BMI1 Inhibition Research Articles

SOX4-BMI1 axis promotes non-small cell lung cancer progression and facilitates angiogenesis by suppressing ZNF24.

The incidence of lung cancer has become the highest among all cancer types globally, also standing as a leading cause of cancer-related deaths. Lung cancer is broadly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter accounting for 85% of total cases. SRY-box transcription factor 4 (SOX4), a crucial transcription factor, has been found to play a key role in the development of various cancers. However, the association between SOX4 and NSCLC is still unclear. This study investigated the clinical relevance of SOX4 and its potential mechanisms in the progression of NSCLC. Analysis of our NSCLC patient cohort revealed a significant increase in SOX4 levels in cancerous tissues, indicating its role as an independent prognostic indicator for NSCLC. In vitro experiments demonstrated that elevated SOX4 expression facilitated NSCLC cell migration, invasion, and EMT. Functionally, SOX4 drives NSCLC progression by enhancing the transcription and expression of B-cell-specific moloney leukemia virus insertion site 1 (BMI1). The oncogenic impact of SOX4-induced BMI1 expression on NSCLC advancement was validated through both in vivo and in vitro studies. In addition, our findings showed that BMI1 promoted the ubiquitination of histone H2A (H2Aub), leading to decreased zinc finger protein 24 (ZNF24) expression, which subsequently triggered vascular endothelial growth factor A (VEGF-A) secretion in NSCLC cells, thereby promoting NSCLC angiogenesis. Moreover, we evaluated the therapeutic potential of a BMI1 inhibitor in combination with Bevacizumab for NSCLC treatment using orthotopic models. The data presented in our study reveal a previously unrecognized role of the SOX4-BMI1 axis in promoting NSCLC progression and angiogenesis. This research significantly contributes to our knowledge of the interplay between SOX4 and BMI1 in NSCLC, potentially paving the way for the development of targeted therapies for this disease.

Gambogic acid impairs the maintenance and therapeutic resistance of glioma stem cells by targeting B-cell-specific Moloney leukemia virus insert site 1

BackgroundGlioblastoma (GBM) is the most common and lethal primary brain tumor with low effectiveness of available treatments. The tumor heterogeneity and therapeutic resistance are largely due to the presence of glioma stem cells (GSCs). Therefore, eliminating GSCs can overcome the progression, relapse, and resistance of GBM. Previous studies have shown that gambogic acid (GA), a natural active ingredient, has anti-glioma properties. Nonetheless, it is still unclear whether it has an inhibitory effect on GSCs and what its target might be. This study aimed to investigate the anti-tumor effects of GA on GSCs. In addition, this study found the target of GA in GSCs and elucidated the potential specific mechanisms by conducting both in vitro and in vivo experiments.B-cell-specific Moloney leukemia virus insert site 1 (BMI1) is a key stem cell factor of the polycomb group (PcG) family with important effects on the development, recurrence, and chemoresistance of several cancers. In both normal and cancer stem cells, BMI1 maintains stem cell self-renewal by regulating the cell cycle, cellular immortalization, and senescence. Its high expression in a variety of cancers correlates with poor clinical prognosis and chemoresistance. These mechanisms of BMI1 make it a potential therapeutic target for cancer therapy, and future studies may further reveal the specific roles of BMI1 mechanism and provide a basis for the development of new cancer therapeutic strategies. PurposeThis study investigated the in vitro and in vivo effects of GA in inducing apoptosis in GSCs and inhibiting GSCs self-renewal, as well as its underlying mechanisms. MethodsThis study synthesized biotinylated gambogic acid for the first time and angled for the target of gambogic acid using LC-MS/MS analysis, which has not been reported previously. Human-derived glioma stem cells GSC123 and GSC111 were used for in vitro studies, analyzing functions and mechanisms via microscale thermophoresis (MST), Annexin V/PI staining, Western blotting, immunofluorescence, and co-immunoprecipitation. The orthotopic glioma mouse model was used to assess the anti-tumor effects of GA in vivo. ResultsThis study demonstrated that GA is a specific inhibitor of BMI1, a key regulator controlling stem cell growth and self-renewal. GA binds to BMI1′s RING domain, accelerating K51-dependent degradation and suppressing H2A ubiquitination. Importantly, GA induces apoptosis, and inhibits GSC self-renewal, but minimally impacts neural progenitor cells (NPCs). GA can also be combined effectively with temozolomide and radiotherapy to increase their sensitivities in resistant cells. Furthermore, exogenous induction of BMI1 expression significantly hinders the disruption of GSCs by GA. In vivo, GA inhibits tumorigenicity, enhances the effect of temozolomide, and reduces BMI1 expression. ConclusionThese findings suggest that GA is a potential candidate for targeting GSCs and therefore be used to treat GBM.

Targeting Bmi1 for Enhancing Anoikis Sensitivity and Inhibiting Metastasis in Colorectal Cancer.

Patients diagnosed with advanced metastatic colorectal cancer (CRC) confront a bleak prognosis characterized by low survival rates. Anoikis, the programmed apoptosis resistance exhibited by metastatic cancer cells, is a crucial factor in this scenario. We employed bulk flow cytometry and RT-qPCR assays, conducted in vivo experiments with mice and zebrafish, and analyzed patient tissues to examine the effects of the B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1)-midkine (MDK) axis on the cellular response to anoikis. Bmi1 is pivotal in tumorigenesis. This study elucidated the involvement of Bmi1 in conferring anoikis resistance in CRC and explored its downstream targets associated with metastasis. Elevated levels of Bmi1 expression correlated with distant metastasis in CRC. Suppression of Bmi1 significantly diminished the metastatic potential of CRC cells. Inhibition of Bmi1 led to an increase in the proportion of apoptotic SW620 cells detached from the matrix. This effect was further enhanced by the addition of irinotecan, a topoisomerase I inhibitor. Furthermore, Bmi1 was found to synergize with MDK in modulating CRC viability, with consistent expression patterns observed in in vivo models and clinical tissue specimens. In summary, Bmi1 acted as a regulator of CRC metastatic capability by conferring anoikis resistance. Additionally, it collaborated with MDK to facilitate invasion and distant metastasis. Targeting Bmi1 may offer a promising adjunctive therapeutic strategy when administering traditional chemotherapy regimens to patients with advanced CRC.

ATRT-07. PRC1/BMI1 ACTIVITY ALTERS CHROMATIN ACCESSIBILITY TO REGULATE DIFFERENTIATION IN ATYPICAL TERATOID RHABDOID TUMORS

Abstract BACKGROUND SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. Atypical Teratoid Rhabdoid Tumor (ATRT) is a highly aggressive pediatric brain tumor and characterized by SMARCB1 deletion. METHODS Genome wide RNAi screen, RNA-seq and ChIP-Seq analysis, colony formation assay, cell cycle, western blotting, immunoprecipitation, mass spectrometry analysis, mouse xenograft model, IVIS and MRI imaging. RESULTS We performed an RNAi-based functional genomic screen to identify key epigenetic regulators that co-operate with SMARCB1 loss and contribute to tumorigenesis in ATRT. 406 genes associated with epigenetic regulation in patient-derived ATRT cell lines were targeted. This screen identified BMI1, a component of the Polycomb Repressive Complex 1 (PRC1), as one of the top targets essential for ATRT cell growth. Using RNA-seq and ChIP-Seq analysis we demonstrated that BMI1 co-operates with SMARCB1 deletion to suppress transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. In SMARCB1 deficient cells BMI1 forms a partial PRC1 complex devoid of DNA binding components. We found that genetic inactivation of BMI1 suppresses ATRT cell clonogenicity and neurosphere formation, leads to S-phase arrest and induces ATRT cell differentiation in vitro. In vivo BMI1 inhibition induces apoptosis, leading to significantly increasing survival of treated mice. By Doxycycline-inducible SMARCB1 re-expression system we revealed activation of proapoptotic proteins p16 and p21. With immunoprecipitation for BMI1, followed by mass spectrometry analysis we demonstrated that re-expression of SMARCB1 activates two PRC1 chromatin localizing components CBX4 and CBX8. Moreover, SMARCB1 re-expression significantly prolongs mice survival and decreases tumor size as evaluated by IVIS imaging system and T2-MRI. CONCLUSION Thus, we demonstrated that SMARCB1 deletion results in reprograming of BMI1 chromatin occupancy away from lineage specification by altering the components of the PRC1 complex and that PRC1/BMI1 inhibition is a novel therapeutic approach in ATRT.

Bmi1 facilitates the progression of cholangiocarcinoma by inhibiting Foxn2 expression dependent on a histone H2A ubiquitination manner

Cholangiocarcinoma (CCA), an exceptionally aggressive malignancy originating from the epithelium of the bile duct, poses a formidable challenge in cancer research and clinical management. Currently, attention is focused on exploring the oncogenic role and prognostic implications associated with Bmi1 in the context of CCA. In our study, we assessed the correlation of Bmi1 and Foxn2 expression across all types of CCA and evaluated their prognostic significance. Our results demonstrated that Bmi1 exhibits significantly upregulated expression in CCA tissues, while Foxn2 expression shows an inverse pattern. Simultaneously, the high expression of Bmi1, coupled with the low expression of Foxn2, indicates an unfavorable prognosis. Through in vitro and in vivo experiments, we confirmed the crucial role of Foxn2 in the proliferation, metastasis, and epithelial-mesenchymal transition (EMT) of CCA. Mechanistically, Bmi1 promotes the ubiquitination of histone H2A (H2AUb), leading to chromatin opening attenuation and a decrease in Foxn2 expression, ultimately driving CCA progression. Additionally, we described the potential value of Bmi1 and H2AUb inhibitors in treating CCA through in vitro experiments and orthotopic models. This study is of significant importance in deepening our understanding of the interaction between Bmi1 and Foxn2 in CCA and has the potential to advance the development of precision therapies for CCA.

Erbb3 regulation of secretory differentiation in the intestinal epithelium

Background: Previous work by our lab showed that intestinal-specific deletion of the ErbB3 receptor tyrosine kinase results in increased ileal Paneth cell numbers. These cells produce host defense peptides and support stem cells with growth factors. The mechanisms by which ErbB3 regulates Paneth cells are not well understood, but a candidate mediator is the polycomb repressor complex 1 component BMI1, which marks secretory progenitor cells. Furthermore, while there are no Paneth cells in the mouse colon, deep crypt secretory (DCS) cells are a distinct Reg4+ population that have some functional overlap with both Paneth and goblet cells; we will also test whether ErbB3 regulates DCS differentiation. Overall we are testing the hypothesis that ErbB3 regulates secretory cell numbers through BMI1. Methods: We bred Villin-Cre;ErbB3flox/flox mice (ErbB3ΔIE) to delete ErbB3 from the intestinal epithelium. Colonic sections from ErbB3ΔIE and ErbB3flox/flox littermates were subjected to RNAscope in situ hybridization for Reg4. RNA from mucosal scrapings (both ileum and colon) was assessed by qPCR for Reg4, Bmi1, and Lgr5. To test signaling mechanisms, we treated HT-29 cells with NRG-1β to activate ErbB3, +/- inhibitors to PI3K (LY294002), MEK/MAPK (U0126), or BMI1 (PTC-209). Colonoids and enteroids from ErbB3ΔIE and ErbB3flox/flox mice were used to confirm responses in a non-transformed system. Results: In ErbB3ΔIE mice, the colonic epithelium showed increased Reg4 levels and more Reg4+ cells compared to littermate controls, similar to our published data for the Paneth cell marker Lyz1 in ileum. BMI1 RNA and protein levels were also increased in both small and large intestinal mucosal scrapings from ErbB3ΔIE mice vs. littermates. ErbB3ΔIE colonoids had increased Reg4 versus control colonoids, and NRG-1β reduced levels in control but not ErbB3ΔIE cultures. Ileal enteroids showed a decrease in Lyz1 expression with Bmi1 inhibition and an increase in Bmi1 expression with PI3K and MAPK blockade. In HT-29 cells, NRG-1β treatment reduced Reg4 and Bmi1 expression. Conversely, Reg4 and Bmi1 were induced by PI3K inhibition with LY294002 (p<0.03) (p<0.0005) or MEK/MAPK inhibition with U0126 (p<0.05) (p<0.0001). Conclusions: NRG1-ErbB3 signaling restricts both ileal Paneth ( Lyz1+) and colonic DCS ( Reg4+) cell numbers. Furthermore, ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling and BMI1 activity promotes secretory cell development. Since loss of DCS or Paneth cells has been shown to disrupt the intestinal stem cell niche and secretory cell markers are altered in several intestinal disease states, these results may point to potential future therapeutic avenues targeting this regulatory mechanism. NIH/NIDDK award R01DK095004. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Abstract PR-012: Proteogenomic analysis of enriched tumor epithelium identifies prognostic signatures and an increased dependency of homologous recombination proficient cells on bmi1 in high grade serous ovarian cancer

Abstract Background: Proteogenomic characterization efforts of high-grade serous ovarian cancer (HGSOC) have largely assessed tumors with high tumor cellularity (“purity”) to enhance detection of cancer-related biomarkers. Comprehensive analyses of cancers independent of purity are needed to reflect a “real-world” spectrum of patient phenotypes. To improve identification of clinically relevant molecular alterations associated with HGSOC independent of tumor purity, we applied cellular enrichment techniques coupled with comprehensive multi-omic analyses in HGSOC patient tumors spanning a broad spectrum of purity. Methods: Seventy HGSOC patient tumors were selected exhibiting low to high tumor purity (20-90%) balanced by clinical outcomes. Whole genome sequencing (WGS), mRNA-seq, quantitative global proteomics, methylation array and reverse phase protein array analyses of Bulk Tumor and laser microdissected (LMD) Enriched Tumor (ET) cells separately for each case. Prognostic and homologous recombination deficient (HRD) expression signatures were validated in two independent cohorts. Results: Analysis of WGS in ET compared to WT resulted in significant increases in sensitivity to identify somatic SNV, indel, structural variants, and neoepitopes (SNV p = 4.8E−3; indel p = 7.7E−5; SV, p = 1.1E−8, neoepitopes, p= 1.0E−10). Following LMD, 63% of cases characterized as mesenchymal subtype (C4) in WT samples were reclassified to other molecular subtypes (1.0E−3) suggesting that historical HGSOC expression subtypes strongly reflect tumor purity. Analysis of paired primary and metastatic tumors demonstrated that WT proteomic profiles largely cluster by anatomic location while ET proteomic profiles co-cluster in a patient centric manner. Hierarchical cluster analysis identified patients with longer progression free survival associated with increased immune signatures and validated proteins correlating with tumor infiltrating lymphocytes (TILs) in 65 tumors collected from an independent cohort of 12 HGSOC patients, as well as with overall survival in an additional cohort of 126 HGSOC patients. We identified that homologous recombination deficient (HRD) tumors express transcriptomic and proteomic pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts, including 69 HRD-positive HGSOC tumors. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells exhibit increased sensitivity to BMI-1 inhibition. Conclusion: Proteogenomic alterations in HGSOC tumors uncovered using enrichment techniques highlight the importance of specimen preparation in the identification of tumor alterations. Our efforts provide insights into low purity HGSOC correlating TILs with improved disease prognosis, expression alterations associated with HRD status, and increased sensitivity of HRP tumor cells to BMI-1 inhibitors. Citation Format: George Larry Maxwell, Nicholas Bateman, Tamara Abulez, Anthony Soltis, Andrew McPherson, Seongmin Choi, Dale Garsed, Chunqiao Tian, Brian Hood, Kelly Conrads, Pang-ning Teng, Julie Oliver, Glenn Gist, Dave Mitchell, Tracy Litzi, Christopher Tarney, Clifton Dalgard, Matthew Wilkerson, Mariaelena Pierobon, Emmanuel Petricoin, Chunhua Yan, Daoud Meerzaman, Clara Bodelon, Nicolas Wentzensen, Jerry S. H. Lee, David Huntsman, Sohrab Shah, Craig Shriver, Neil Phippen, Kathleen Darcy, David Bowtell, Thomas Conrads. Proteogenomic analysis of enriched tumor epithelium identifies prognostic signatures and an increased dependency of homologous recombination proficient cells on bmi1 in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-012.

PTC596-Induced BMI-1 Inhibition Fights Neuroblastoma Multidrug Resistance by Inducing Ferroptosis.

Neuroblastoma (NB) is a paediatric cancer with noteworthy heterogeneity ranging from spontaneous regression to high-risk forms that are characterised by cancer relapse and the acquisition of drug resistance. The most-used anticancer drugs exert their cytotoxic effect by inducing oxidative stress, and long-term therapy has been demonstrated to cause chemoresistance by enhancing the antioxidant response of NB cells. Taking advantage of an in vitro model of multidrug-resistant (MDR) NB cells, characterised by high levels of glutathione (GSH), the overexpression of the oncoprotein BMI-1, and the presence of a mutant P53 protein, we investigated a new potential strategy to fight chemoresistance. Our results show that PTC596, an inhibitor of BMI-1, exerted a high cytotoxic effect on MDR NB cells, while PRIMA-1MET, a compound able to reactivate mutant P53, had no effect on the viability of MDR cells. Furthermore, both PTC596 and PRIMA-1MET markedly reduced the expression of epithelial-mesenchymal transition proteins and limited the clonogenic potential and the cancer stemness of MDR cells. Of particular interest is the observation that PTC596, alone or in combination with PRIMA-1MET and etoposide, significantly reduced GSH levels, increased peroxide production, stimulated lipid peroxidation, and induced ferroptosis. Therefore, these findings suggest that PTC596, by inhibiting BMI-1 and triggering ferroptosis, could be a promising approach to fight chemoresistance.

Abstract A158: Rational development of synergistic therapies alongside BMI1 Inhibition for group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Of its four distinct molecular subgroups, Group 3 MBs are associated with increased risk of recurrence, metastasis and overall poor patient outcome. In recent years, small molecule inhibitors that decrease BMI1 expression have shown to be efficacious against several types of malignant tumors including pediatric MB. Although in vivo studies provide a promising proof-of-concept for the therapeutic targeting of BMI1 in Group 3 MB, mice that receive treatment eventually succumb to their disease. These results suggest that additional mechanisms may underlie the maintenance of MB and underscore the main obstacle in treating a constantly evolving tumor. After initial preclinical validation of BMI1 inhibitor PTC-596, DNA barcoding clonal tracking technology was leveraged to profile in vivo clonal dynamics of Group 3 MB in response to the established chemoradiotherapy regimen alone and in combination with PTC-596. Comparison of clonal composition of tumors extracted from brains and spines post-treatment revealed the persistence of a small number of clones with the ability to escape therapy and drive subsequent tumor expansion. In order to better understand molecular susceptibilities of MB cells post-BMI1 inhibition, we undertook an in vitro genome-wide CRISPR/Cas9 screening to identify context-specific MB regulatory pathways that could be synergistically targeted along with BMI1. By comparing the results of the in vitro genome wide CRISPR/Cas9 screen to the essential genes in human neural stem cells (hNSCs), we identified several context specific regulators of mTOR, AKT and PLK1 pathways. Combination treatment alongside PTC-596 has demonstrated synergistic efficacy against MB cells with minimal toxicity to hNSCs in vitro and in preclinical in vivo studies. This study provides the foundation for clinical validation of small-molecule inhibitors synergistic with PTC-596 to improve the durability of remissions and extend survival of patients with treatment-refractory Group 3 MB. Citation Format: David Bakhshinyan, Stefan Custers, Ashley Adile, Kevin Brown, Maleeha Qazi, Chirayu Chokshi, Laura Escudero, Yujin Suk, Chitra Venugopal, Jason Moffat, Sheila Singh. Rational development of synergistic therapies alongside BMI1 Inhibition for group 3 medulloblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A158.

Core-shell cisplatin/SiO2 nanocapsules combined with PTC-209 overcome chemotherapy–Acquired and intrinsic resistance in hepatocellular carcinoma

The primary cause of cisplatin resistance in liver cancer is reduced intracellular drug accumulation and altered DNA repair/apoptosis signaling. Existing strategies to reverse cisplatin resistance have limited efficacy, as they target individual factors. This study proposes a drug delivery system consisting of a cisplatin core, a silica shell with a tetra-sulfide bond, and a PEG-coated surface (Core/shell-PGCN). The system is designed to consume glutathione (GSH) and reduce cisplatin excretion from cells, thereby overcoming acquired cisplatin resistance. In addition, Core/shell-PGCN incorporates PTC-209 (Core/shell-PGCN@PTC-209), a Bmi1 inhibitor that suppresses liver cancer stem cells (CSC), to mitigate DNA repair/apoptosis signaling and reverse intrinsic cisplatin resistance. In vivo and in vitro results demonstrate that Core/shell-PGCN@PTC-209 can comprehensively regulate GSH and CSC, reverse intrinsic and acquired cisplatin resistance, and enhance the efficacy of cisplatin in treating liver cancer. This "inner cultivation, outer action" approach may offer a new strategy for reversing cisplatin resistance in liver cancer. Statement of significanceCisplatin resistance is widely observed in liver cancer (HCC) chemotherapy, with two mechanisms identified: acquired and intrinsic. Most strategies aimed at overcoming cisplatin resistance focus on a single perspective. This study introduces a core-shell drug delivery system (DDS) combined with HCC stem cell inhibitors, which can effectively address cisplatin resistance in HCC by targeting both acquisition and internality. Specifically, the core-shell drug delivery system can impede cisplatin efflux by neutralizing the acquired resistance factor (GSH), thus overcoming acquired resistance. Additionally, HCC stem cell inhibitors can reverse intrinsic resistance by inhibiting HCC stem cells. Therefore, this study contributes to the application of DDS in combating drug resistance in HCC and enhances its potential for clinical implementation.

BMI1-induced CD127+KLRG1+ memory T cells enhance the efficacy of liver cancer immunotherapy

The efficacy of HCC (hepatocellular carcinoma) immunotherapy is hindered by the limited reactivity and short duration of tumor-infiltrating T cells. These deficiencies may be ascribed to the proliferative ability of T cells. The primary objective of this study was to identify the key factor regulating tumor-infiltrating lymphocytes (TIL) proliferation within the HCC microenvironment. Through the utilization of tissue-infiltrated T cell proteomics and fraction proteomics, we analyzed the differential proteins in T cells among HCC, liver fibrosis, and hemangioma (serving as controls) groups. Additionally, we examined the differential regulatory TFs of T cells between the HCC and VH (volunteer healthy, as a control) groups. Using cyTOF and flow cytometry technologies, as well as generating CD8+ T-specific BMI1 knockout mice, we confirmed that BMI1 controls CD127+KLRG1+ memory cell differentiation. Through RNA-seq and MeRIP-seq, we verified that BMI1 regulates TCF1 expression independently of its classical function. Furthermore, by conducting Tyramide signal amplification (TSA) IHC analysis, employing a hydrodynamic mouse HCC model, and utilizing liver-specific nanoparticle targeting therapy, we demonstrated that BMI1 in HCC influences the proliferation of infiltrating CD8+T. BMI1 inhibition promotes effector T cell differentiation while suppressing memory T cell differentiation. Moreover, liver-specific BMI1 knockdown proves beneficial in ameliorating T cell dysfunction and decelerating HCC progression. Our research group has pioneered the exploration of the proteomics of HCC-infiltrated T cells, shedding light on the pivotal role of BMI1 in controlling CD127+KLRG1+ memory CD8+ T cell differentiation, which serves as the cornerstone for achieving immunotherapy efficacy in HCC.

Therapeutic inhibition of Bmi-1 ablates chemoresistant cancer stem cells in adenoid cystic carcinoma

ObjectivesAdenoid Cystic Carcinomas (ACC) typically show modest responseto cytotoxic therapy. Cancer stem cells (CSC) have been implicated in chemoresistance and tumor relapse. However, their role in ACC remains unknown. The purpose of this work was to evaluate the impact of targeting ACC CSCs with Bmi-1 inhibitors on resistance to cytotoxic therapy and tumor relapse. Materials and methodsTherapeutic efficacy of a small molecule inhibitor of Bmi-1 (PTC596; Unesbulin) and/or Cisplatin on ACC stemness was evaluated in immunodeficient mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in human ACC cell-lines (UM-HACC-2A,-14) or low passage primary human ACC cells (UM-HACC-6). The effect of therapy on stemness was examined by salisphere assays, flow cytometry for ALDH activity and CD44 expression, and Western blots for Bmi-1 (self-renewal marker) and Oct4 (embryonic stem cell marker) expression. ResultsPlatinum-based agents (Cisplatin, Carboplatin) induced Bmi-1 and Oct4 expression, increased salisphere formation and the CSC fraction in vitro and in vivo. In contrast, PTC596 inhibited expression of Bmi-1, Oct4 and pro-survival proteins Mcl-1 and Claspin; decreased the number of salispheres, and the fraction of ACC CSCs in vitro. Silencing Claspin decreased salisphere formation and CSC fraction. Both, single agent PTC596 and PTC596/Cisplatin combination decreased the CSC fraction in PDX ACC tumors. Notably, short-term combination therapy (2 weeks) with PTC596/Cisplatin prevented tumor relapse for 150 days in a preclinical trial in mice. ConclusionTherapeutic inhibition of Bmi-1 ablates chemoresistant CSCs and prevents ACC tumor relapse. Collectively, these results suggest that ACC patients might benefit from Bmi-1-targeted therapies.

A transverse colon lipoma complicated with an adenoma is successfully and safely resected en bloc by endoscopic mucosal resection during water immersion

The B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs.We assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases.BMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues.Our study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

Alpha-tocopherol enhances spermatogonial stem cell proliferation and restores mouse spermatogenesis by up-regulating BMI1.

Spermatogonial stem cells (SSCs) are essential for maintaining reproductive function in males. B-lymphoma Mo-MLV insertion region 1 (BMI1) is a vital transcription repressor that regulates cell proliferation and differentiation. However, little is known about the role of BMI1 in mediating the fate of mammalian SSCs and in male reproduction. This study investigated whether BMI1 is essential for male reproduction and the role of alpha-tocopherol (α-tocopherol), a protective agent for male fertility, as a modulator of BMI1 both in vitro and in vivo. Methyl thiazolyl tetrazolium (MTT) and 5-ethynyl-2'-deoxyuridine (EDU) assays were used to assess the effect of BMI1 on the proliferative ability of the mouse SSC line C18-4. Real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence were applied to investigate changes in the mRNA and protein expression levels of BMI1. Male mice were used to investigate the effect of α-tocopherol and a BMI1 inhibitor on reproduction-associated functionality in vivo. Analysis revealed that BMI1 was expressed at high levels in testicular tissues and spermatogonia in mice. The silencing of BMI1 inhibited the proliferation of SSCs and DNA synthesis and enhanced the levels of γ-H2AX. α-tocopherol enhanced the proliferation and DNA synthesis of C18-4 cells, and increased the levels of BMI1. Notably, α-tocopherol rescued the inhibition of cell proliferation and DNA damage in C18-4 cells caused by the silencing of BMI1. Furthermore, α-tocopherol restored sperm count (Ctrl vs. PTC-209, p = 0.0034; Ctrl vs. PTC-209 + α-tocopherol, p = 0.7293) and normalized sperm malformation such as broken heads, irregular heads, lost and curled tails in vivo, as demonstrated by its antagonism with the BMI1 inhibitor PTC-209. Analysis demonstrated that α-tocopherol is a potent in vitro and in vivo modulator of BMI1, a transcription factor that plays an important role in in SSC proliferation and spermatogenesis. Our findings identify a new target and strategy for treating male infertility that deserves further pre-clinical investigation.

B-cell-specific Moloney murine leukemia virus integration site 1 knockdown impairs adriamycin resistance of gastric cancer cells

Background and study aimsThe B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs. Patients and methodsWe assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases. ResultsBMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues. ConclusionOur study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

BMI-1 inhibition fights cancer chemoresistance by altering redox homeostasis and inducing lipid peroxidation

BMI-1 inhibition fights cancer chemoresistance by altering redox homeostasis and inducing lipid peroxidation

Repurposing the Bis-Biguanide Alexidine in Combination with Tyrosine Kinase Inhibitors to Eliminate Leukemic Stem/Progenitor Cells in Chronic Myeloid Leukemia.

In CML, Leukemic Stem Cells (LSCs) that are insensitive to Tyrosine Kinase Inhibitors are responsible for leukemia maintenance and relapses upon TKI treatment arrest. We previously showed that downregulation of the BMI1 polycomb protein that is crucial for stem/progenitor cells self-renewal induced a CCNG2/dependent proliferation arrest leading to elimination of Chronic Myeloid Leukemia (CML) cells. Unfortunately, as of today, pharmacological inhibition of BMI1 has not made its way to the clinic. We used the Connectivity Map bioinformatic database to identify pharmacological molecules that could mimick BMI1 silencing, to induce CML cell death. We selected the bis-biguanide Alexidin (ALX) that produced a transcriptomic profile positively correlating with the one obtained after BMI silencing in K562 CML cells. We then evaluated the efficiency of ALX in combination with TKI on CML cells. Here we report that cell growth and clonogenic activity of K562 and LAMA-84 CML cell lines were strongly inhibited by ALX. ALX didn't modify BCR::ABL1 phosphorylation and didn't affect BMI1 expression but was able to increase CCNG2 expression leading to autophagic processes that preceed cell death. Besides, ALX could enhance the apoptotic response induced by any Tyrosine Kinase Inhibitors (TKI) of the three generations. We also noted a strong synergism between ALX and TKIs to increase expression of caspase-9 and caspase-3 and induce PARP cleavage, Bad expression and significantly decreased Bcl-xL family member expression. We also observed that the blockage of the mitochondrial respiratory chain by ALX can be associated with inhibition of glycolysis by 2-DG to achieve an enhanced inhibition of K562 proliferation and clonogenicity. ALX specifically affected the differentiation of BCR::ABL1-transduced healthy CD34+ cells but not of mock-infected healthy CD34+ control cells. Importantly, ALX strongly synergized with TKIs to inhibit clonogenicity of primary CML CD34+ cells from diagnosed patients. Long Term Culture of Initiating Cell (LTC-IC) and dilution of the fluorescent marker CFSE allowed us to observe that ALX and Imatinib (IM) partially reduced the number of LSCs by themselves but that the ALX/IM combination drastically reduced this cell compartment. Using an in vivo model of NSG mice intravenously injected with K562-Luciferase transduced CML cells, we showed that ALX combined with IM improved mice survival. Collectively, our results validate the use of ALX bis-biguanide to potentiate the action of conventional TKI treatment as a potential new therapeutic solution to eradicate CML LSCs.

Targeting BMI-1 to deplete antibody-secreting cells in autoimmunity.

B cells drive the production of autoreactive antibody-secreting cells (ASCs) in autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Sjögren's syndrome, causing long-term organ damage. Current treatments for antibody-mediated autoimmune diseases target B cells or broadly suppress the immune system. However, pre-existing long-lived ASCs are often refractory to treatment, leaving a reservoir of autoreactive cells that continue to produce antibodies. Therefore, the development of novel treatment methods targeting ASCs is vital to improve patient outcomes. Our objective was to test whether targeting the epigenetic regulator BMI-1 could deplete ASCs in autoimmune conditions in vivo and in vitro. Use of a BMI-1 inhibitor in both mouse and human autoimmune settings was investigated. Lyn -/- mice, a model of SLE, were treated with the BMI-1 small molecule inhibitor PTC-028, before assessment of ASCs, serum antibody and immune complexes. To examine human ASC survival, a novel human fibroblast-based assay was established, and the impact of PTC-028 on ASCs derived from Sjögren's syndrome patients was evaluated. BMI-1 inhibition significantly decreased splenic and bone marrow ASCs in Lyn -/- mice. The decline in ASCs was linked to aberrant cell cycle gene expression and led to a significant decrease in serum IgG3, immune complexes and anti-DNA IgG. PTC-028 was also efficacious in reducing ex vivo plasma cell survival from both Sjögren's syndrome patients and age-matched healthy donors. These data provide evidence that inhibiting BMI-1 can deplete ASC in a variety of contexts and thus BMI-1 is a viable therapeutic target for antibody-mediated autoimmune diseases.

Glucose and Cell Context-Dependent Impact of BMI-1 Inhibitor PTC-209 on AKT Pathway in Endometrial Cancer Cells

In our study, the glucose and cell context-dependent impact of the BMI-1 inhibitor PTC-209 on the AKT pathway in endometrial cancer cells was determined. The expression of BMI-1 was inhibited by PTC-209 in endometrial cancer cells HEC-1A and Ishikawa stimulated with insulin and grown in different glucose concentrations. The migration, invasion, viability, and proliferative potential after PTC-209 treatment was assessed using wound-healing, Transwell assay, Matrigel-coated inserts, and MTT tests. Chromatin immunoprecipitation was used to determine the localization of BMI-1 protein at promoter sites of the genes tested. BMI-1 inhibition caused an increase in PHLPP1/2 expression and a decrease in phospho-AKT level in both cell lines. The glucose concentration and insulin stimulation differentially impact the AKT pathway through BMI-1 in cells differing in PTEN statuses. The expression of BMI-1 is dependent on the glucose concentration and insulin stimulation mostly in PTEN positive HEC-1A cells. In high glucose concentrations, BMI-1 affects AKT activity through PHLPPs and in hypoglycemia mostly through PTEN. BMI-1 inhibition impacts on genes involved in SNAIL, SLUG, and CDH1 and reduces endometrial cancer cells' migratory and invasive potential. Our results indicate that the relationship between BMI-1 and phosphatases involved in AKT regulation depends on the glucose concentration and insulin stimulation.