IDO1 inhibitor enhances the effectiveness of PD-1 blockade in microsatellite stable colorectal cancer by promoting macrophage pro-inflammatory phenotype polarization.

Microsatellite stable (MSS) colorectal cancer (CRC) is a subtype of CRC that generally exhibits resistance to immunotherapy, particularly immune checkpoint inhibitors such as PD-1 blockade. This study investigates the effects and underlying mechanisms of combining PD-1 blockade with IDO1 inhibition in MSS CRC. Bioinformatics analyses of TCGA-COAD and TCGA-READ cohorts revealed significantly elevated IDO1 expression in CRC tumors, correlating with tumor mutation burden across TCGA datasets. In vivo experiments demonstrated that the combination of IDO1 inhibition and PD-1 blockade significantly reduced tumor growth and increased immune cell infiltration, particularly pro-inflammatory macrophages and CD8+ T cells. IDO1 knockdown in CRC cell lines impaired tolerance to interferon-γ and increased apoptosis in vitro, which were rescued by the application of kynurenine, the end product of IDO1. IDO1 knockdown in MSS CRC enhanced the effectiveness of PD-1 blockade therapy in vivo. IDO1 knockdown cancer cells promoted pro-inflammatory macrophage polarization and enhanced phagocytic activity in vitro, associated with the upregulation of JAK2-STAT3-IL6 signaling pathway. These findings highlight the role of IDO1 in modulating the tumor immune microenvironment in MSS CRC and suggest that combining PD-1 blockade with IDO1 inhibition could enhance therapeutic efficacy by promoting macrophage pro-inflammatory polarization and infiltration through the JAK2-STAT3-IL6 pathway.

Guangzhao L ，Xin W ，Miaoqing W ，Wenjuan M ，Ranyi L ，Zhizhong P ，Rongxin Z ，Gong C ... -  《-》

HDAC and MEK inhibition synergistically suppresses HOXC6 and enhances PD-1 blockade efficacy in BRAF(V600E)-mutant microsatellite stable colorectal cancer.

B-Raf proto-oncogene, serine/threonine kinase (BRAF)V600E-mutant microsatellite stable (MSS) colorectal cancer (CRC) constitutes a distinct CRC subgroup, traditionally perceived as minimally responsive to standard therapies. Recent clinical attempts, such as BRAF inhibitors (BRAFi) monotherapy and combining BRAFi with other inhibitors, have yielded unsatisfactory efficacy. This study aims to identify a novel therapeutic strategy for this challenging subgroup. We first performed a large-scale drug screening using patient-derived organoid models and cell lines to pinpoint potential therapies. Subsequently, we investigated the synergistic effects of identified effective inhibitors and probed their cooperative mechanisms. Concurrently, we explored the immune characteristics of BRAFV600E MSS CRC using RNA sequencing and multiplex immunohistochemistry. Finally, we established a CT26 BRAFV637E mouse cell line and validated the efficacy of combining these inhibitors and programmed death 1 (PD-1) blockades in immunocompetent mice. Drug screening identified histone deacetylase (HDAC) inhibitor and mitogen-activated protein kinase kinase (MEK) inhibitor as significantly effective against BRAFV600E MSS CRC. Further research revealed that these two inhibitors have superior synergistic effects by comprehensively inhibiting the activation of the epidermal growth factor receptor, mitogen-activated protein kinase, and phosphoinositide 3-kinase-protein kinase B pathways and suppressing the key target homeobox C6 (HOXC6). HOXC6, overexpressed in BRAFV600E MSS CRC, regulates the MYC gene and contributes to treatment resistance, tumor growth, and metastasis. Moreover, the combination therapy demonstrated the ability to enhance antitumor immunity by synergistically upregulating the expression of immune activation-related genes, activating the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon genes (cGAS/STING) pathway, and diminishing the tumor cells' DNA mismatch repair capacity. Notably, BRAFV600E MSS CRC was identified to exhibit a distinct immune microenvironment with increased PD-1+ cell infiltration and potential responsiveness to immunotherapy. Echoing the above findings, in vivo, HDAC and MEK inhibitors significantly improved PD-1 blockade efficacy, accompanied by increased CD8+ T-cell infiltration. Our findings indicate that combining HDAC inhibitor, MEK inhibitor, and PD-1 blockade is a potential strategy for treating BRAFV600E-mutant MSS CRC, warranting further investigation in clinical settings.

Sun Z ，Shi M ，Xia J ，Li X ，Chen N ，Wang H ，Gao Z ，Jia J ，Yang P ，Ji D ，Gu J ... -  《Journal for ImmunoTherapy of Cancer》

HDACi combination therapy with IDO1i remodels the tumor microenvironment and boosts antitumor efficacy in colorectal cancer with microsatellite stability.

Immunotherapy for colorectal cancer (CRC) with microsatellite stability (MSS) and mismatch repair proficiency (pMMR) has shown limited success in clinical trials. The combination of immunomodulators and immune checkpoint inhibitors (ICIs) is a potential strategy for treating CRC. Histone deacetylase (HDAC) and indoleamine 2,3-dioxygenase 1 (IDO1) expression in CRC tissues and adjacent normal tissues was analyzed via database analysis, immunohistochemistry, and western blotting. A nanodrug designated as NP-I/P was subsequently formulated, encapsulating an IDO1 inhibitor (IDO1i; namely, epacadostat) and an immunomodulatory HDAC inhibitor (HDACi; namely, panobinostat). The antitumor efficacy of the nanoparticles and their effects on tumor microenvironment features were evaluated via in vitro and in vivo experiments. In the present study, we found that HDAC overexpression and IDO1 expression were attenuated in MSS/pMMR CRC. Thus, a nanodrug designated as NP-I/P was formulated to encapsulate epacadostat and panobinostat. In vitro, NP-I/P treatment promoted the apoptosis of tumor cells and induced the release of damage-associated molecular patterns, thereby leading to cell death-associated immune activation. The in vivo results revealed that NP-I/P treatment reversed the immunosuppressive phenotype of the microenvironment by inducing tumor immunogenic cell death (ICD), promoting CD8+ T cell infiltration, and reducing the numbers of Tregs, tumor-associated macrophages, and myeloid-derived suppressor cells. Finally, the results of the patient-derived xenograft and patient-derived organoid models demonstrated that NP-I/P treatment triggered tumor cell death and modulated the immune microenvironment in human CRC. The combination of IDO1 and HDAC inhibitors represents a promising strategy for CRC treatment, and NP-I/P is a candidate for clinical trials.

Liang R ，Ding D ，Li Y ，Lan T ，Ryabtseva S ，Huang S ，Ren J ，Huang H ，Wei B ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Icariin promoted ferroptosis by activating mitochondrial dysfunction to inhibit colorectal cancer and synergistically enhanced the efficacy of PD-1 inhibitors.

A controlled type of cell death called ferroptosis is linked to increased reactive oxygen species (ROS), lipid peroxidation, and iron buildup. Furthermore, evidence indicates that ferroptosis may act as an immunogenic form of cell death with potential physiological functions in tumors and immunosuppression. Inducing ferroptosis in tumor cells may have the potential to complement cancer immunotherapy strategies. The development of colorectal cancer (CRC) and the poor efficacy of immunotherapy are associated with the crosstalk of cellular ferroptosis. Currently, Icariin (ICA), the main bioactive component extracted from Epimedium, has been shown to inhibit a variety of cancers. However, the specific role and potential mechanism of ICA in regulating ferroptosis in CRC remains unclear. The aim of this investigation was to clarify the mechanism underlying the anti-CRC cancer properties of ICA and how it induces ferroptosis to enhance immunotherapy. To evaluate cell viability, the Cell Counting Kit-8 (CCK-8) test was utilized. The transwell test and the wound healing assay were used to assess cell migration. A subcutaneous graft tumor model was constructed with C57BL/6 mice using MC38 colorectal cancer cell lines. The inhibitory effect of ICA on CRC, ferroptosis level and immunomodulatory effects were detected by serum biochemical assay, cytokine assay, hematoxylin-eosin (H&E) staining, immunofluorescence staining, CyTOF mass spectrometry flow screening and Western blotting. Western blotting, proteomics, molecular docking and microscale thermophoresis (MST) were used to forecast and confirm ICA's binding and interaction with HMGA2, STAT3, and HIF-1α. Moreover, the levels of lipid peroxidation and ferroptosis were assessed through the use of the C11-BODIPY fluorescent probe, the FerroOrange fluorescent probe, the iron level, the malondialdehyde (MDA) and reduced glutathione (GSH) assay kit, and Western blotting analysis. To assess alterations in mitochondrial structure and membrane potential, transmission electron microscopy (TEM) and JC-1 immunofluorescence were employed. It was demonstrated in the current study that ICA treatment inhibits CRC and enhances anti-PD-1 therapy efficacy by inciting ferroptosis. As shown in vitro, ICA inhibits CRC cell proliferation, migration, and apoptosis. As demonstrated in vivo, ICA has a dose-dependent tumor suppressor effect when combined with anti-PD-1, it can significantly inhibit tumor growth, increase the expression of serum TNF-α, IFN-γ, and granzyme B, and promote CD69+CD8+ T, CD69+CD8+Tem, CD69+CD8+Teff, TCRβ+CD8+ T, TCRβ+CD8+ T, TCRβ+CD8+Tem, TCRβ+CD8+Teff. The inhibitory effect of ICA on CRC was associated with the binding of HMGA2, STAT3, and HIF-1α proteins, which inhibited CRC by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), promoting the accumulation of iron (Fe2+), depletion of reduced glutathione (GSH), inhibiting SLC7A11 and GPX4 expressions, thereby inducing ferroptosis in CRC. As a consequence of ICA-induced ferroptosis, mitochondria are dysfunctional, with increased ROS production, membrane potential depolarization (MMP), and ATP production reduced. This process can be efficiently reversed by the mitochondria-targeted antioxidant Mito-Q. It is noteworthy that the ferroptosis inhibitor liproxstatin-1 (lip-1), anti-CD8, and anti-IFN-γ exhibited a significant inhibitory effect on the level of ferroptosis and antitumor capacity of ICA combined with anti-PD-1. This finding suggests that the antitumor immunopotentiating effect of ICA on anti-PD-1 is dependent on the secretion of IFN-γ-induced ferroptosis of CRC cells by the CD8+ T cell. Our study represents the inaugural demonstration of the mechanism whereby ICA exerts anti-CRC effects and synergistically enhances the efficacy of anti-PD-1, inducing mitochondrial damage and leading to ferroptosis. ICA promotes ferroptosis of CRC cells by inducing mitochondrial dysfunction, and ICA combined with anti-PD-1 significantly promotes CD69, TCRβ signalling, activates effector CD8+ T cells to secrete IFN-γ, and achieves immunopotentiation by promoting ferroptosis of CRC cells, thus inhibiting CRC development. This study is built upon existing research into the pharmacodynamic mechanisms of ICA in the context of CRC, and offers a novel therapeutic approach in addressing the issue of CRC immunotherapy potentiation.

Haoyue W ，Kexiang S ，Shan TW ，Jiamin G ，Luyun Y ，Junkai W ，Wanli D ... -  《-》

Euphorbia Pekinensis Rupr. sensitizes colorectal cancer to PD-1 blockade by remodeling the tumor microenvironment and enhancing peripheral immunity.

Immune checkpoint blockade, such as monoclonal antibodies targeting programmed cell death protein 1 (PD-1), has been a major breakthrough in the treatment of several cancers, but has limited effect in colorectal cancer (CRC), which is a highly prevalent cancer worldwide. Current chemotherapy-based strategies to boost PD-1 response have many limitations. And the role of peripheral immunity in boosting PD-1 response continues to attract attention. Therefore, candidate combinations of PD-1 blockade need to be drugs with multi-targets and multi-modulatory functions. However, it is still unknown whether traditional Chinese medicines with such property can enhance the applicability and efficacy of PD-1 blockade in colorectal cancer. Euphorbia Pekinensis extract (EP) was prepared and the constituents were analyzed by HPLC. CRC cells were used for in vitro experiments, including cell viability assay, colony formation assay, flow cytometry for 7-AAD staining, western blotting for caspase 3 and caspase 7, HMGB1 and ATP detection. An orthotopic CT26 mouse model was subsequently used to investigate the combination of EP and PD-1 blockade therapy. Tumor volume and tumor weight were assessed, tumor tissues were subjected to histopathological HE staining and TUNEL staining, and tumor-infiltrating immune cells were evaluated by immunofluorescence staining. RNA-sequencing, target prediction and pathway analysis were further employed to explore the mechanism. Molecular docking and cellular thermal shift assay (CETSA) were utilized to verify the direct target of the core component of EP. And, loss-of-function analysis was carried to confirm the upstream-downstream relationship. Flow cytometry was employed to analyze CD8+ T cells in the peripheral blood and spleen. The main constituents of EP are diterpenoids and flavonoids. EP dramatically suppresses CRC cell growth and exerts its cytotoxic effect by triggering immunogenic cell death in vitro. Moreover, EP synergizes with PD-1 blockade to inhibit tumorigenesis in tumor-bearing mice. Disruption of ISX nuclear localization by helioscopinolide E is a central mechanism of EP-induced apoptosis in CRC cell. Meanwhile, EP activates immune response by upregulating Phox2b to reshape the immune microenvironment. In addition, EP regulates peripheral immunity by regulating the T cell activation and proliferation, and the ratio of CD8+ T cells in peripheral blood is drastically increased, thereby enhancing the therapeutic efficacy of anti-PD1 immunotherapy. EP triggers intra-tumor immunogenic cell death and modulates the immunoregulatory signaling to elicit the tumor immunogenicity. Moreover, EP participates in transcriptional activation of immune response-related pathways. Consequently, multiple stimulating functions of EP on macro- and micro-immune potentiates the anti-tumor effect of PD-1 blockade in CRC.

Chen YY ，Zeng XT ，Gong ZC ，Zhang MM ，Wang KQ ，Tang YP ，Huang ZH ... -  《-》