CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma.

Rationale: The large-scale genomic analysis classifies glioblastoma (GBM) into three major subtypes, including classical (CL), proneural (PN), and mesenchymal (MES) subtypes. Each of these subtypes exhibits a varying degree of sensitivity to the temozolomide (TMZ) treatment, while the prognosis corresponds to the molecular and genetic characteristics of the tumor cell type. Tumors with MES features are predominantly characterized by the NF1 deletion/alteration, leading to sustained activation of the RAS and PI3K-AKT signaling pathways in GBM and tend to acquire drug resistance, resulting in the worst prognosis compared to other subtypes (PN and CL). Here, we used the CRISPR/Cas9 library screening technique to detect TMZ-related gene targets that might play roles in acquiring drug resistance, using overexpressed KRAS-G12C mutant GBM cell lines. The study identified a key therapeutic strategy to address the chemoresistance against the MES subtype of GBM. Methods: The CRISPR-Cas9 library screening was used to discover genes associated with TMZ resistance in the U87-KRAS (U87-MG which is overexpressed KRAS-G12C mutant) cells. The patient-derived GBM primary cell line TBD0220 was used for experimental validations in vivo and in vitro. Chromatin isolation by RNA purification (ChIRP) and chromatin immunoprecipitation (ChIP) assays were used to elucidate the silencing mechanism of tumor suppressor genes in the MES-GBM subtype. The small-molecule inhibitor EPIC-0412 was obtained through high-throughput screening. Transmission electron microscopy (TEM) was used to characterize the exosomes (Exos) secreted by GBM cells after TMZ treatment. Blood-derived Exos-based targeted delivery of siRNA, TMZ, and EPIC-0412 was optimized to tailor personalized therapy in vivo. Results: Using the genome-wide CRISPR-Cas9 library screening, we found that the ERBIN gene could be epigenetically regulated in the U87-KRAS cells. ERBIN overexpression inhibited the RAS signaling and downstream proliferation and invasion effects of GBM tumor cells. EPIC-0412 treatment inhibited tumor proliferation and EMT progression by upregulating the ERBIN expression both in vitro and in vivo. Genome-wide CRISPR-Cas9 screening also identified RASGRP1(Ras guanine nucleotide-releasing protein 1) and VPS28(Vacuolar protein sorting-associated protein 28) genes as synthetically lethal in response to TMZ treatment in the U87-KRAS cells. We found that RASGRP1 activated the RAS-mediated DDR pathway by promoting the RAS-GTP transformation. VPS28 promoted the Exos secretion and decreased intracellular TMZ concentration in GBM cells. The targeted Exos delivery system encapsulating drugs and siRNAs together showed a powerful therapeutic effect against GBM in vivo. Conclusions: We demonstrate a new mechanism by which ERBIN is epigenetically silenced by the RAS signaling in the MES subtype of GBM. Restoration of the ERBIN expression with EPIC-0412 significantly inhibits the RAS signaling downstream. RASGRP1 and VPS28 genes are associated with the promotion of TMZ resistance through RAS-GDP to RAS-GTP transformation and TMZ efflux, as well. A quadruple combination therapy based on a targeted Exos delivery system demonstrated significantly reduced tumor burden in vivo. Therefore, our study provides new insights and therapeutic approaches for regulating tumor progression and TMZ resistance in the MES-GBM subtype.

Retraction of "Universal theranostic CRISPR/Cas13a RNA-editing system for glioma".

[This retracts the article DOI: 10.7150/thno.84429.].

Stressing the Role of CCL3 in Reversing the Immunosuppressive Microenvironment in Gliomas.

Patients with gliomas often experience mental health problems, such as depression and anxiety, that lead to worsening tumor progression and shortened survival. In this issue, Wang and colleagues report a novel mechanism underlying this, finding that chronic stress reduces secretion of the chemokine CCL3, which leads to an immunosuppressive glioma microenvironment. CCL3 administration enhances the infiltration of antitumor immune cells, providing rationale for a potential new therapeutic approach. See related article by Wang et al., p. 516 (4).

Deep-targeted gene sequencing reveals ARID1A mutation as an important driver of glioblastoma.

AIMS: To investigate the key factors influencing glioma progression and the emergence of treatment resistance by examining the intrinsic connection between mutations in DNA damage and repair-related genes and the development of chemoresistance in gliomas. METHODS: We conducted a comprehensive analysis of deep-targeted gene sequencing data from 228 glioma samples. This involved identifying differentially mutated genes across various glioma grades, assessing their functions, and employing I-TASSER for homology modeling. We elucidated the functional changes induced by high-frequency site mutations in these genes and investigated their impact on glioma progression. RESULTS: The analysis of sequencing mutation results of deep targeted genes in integration revealed that ARID1A gene mutation occurs frequently in glioblastoma and alteration of ARID1A could affect the tolerance of glioma cells to temozolomide treatment. The deletion of proline at position 16 in the ARID1A protein affected the stability of binding of the SWI/SNF core subunit BRG1, which in turn affected the stability of the SWI/SNF complex and led to altered histone modifications in the CDKN1A promoter region, thereby affecting the biological activity of glioma cells, as inferred from modeling and protein interaction analysis. CONCLUSION: The ARID1A gene is a critical predictive biomarker for glioma. Mutations at the ARID1A locus alter the stability of the SWI/SNF complex, leading to changes in transcriptional regulation in glioma cells. This contributes to an increased malignant phenotype of GBM and plays a pivotal role in mediating chemoresistance.

HIF1α/ATF3 partake PGK1 K191/K192 succinylation by modulating P4HA1/succinate signaling in glioblastoma.

BACKGROUND: Hypoxia is a pathological hallmark in most cancers, including glioblastoma (GBM). Hypoxic signaling activation and posttranslational modification (PTM) of oncogenic proteins are well-studied in cancers. Accumulating studies indicate glycolytic enzyme PGK1 plays a crucial role in tumorigenesis, yet the underlying mechanisms remain unknown. METHODS: We first used ChIP assays to uncover the crosstalk between HIF1α and ATF3 and their roles in P4HA1 regulation. Protein degradation analysis, LC-MS/MS, and in vitro succinate production assays were performed to examine the effect of protein succinylation on GBM pathology. Seahorse assay measured the effects of PGK1 succinylation at K191/192 or its mutants on glucose metabolism. We utilized an in vivo intracranial mouse model for biochemical studies to elucidate the impact of ATF3 and P4HA1 on aerobic glycolysis and the tumor immune microenvironment. RESULTS: We demonstrated that HIF1α and ATF3 positively and negatively regulate the transcription of P4HA1, respectively, leading to an increased succinate production and increased activation of HIF1α signaling. P4HA1 expression elevated the succinate concentration, resulting in the enhanced succinylation of PGK1 at the K191 and K192 sites. Inhibition of proteasomal degradation of PGK1 by succinylation significantly increased aerobic glycolysis to generate lactate. Furthermore, ATF3 overexpression and P4HA1 knockdown reduced succinate and lactate levels in GBM cells, inhibiting immune responses and tumor growth. CONCLUSION: Together, our study demonstrates that HIF1α/ATF3 participated in P4HA1/succinate signaling, which is the major regulator of succinate biosynthesis and PGK1 succinylation at K191 and K192 sites in GBM. The P4HA1/succinate pathway might be a novel and promising target for aerobic glycolysis in GBM.

EPIC-0628 abrogates HOTAIR/EZH2 interaction and enhances the temozolomide efficacy via promoting ATF3 expression and inhibiting DNA damage repair in glioblastoma.

The efficacy of temozolomide (TMZ) treatment in glioblastoma (GBM) is influenced by various mechanisms, mainly including the level of O6-methylguanine-DNA methyltransferase (MGMT) and the activity of DNA damage repair (DDR) pathways. In our previous study, we had proved that long non-coding RNA HOTAIR regulated the GBM progression and mediated DDR by interacting with EZH2, the catalytic subunit of PRC2. In this study, we developed a small-molecule inhibitor called EPIC-0628 that selectively disrupted the HOTAIR-EZH2 interaction and promoted ATF3 expression. The upregulation of ATF3 inhibited the recruitment of p300, p-p65, p-Stat3 and SP1 to the MGMT promoter. Hence, EPIC-0628 silenced MGMT expression. Besides, EPIC-0628 induced cell cycle arrest by increasing the expression of CDKN1A and impaired DNA double-strand break repair via suppressing the ATF3-p38-E2F1 pathway. Lastly, EPIC-0628 enhanced TMZ efficacy in GBM in vitro and vivo. Hence, this study provided evidence for the combination of epigenetic drugs EPIC-0628 with TMZ for GBM treatment through the above mechanisms.

RUNX1/NPM1/H3K4me3 complex contributes to extracellular matrix remodeling via enhancing FOSL2 transcriptional activation in glioblastoma.

Extracellular matrix (ECM) remodeling has been implicated in the tumor malignant progression and immune escape in glioblastoma (GBM). Runt-related transcription factor 1 (RUNX1) is a vital transcriptional factor for promoting tumorigenesis and invasion in mesenchymal subtype of GBM. But the correlation between RUNX1 and ECM genes expression and regulatory mechanism of RUNX1 on ECM genes expression remain poorly understood to date. In this study, by using integral analysis of chromatin immunoprecipitation-sequencing and RNA sequencing, we reported that RUNX1 positively regulated the expression of various ECM-related genes, including Fibronectin 1 (FN1), Collagen type IV alpha 1 chain (COL4A1), and Lumican (LUM), in GBM. Mechanistically, we demonstrated that RUNX1 interacted with Nucleophosmin 1 (NPM1) to maintain the chromatin accessibility and facilitate FOS Like 2, AP-1 Transcription Factor Subunit (FOSL2)-mediated transcriptional activation of ECM-related genes, which was independent of RUNX1's transcriptional function. ECM remodeling driven by RUNX1 promoted immunosuppressive microenvironment in GBM. In conclusion, this study provides a novel mechanism of RUNX1 binding to NPM1 in driving the ECM remodeling and GBM progression.

Levetiracetam: A Potent Sword against Microglia Polarization in Gliomas.

Crosstalk between tumor cells and peritumoral cells contributes to immunosuppressive microenvironment formation in glioblastomas (GBM). A recent study revealed that glioma stem cells activated neuronal activity to promote microglial M2 polarization, leading to GBM progression, which could be pharmacologically blocked by levetiracetam, providing a practical strategy for GBM immunotherapy. See related article by Guo et al., p. 1160.

EPIC-1042 as a potent PTRF/Cavin1-caveolin-1 interaction inhibitor to induce PARP1 autophagic degradation and suppress temozolomide efflux for glioblastoma.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. METHODS: EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. RESULTS: EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. CONCLUSION: EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.

[Retracted] Role of the AKT pathway in microRNA expression of human U251 glioblastoma cells.

Following the publication of the above article, a concerned reader drew to the Editor's attention that, regarding the western blots featured in Fig. 3B on p. 670, the bands featured in the U251 and U251­MC lanes for the miR­21 and U6 experiments appeared to be duplicates of each other. Moreover, certain of these data were strikingly similar to data that appeared in another article published at around the same time featuring some of the same authors (again, with apparent duplications of bands within the same gel slices, as they were presented). After having conducted an internal investigation of this matter, the Editor of International Journal of Oncology has judged that the apparently anomalous grouping of the data could not have been attributed to pure coincidence. Therefore, the Editor has decided that this article should be retracted from the publication on the grounds of an overall lack of confidence in the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor sincerely apologizes to the readership for any incovenience caused, and we thank the reader for bringing this matter to our attention. [International Journal of Oncology 36: 665­672, 2010; DOI: 10.3892/ijo_00000542].

Universal theranostic CRISPR/Cas13a RNA-editing system for glioma.

Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for in vivo molecular diagnoses. Methods: A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and in vitro biological validation methods. For in vivo imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice. Results: The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us in vivo imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice. Conclusions: Our work demonstrated the clinical potential of a Cas13a-based in vivo imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma.

Blockage of EGFR/AKT and mevalonate pathways synergize the antitumor effect of temozolomide by reprogramming energy metabolism in glioblastoma.

BACKGROUND: Metabolism reprogramming plays a vital role in glioblastoma (GBM) progression and recurrence by producing enough energy for highly proliferating tumor cells. In addition, metabolic reprogramming is crucial for tumor growth and immune-escape mechanisms. Epidermal growth factor receptor (EGFR) amplification and EGFR-vIII mutation are often detected in GBM cells, contributing to the malignant behavior. This study aimed to investigate the functional role of the EGFR pathway on fatty acid metabolism remodeling and energy generation. METHODS: Clinical GBM specimens were selected for single-cell RNA sequencing and untargeted metabolomics analysis. A metabolism-associated RTK-fatty acid-gene signature was constructed and verified. MK-2206 and MK-803 were utilized to block the RTK pathway and mevalonate pathway induced abnormal metabolism. Energy metabolism in GBM with activated EGFR pathway was monitored. The antitumor effect of Osimertinib and Atorvastatin assisted by temozolomide (TMZ) was analyzed by an intracranial tumor model in vivo. RESULTS: GBM with high EGFR expression had characteristics of lipid remodeling and maintaining high cholesterol levels, supported by the single-cell RNA sequencing and metabolomics of clinical GBM samples. Inhibition of the EGFR/AKT and mevalonate pathways could remodel energy metabolism by repressing the tricarboxylic acid cycle and modulating ATP production. Mechanistically, the EGFR/AKT pathway upregulated the expressions of acyl-CoA synthetase short-chain family member 3 (ACSS3), acyl-CoA synthetase long-chain family member 3 (ACSL3), and long-chain fatty acid elongation-related gene ELOVL fatty acid elongase 2 (ELOVL2) in an NF-κB-dependent manner. Moreover, inhibition of the mevalonate pathway reduced the EGFR level on the cell membranes, thereby affecting the signal transduction of the EGFR/AKT pathway. Therefore, targeting the EGFR/AKT and mevalonate pathways enhanced the antitumor effect of TMZ in GBM cells and animal models. CONCLUSIONS: Our findings not only uncovered the mechanism of metabolic reprogramming in EGFR-activated GBM but also provided a combinatorial therapeutic strategy for clinical GBM management.

Compound AC1Q3QWB upregulates CDKN1A and SOX17 by interrupting the HOTAIR-EZH2 interaction and enhances the efficacy of tazemetostat in endometrial cancer.

Endometrial cancer (EC) is a common malignancy of the female reproductive system, with an escalating incidence. Recurrent/metastatic EC presents a poor prognosis. The interaction between the long non-coding RNA (lncRNA) HOTAIR and the polycomb repressive complex 2 (PRC2) induces abnormal silencing of tumor suppressor genes, exerting a pivotal role in tumorigenesis. We have previously discovered AC1Q3QWB (AQB), a small-molecule compound targeting HOTAIR-EZH2 interaction. In the present study, we unveil that AQB selectively hampers the interaction between HOTAIR and EZH2 within EC cells, thus reversing the epigenetic suppression of tumor suppressor genes. Furthermore, our findings demonstrate AQB's synergistic effect with tazemetostat (TAZ), an EZH2 inhibitor, significantly boosting the expression of CDKN1A and SOX17. This, in turn, induces cell cycle arrest and impedes EC cell proliferation, migration, and invasion. In vivo experiments further validate AQB's potential by enhancing TAZ's anti-tumor efficacy at lower doses. Our results advocate AQB, a recently discovered small-molecule inhibitor, as a promising agent against EC cells. When combined with TAZ, it offers a novel therapeutic strategy for EC treatment.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

OBJECTIVE: Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. METHODS: CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). RESULTS: Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. CONCLUSIONS: Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

EPIC-0307-mediated selective disruption of PRADX-EZH2 interaction and enhancement of temozolomide sensitivity to glioblastoma via inhibiting DNA repair and MGMT.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) has been limited by resistance. The level of O-6-methylguanine-DNA methyltransferase (MGMT) and intrinsic DNA damage repair factors are important for the TMZ response in patients. Here, we reported a novel compound, called EPIC-0307, that increased TMZ sensitivity by inhibiting specific DNA damage repair proteins and MGMT expression. METHODS: EPIC-0307 was derived by molecular docking screening. RNA immunoprecipitation (RIP), and chromatin immunoprecipitation by RNA (ChIRP) assays were used to verify the blocking effect. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) assays were performed to explore the mechanism of EPIC-0307. A series of in vivo and in vitro experiments were designed to evaluate the efficacy of EPIC-0307 in sensitizing GBM cells to TMZ. RESULTS: EPIC-0307 selectively disrupted the binding of PRADX to EZH2 and upregulated the expression of P21 and PUMA, leading to cell cycle arrest and apoptosis in GBM cells. EPIC-0307 exhibited a synergistic inhibitory effect on GBM when combined with TMZ by downregulating TMZ-induced DNA damage repair responses and epigenetically silencing MGMT expression through modulating the recruitment of ATF3-pSTAT3-HDAC1 regulatory complex to the MGMT promoter. EPIC-0307 demonstrated significant efficacy in suppressing the tumorigenesis of GBM cells, restoring TMZ sensitivity. CONCLUSION: This study identified a potential small-molecule inhibitor (SMI) EPIC-0307 that selectively disrupted the PRADX-EZH2 interaction to upregulate expressions of tumor suppressor genes, thereby exerting its antitumor effects on GBM cells. EPIC-0307 treatment also increased the chemotherapeutic efficacy of TMZ by epigenetically downregulating DNA repair-associate genes and MGMT expression in GBM cells.

Association between behavioural risks and Alzheimer's disease: Elucidated with an integrated analysis of gene expression patterns and molecular mechanisms.

Alzheimer's disease (AD) remains a global health challenge. Previous studies have reported linkages between AD and multiple behavioural risk exposures, however, the underlying biological mechanisms and crucial genes of gene expression patterns driven by behavioural risks on the onset or progression of AD remains ambiguous. In this study, we performed an integrated analysis on the influence of behavioural risks including smoking, excessive alcohol consumption, physical inactivity, and non-healthy dietary pattern on AD with a comprehensive strategy. Our results demonstrated that multiple behavioural risk exposures could independently or collectively influence diverse hierarchical levels of gene expression patterns through multiple biological mechanisms such as Wnt, mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK), nuclear factor (NF)-κB, phosphatidylinositol 3-kinase (PI3K)-Akt, and insulin (INS) signalling pathways-mediated pathological processes, thereby prodromally or intermediately impacting AD. Our study provided insights into understanding the association of behavioural risk exposures with AD and informative support for further studies.

Expert opinion on translational research for advanced glioblastoma treatment.

Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern, rapid progression, and poor prognosis. Many antitumor drugs are not ideal for the treatment of gliomas due to the blood-brain barrier. Temozolomide (TMZ) is a DNA alkylating agent that can cross the blood-brain barrier. As the only first-line chemotherapeutic drug for malignant gliomas at present, TMZ is widely utilized to provide a survival benefit; however, some patients are inherently insensitive to TMZ. In addition, patients could develop acquired resistance during TMZ treatment, which limits antitumor efficacy. To clarify the mechanism underlying TMZ resistance, numerous studies have provided multilevel solutions, such as improving the effective concentration of TMZ in tumors and developing novel small molecule drugs. This review discusses the in-depth mechanisms underlying TMZ drug resistance, thus aiming to provide possibilities for the establishment of personalized therapeutic strategies against malignant gliomas and the accelerated development and transformation of new targeted drugs.

AC1Q3QWB inhibits colorectal cancer progression by modulating the immune response and balancing the structure of the intestinal microbiota.

AC1Q3QWB enhances CD8 + T cell response and triggers accumulation of Tregs and B cells. BACKGROUND: Colorectal cancer (CRC) is a common malignancy with high mortality and few effective therapeutic measures. Gut microbiota dysbiosis and chronic inflammation might contribute to the development of CRC. The present study aimed to explore the effect of AC1Q3QWB (AQB) on colon carcinogenesis in vivo. METHODS: A mouse colon cancer model was constructed by intraperitoneal injection of 10 mg/kg of Azoxymethane (AOM) and 2 % dextran sodium sulfate (DSS) in drinking water. Mice were randomly assigned to four groups: normal control (NC), AOM/DSS (model control, MC), DMSO + AOM/DSS (DMSO), and AQB + AOM/DSS (AQB). Mice in the AQB group were treated with an intraperitoneal injection of AQB (50 mg/kg) after successful modeling. Then, the disease activity index (DAI) of colitis was analyzed. Colon tissues were collected for hematoxylin-eosin, immunohistochemistry, and microscopic and histological evaluation. Stool samples were collected for microbiota analysis by 16S rRNA sequencing. Blood samples were analyzed by flow cytometry to investigate the inflammatory response. RESULTS: In AOM/DSS-induced CRC mouse model, AQB treatment dramatically reduced the number and size of colon tumors. AQB treatment enhances CD8++T cell response and triggers the accumulation of CD4++CD25++Foxp3++Regulatory T cells (Tregs) and B cells. AQB regulated the structure and composition of the gut microbiota, which decreased the Firmicutes/Bacteroidetes ratio at the phylum level and increased the abundance of probiotics. CONCLUSIONS: AQB has potent antitumor activity against colorectal cancer in vivo by a mechanism that might involve modulation of the immune system and alteration of the intestinal microbiota.