Small-molecule inhibitors targeting Polycomb repressive complex 1 RING domain.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-modifying complex that monoubiquitinates histone H2A and is involved in maintaining the repressed chromatin state. Emerging evidence suggests PRC1 activity in various cancers, rationalizing the need for small-molecule inhibitors with well-defined mechanisms of action. Here, we describe the development of compounds that directly bind to RING1B-BMI1, the heterodimeric complex constituting the E3 ligase activity of PRC1. These compounds block the association of RING1B-BMI1 with chromatin and inhibit H2A ubiquitination. Structural studies demonstrate that these inhibitors bind to RING1B by inducing the formation of a hydrophobic pocket in the RING domain. Our PRC1 inhibitor, RB-3, decreases the global level of H2A ubiquitination and induces differentiation in leukemia cell lines and primary acute myeloid leukemia (AML) samples. In summary, we demonstrate that targeting the PRC1 RING domain with small molecules is feasible, and RB-3 represents a valuable chemical tool to study PRC1 biology.

Disulfiram/Copper Suppresses Cancer Stem Cell Activity in Differentiated Thyroid Cancer Cells by Inhibiting BMI1 Expression.

Differentiated thyroid carcinomas (DTCs), which have papillary and follicular types, are common endocrine malignancies worldwide. Cancer stem cells (CSCs) are a particular type of cancer cells within bulk tumors involved in cancer initiation, drug resistance, and metastasis. Cells with high intracellular aldehyde hydrogenase (ALDH) activity are a population of CSCs in DTCs. Disulfiram (DSF), an ALDH inhibitor used for the treatment of alcoholism, reportedly targets CSCs in various cancers when combined with copper. This study reported for the first time that DSF/copper can inhibit the proliferation of papillary and follicular DTC lines. DSF/copper suppressed thyrosphere formation, indicating the inhibition of CSC activity. Molecular mechanisms of DSF/copper involved downregulating the expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and cell cycle-related proteins, including cyclin B2, cyclin-dependent kinase (CDK) 2, and CDK4, in a dose-dependent manner. BMI1 overexpression diminished the inhibitory effect of DSF/copper in the thyrosphere formation of DTC cells. BMI1 knockdown by RNA interference in DTC cells also suppressed the self-renewal capability. DSF/copper could inhibit the nuclear localization and transcriptional activity of c-Myc and the binding of E2F1 to the BMI1 promoter. Overexpression of c-Myc or E2F1 further abolished the inhibitory effect of DSF/copper on BMI1 expression, suggesting that the suppression of c-Myc and E2F1 by DSF/copper was involved in the downregulation of BMI1 expression. In conclusion, DSF/copper targets CSCs in DTCs by inhibiting c-Myc- or E2F1-mediated BMI1 expression. Therefore, DSF is a potential therapeutic agent for future therapy in DTCs.

Remodeling and destabilization of chromosome 1 pericentromeric heterochromatin by SSX proteins.

Rearrangement of the 1q12 pericentromeric heterochromatin and subsequent amplification of the 1q arm is commonly associated with cancer development and progression and may result from epigenetic deregulation. In many premalignant and malignant cells, loss of 1q12 satellite DNA methylation causes the deposition of polycomb factors and formation of large polycomb aggregates referred to as polycomb bodies. Here, we show that SSX proteins can destabilize 1q12 pericentromeric heterochromatin in melanoma cells when it is present in the context of polycomb bodies. We found that SSX proteins deplete polycomb bodies and promote the unfolding and derepression of 1q12 heterochromatin during replication. This further leads to segregation abnormalities during anaphase and generation of micronuclei. The structural rearrangement of 1q12 pericentromeric heterochromatin triggered by SSX2 is associated with loss of polycomb factors, but is not mediated by diminished polycomb repression. Instead, our studies suggest a direct effect of SSX proteins facilitated though a DNA/chromatin binding, zinc finger-like domain and a KRAB-like domain that may recruit chromatin modifiers or activate satellite transcription. Our results demonstrate a novel mechanism for generation of 1q12-associated genomic instability in cancer cells.

Inhibition of BMI-1 Induces Apoptosis through Downregulation of DUB3-Mediated Mcl-1 Stabilization.

BMI-1, a polycomb ring finger oncogene, is highly expressed in multiple cancer cells and is involved in cancer cell proliferation, invasion, and apoptosis. BMI-1 represents a cancer stemness marker that is associated with the regulation of stem cell self-renewal. In this study, pharmacological inhibition (PTC596) or knockdown (siRNA) of BMI-1 reduced cancer stem-like cells and enhanced cancer cell death. Mechanistically, the inhibition of BMI-1 induced the downregulation of Mcl-1 protein, but not Mcl-1 mRNA. PTC596 downregulated Mcl-1 protein expression at the post-translational level through the proteasome-ubiquitin system. PTC596 and BMI-1 siRNA induced downregulation of DUB3 deubiquitinase, which was strongly linked to Mcl-1 destabilization. Furthermore, overexpression of Mcl-1 or DUB3 inhibited apoptosis by PTC596. Taken together, our findings reveal that the inhibition of BMI-1 induces Mcl-1 destabilization through downregulation of DUB3, resulting in the induction of cancer cell death.

CBX7 suppression prevents ischemia-reperfusion injury-induced endoplasmic reticulum stress through the Nrf-2/HO-1 pathway.

Renal ischemia-reperfusion injury (I/R) usually occurs in renal transplantation and partial nephrectomy, which could lead to acute kidney injury. However, the effective treatment for renal I/R still remains limited. In the present study, we investigated whether inhibition of chromobox 7 (CBX7) could attenuate renal I/R injury in vivo and in vitro as well as the potential mechanisms. Adult male mice were subjected to right renal ischemia and reperfusion for different periods, both with and without the CBX7 inhibitor UNC3866. In addition, human kidney cells (HK-2) were subjected to a hypoxia/reoxygenation (H/R) process for different periods, both with or without the CBX7 inhibitor or siRNA for CBX7. The results showed that expression of CBX7, glucose regulator protein-78 (GRP78), phosphorylated eukaryotic translation initiation factor-2α (p-eIF2α), and C/EBP homologous protein (CHOP) were increased after extension of I/R and H/R periods. Moreover, overexpression of CBX7 could elevate the expression of CBX7, GRP78, p-eIF2α, and CHOP. However, CBX7 inhibition with either UNC3866 or genetic knockdown led to reduced expression of GRP78, p-eIF2α, and CHOP through nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 activation in I/R and H/R injury. Furthermore, ML385, the Nrf2 inhibitor, could elevate endoplasmic reticulum stress levels, abrogating the protective effects of UNC3866 against renal I/R injury. In conclusion, our results demonstrated that CBX7 inhibition alleviated acute kidney injury by preventing endoplasmic reticulum stress via the Nrf2/HO-1 pathway, indicating that CBX7 inhibitor could be a potential therapeutic target for renal I/R injury.

The anti-mitotic agents PTC-028 and PTC596 display potent activity in pre-clinical models of multiple myeloma but challenge the role of BMI-1 as an essential tumour gene.

Future progress in the treatment of multiple myeloma (MM) requires both the characterisation of key drivers of the disease and novel, innovative approaches to tackle these vulnerabilities. The present study focussed on the pre-clinical evaluation of a novel drug class, BMI-1 modulators, in MM. We demonstrate potent activity of PTC-028 and PTC596 in a comprehensive set of in vitro and in vivo models, including models of drug resistance and stromal support. Treatment of MM cells with PTC-028 and PTC596 downregulated BMI-1 protein levels, which was found to correlate with drug activity. Surprisingly, BMI-1 was dispensable for the activity of BMI-1 modulators and MM cell growth. Our data rather point to mitotic arrest accompanied by myeloid cell leukaemia-1 (MCL-1) loss as key anti-MM mechanisms and reveal impaired MYC and AKT signalling activity due to BMI-1 modulator treatment. Moreover, we observed a complete eradication of MM after PTC596 treatment in the 5TGM.1 in vivo model and define epigenetic compounds and B cell leukaemia/lymphoma 2 homology domain 3 (BH3) mimetics as promising combination partners. These results bring into question the postulated role of BMI-1 as an essential MM gene and confirm BMI-1 modulators as potent anti-mitotic agents with encouraging pre-clinical activity that supports their rapid translation into clinical trials.

Chromatin remodeling controls Kaposi's sarcoma-associated herpesvirus reactivation from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, the endothelial cell cancer Kaposi's sarcoma, and two B cell cancers, Primary Effusion Lymphoma and multicentric Castleman's disease. KSHV has latent and lytic phases of the viral life cycle, and while both contribute to viral pathogenesis, lytic proteins contribute to KSHV-mediated oncogenesis. Reactivation from latency is driven by the KSHV lytic gene transactivator RTA, and RTA transcription is controlled by epigenetic modifications. To identify host chromatin-modifying proteins that are involved in the latent to lytic transition, we screened a panel of inhibitors that target epigenetic regulatory proteins for their ability to stimulate KSHV reactivation. We found several novel regulators of viral reactivation: an inhibitor of Bmi1, PTC-209, two additional histone deacetylase inhibitors, Romidepsin and Panobinostat, and the bromodomain inhibitor (+)-JQ1. All of these compounds stimulate lytic gene expression, viral genome replication, and release of infectious virions. Treatment with Romidepsin, Panobinostat, and PTC-209 induces histone modifications at the RTA promoter, and results in nucleosome depletion at this locus. Finally, silencing Bmi1 induces KSHV reactivation, indicating that Bmi1, a member of the Polycomb repressive complex 1, is critical for maintaining KSHV latency.

Inhibition of RING1B alters lineage specificity in human embryonic stem cells.

Polycomb group (PcG) proteins are histone modifiers which are known to perform transcriptional repression and have been shown to be critical during murine embryonic development. PcGs are broadly characterized into polycomb repressive complex 1 (PRC1) and 2 and (PRC2). RING1B, core catalytic unit of PRC1 performs H2AK119 monoubiquitination leading to transcriptional repression. We used human embryonic stem cell (hESC) line to study the fate of pluripotent stem cells (PSCs) under inhibition of RING1B, as its role in human development is still to be completely explored. Embryoid bodies (EBs) were generated to differentiate hESCs using hanging drop method. PRT4165 (synthetic RING1B catalytic activity inhibitor) was added to undifferentiated and differentiated cells for 24 h. When we inhibited RING1B in undifferentiated cells, OCT4 levels remained unchanged indicating RING1B does not regulate pluripotency. The drug when added to differentiated cells led to decrease in the levels of RING1B, BMI1, and H2AK119ub1. Interestingly, we also report that the differentiated cells show an increased expression of neuroectodermal markers: SOX1 and PAX6 as well as expression of other neuroectodermal markers such as TUJ1, FOXG1, and NCAM. However, there was reduction in expression of endodermal (SOX17 and FOXA2) mesodermal marker BRACHYURY and TBX5 in treated EBs compared with control EBs. In summary, alteration of RING1B catalytic activity in hESCs during differentiation promotes neuroectodermal differentiation thus, we demonstrate critical role of RING1B in regulating neural differentiation. The strategy of inhibiting RING1B could be used to direct PSCs towards early neuronal fate.

Cbx2, a PcG Family Gene, Plays a Regulatory Role in Medaka Gonadal Development.

Chromobox homolog 2 (CBX2), a key member of the polycomb group (PcG) family, is essential for gonadal development in mammals. A functional deficiency or genetic mutation in cbx2 can lead to sex reversal in mice and humans. However, little is known about the function of cbx2 in gonadal development in fish. In this study, the cbx2 gene was identified in medaka, which is a model species for the study of gonadal development in fish. Transcription of cbx2 was abundant in the gonads, with testicular levels relatively higher than ovarian levels. In situ hybridization (ISH) revealed that cbx2 mRNA was predominately localized in spermatogonia and spermatocytes, and was also observed in oocytes at stages I, II, and III. Furthermore, cbx2 and vasa (a marker gene) were co-localized in germ cells by fluorescent in situ hybridization (FISH). After cbx2 knockdown in the gonads by RNA interference (RNAi), the sex-related genes, including sox9 and foxl2, were influenced. These results suggest that cbx2 not only plays a positive role in spermatogenesis and oogenesis but is also involved in gonadal differentiation through regulating the expression levels of sex-related genes in fish.

Identification of BMI1 promoter inhibitors from Streptomyces sp. IFM-11958.

B-cell-specific Moloney murine leukemia virus region 1 (BMI1) is a central component of polycomb repressive complex 1 (PRC1), which maintains epigenetic repression of genes expression via chromatin condensation. BMI1 overexpression downregulates the expression of tumor suppressor genes, such as p16Ink4a and PTEN. BMI1 expression is upregulated in cancer stem cells (CSCs). Therefore, inhibitors of BMI1 expression have potential as therapeutic agents for cancer. This study aimed to identify BMI1 promoter inhibitors from actinomycetes. Using a recently constructed BMI1 promoter assay, we isolated three known compounds, elaiophylin (1), 2-methylelaiophylin (2), and nocardamin (3), from Streptomyces sp. IFM-11958 that inhibited BMI1 promoter activity with IC50 values of 30 nM, 447 nM, 22 µM, respectively. Elaiophylin (1) was the most potent. Western blot and PCR analyses revealed that elaiophylin (1) inhibited BMI1 expression at the mRNA level in human prostate cancer cells (DU145). Elaiophylin (1) also inhibited the sphere-forming activity of human hepatocellular carcinoma cells (Huh7), indicating that elaiophylin (1) suppresses the self-renewal capacity of CSCs. Elaiophylin (1) is the first BMI1 promoter inhibitor isolated from actinomycete metabolites.

Long Intergenic Noncoding RNA 00152 Promotes Glioma Cell Proliferation and Invasion by Interacting with MiR-16.

BACKGROUND/AIMS: Long noncoding RNAs (lncRNAs) are a novel class of protein-noncoding transcripts that are aberrantly expressed in multiple diseases including cancers. LINC00152 has been identified as an oncogene involved in many kinds of cancer; however, its expression pattern and function in human glioma remain unclear. METHODS: Quantitative real-time polymerase chain reaction was carried out to measure LINC00152 expression in human glioma cell lines and tissues. CCK-8 and EdU assays were performed to assess cell proliferation, and scratch assays and Transwell assays were used to assess cell migration and invasion, respectively. Luciferase reporter assays were carried out to determine the interaction between miR-16 and LINC00152. In vivo experiments were conducted to assess tumor formation. RESULTS: LINC00152 was found to be significantly upregulated in human glioma cell lines and clinical samples. Knockdown of LINC00152 suppressed glioma cell proliferation, migration, and invasion in vitro. In vivo assays in nude mice confirmed that LINC00152 knockdown inhibits tumor growth. Furthermore, mechanistic investigation showed that LINC00152 binds to miR-16 in a sequence-specific manner and suppresses its expression. miR-16 inhibition strongly attenuated LINC00152 knockdown-mediated suppressive effects on proliferation, migration, and invasion. Moreover, LINC00152 induced BMI1 expression by sponging miR-16; this effect further promoted glioma cell proliferation and invasion. CONCLUSION: We regard LINC00152 as an oncogenic lncRNA promoting glioma cell proliferation and invasion and as a potential target for human glioma treatment.

BMI1 activates WNT signaling in colon cancer by negatively regulating the WNT antagonist IDAX.

Aberrant activation of Wnt signaling plays a critical role in the development of colon cancer. BMI, a component of the polycomb repressive complex (PRC1), is upregulated in various types of cancer and contributes to epigenetic silencing of tumor suppressors. In this study, we showed that BMI1 is upregulated in colon cancer tissues and cell lines. Overexpression of BMI1 in primary epithelial colon cells promotes cellular growth and activates WNT pathway, while BMI1 silencing in colon cancer cells represses these effects. We also found that BMI1 binds to the promoter of IDAX, a Wnt antagonist, and decreases its transcription. Expression of IDAX is downregulated in colon cancer tissues and cell lines and negatively correlated with BMI1 in colon cancer tissues. Furthermore, Silencing of IDAX counteracts the effects of BMI1 suppression, while its overexpression reverses oncogenic effects of BMI1. Together, these findings indicate that BMI1-mediated IDAX epigenetic suppression is crucial for enhancement of colon carcinogenesis, suggesting that BMI1∖IDAX axis as a potential novel diagnostic and therapeutic target of colon cancer.

A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1.

We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a K(d) of ∼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.

RING1A and BMI1 bookmark active genes via ubiquitination of chromatin-associated proteins.

During mitosis the chromatin undergoes dramatic architectural changes with the halting of the transcriptional processes and evacuation of nearly all transcription associated machinery from genes and promoters. Molecular bookmarking of genes during mitosis is a mechanism of faithfully transmitting cell-specific transcription patterns through cell division. We previously discovered chromatin ubiquitination at active promoters as a potential mitotic bookmark. In this study, we identify the enzymes involved in the deposition of ubiquitin before mitosis. We find that the polycomb complex proteins BMI1 and RING1A regulate the ubiquitination of chromatin associated proteins bound to promoters, and this modification is necessary for the expression of marked genes once the cells enter G1. Depletion of RING1A, and thus inactivation of mitotic bookmarking by ubiquitination, is deleterious to progression through G1, cell survival and proliferation. Though the polycomb complex proteins are thought to primarily regulate gene expression by transcriptional repression, in this study, we discover that these two polycomb proteins regulate the transcription of active genes during the mitosis to G1 transition.

Enhancing the therapeutic effect via elimination of hepatocellular carcinoma stem cells using Bmi1 siRNA delivered by cationic cisplatin nanocapsules.

Resistance of hepatocellular carcinoma (HCC) to systemic chemotherapy is partially due to presence of drug-resistant cancer stem cells. Bmi1 protein is essential for survival and proliferation of HCC cancer stem cells (CSCs). Here, we report that Bmi1 siRNA (Bmi1siR) loaded in cationic nanocapsules of cisplatin (NPC) eliminated stem cells in situ HCC in mice. NPC/Bmi1siR was fabricated via electrostatic complexation of Bmi1 siRNA to NPCs, which had cores composed of cisplatin and were coated with cationic lipids. In vivo, NPC/Bmi1siR showed higher anti-tumor activity in HCC bearing mice compared with cisplatin or NPC. Critically, both flow cytometry (FACS) analysis in vitro and histological examination in vivo revealed that side population or CD133+ HCC cells were dramatically decreased by NPC/Bmi1siR treatment, suggesting that HCC CSCs were eliminated. Altogether, our results suggest that drug resistance of HCC can be overcome by co-delivering Bmi1 siRNA with cisplatin in cationic nanocapsules.

Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of Histone H3 Lysine 27 (H3K27) Deacetylation and Methylation.

Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity, and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of the brown adipocyte thermogenic program. The Hdac1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or ß3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing Hdac1 profoundly blocks, whereas deleting Hdac1 significantly enhances, ß-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. ß-Adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor γ co-activator 1α (Pgc1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from (and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to) Ucp1 and Pgc1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates the brown adipocyte thermogenic program, and inhibiting Hdac1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching the transcriptional repressive state to the active state at the promoters of Ucp1 and Pgc1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis.

Aza-amino acid scanning of chromobox homolog 7 (CBX7) ligands.

An aza-amino acid scan of peptide inhibitors of the chromobox homolog 7 (CBX7) was performed to study the conformational requirements for affinity to the methyllysine reader protein. Twelve azapeptide analogues were prepared using three different approaches employing respectively N-(Fmoc)aza-amino acid chlorides and submonomer azapeptide synthesis to install systematically aza-residues at the first four residues of the peptide, as well as to provide aza-lysine residues possessing saturated and unsaturated side chains. The aza-peptide ligands were evaluated in a chromobox homolog 7 binding assay, providing useful insight into structural requirements for affinity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

MicroRNA-31 is a transcriptional target of histone deacetylase inhibitors and a regulator of cellular senescence.

MicroRNAs (miRNAs) have emerged as important regulators of tumorigenesis. Several miRNAs, which can function either as oncomiRs or tumor suppressive miRs are deregulated in cancer cells. The microRNA-31 (miR-31) has been shown to be overexpressed in metastatic breast cancer. It promotes multiple oncogenic phenotypes, including proliferation, motility, and invasion of cancer cells. Using a breast cancer-related miRNA array analysis, we identified miR-31 as a novel target of histone deacetylase inhibitors (HDACi) in breast cancer cells. Specifically, we show that sodium butyrate (NaB) and panobinostat (LBH589), two broad-spectrum HDAC inhibitors up-regulate hsa-miR-31 (miR-31). The up-regulation of miR-31 was accompanied by repression of the polycomb group (PcG) protein BMI1 and induction of cellular senescence. We further show that inhibition of miR-31 overcomes the senescence-inducing effect of HDACi, and restores expression of the PcG protein BMI1. Interestingly, BMI1 also acts as a repressor of miR-31 transcription, suggesting a cross-negative feedback loop between the expression of miR-31 and BMI1. Our data suggest that miR-31 is an important physiological target of HDACi, and that it is an important regulator of senescence relevant to cancer. These studies further suggest that manipulation of miR-31 expression can be used to modulate senescence-related pathological conditions such as cancer, and the aging process.

Potential role of Shh-Gli1-BMI1 signaling pathway nexus in glioma chemoresistance.

Chemoresistance is a common hurdle for the proper treatment of gliomas. The role of Shh-Gli1 signaling in glioma progression has been reported. However, its role in glioma chemoresistance has not been well studied yet. In this work, we found that Shh-Gli1 signaling regulates the expression of one stem cell marker, BMI1 (B cell-specific Moloney murine leukemia virus), in glioma. Interestingly, we also demonstrated high expression of MRP1 (multi-drug resistance protein 1) in glioma. MRP1 expression was decreased by BMI1 siRNA and Shh-Gli1 cell signaling specific inhibitor GANT61 in our experiments. GANT61 very efficiently inhibited cell colony growth in glioma cell lines, compared to temozolomide. Moreover, a synergic effect of GANT61 and temozolomide drastically decreased the LD50 of temozolomide in the cell colony experiments. Therefore, our results suggest that there is a potential nexus of Shh-Gli1-BMI1 cell signaling to regulate MRP1 and to promote chemoresistance in glioma. Henceforth, our study opens the possibility of facing new targets, Gli1 and BMI1, for the effective treatment of glioma suppression of chemoresistance with adjuvant therapy of GANT61 and temozolomide.