immune cell types due to the limited numbers of fluorophore used in the traditional FACS technique. In our current study, we utilized the novel technique mass cytometry (CyTOF) to analyze multiple immune cell types. We detected these immune cells from the ischemic brain, peripheral blood, spleen, and bone marrow at different time courses after stroke. Our data showed (1) dynamic changes in the immune cell numbers in the ischemic brain and peripheral organs. (2) The expression levels of cell surface markers indicate the inflammation response status after stroke. Interestingly, CD62L, a key adhesion molecule, regulates the migration of leukocytes from blood vessels into secondary lymphoid tissues and peripheral tissues. (3) A strong leukocyte network across the brain and peripheral immune organs was identified using the R program at day 1 after ischemia, suggesting that the peripheral immune cells dramatically migrated into the ischemic areas after stroke. This study provides a systematic, wide view of the immune components in the brain and peripheral organs for a deep understanding of the immune response after ischemic stroke.