Role Of BMI1 Research Articles

A new digital droplet PCR method for looking at epigenetics in diffuse large B-cell lymphomas: The role of BMI1, EZH2, and USP22 genes.

Epigenetics has been shown to be relevant in oncology: BMI1 overexpression has been reported in leukemias, EZH2 mutations have been found in follicular lymphoma, and USP22 seems to stabilize BMI1 protein. In this study, we measured the expression of BMI1, EZH2, and USP22 in lymph nodes from 56 diffuse large B-cell lymphoma (DLBCL) patients. A new multiplex digital droplet PCR (ddPCR) has been set up to measure the expression of 4 genes (BMI1, EZH2, USP22, and GAPDH) in the same reaction on RNA extracted from paraffin-embedded tissues. The specificity of ddPCR was confirmed by a 100% alignment on the BLAST platform and its repeatability demonstrated by duplicates. A strict correlation between expression of BMI1 and EZH2 and BMI1 and USP22 has been found, and high expression of these genes was correlated with extra-nodal lymphomas. Progression-free survival (PFS) and overall survival (OS) were conditioned by IPI, bone marrow infiltration, and the complete response achievement. High levels of BMI1 and USP22 did not condition the response to therapy, but impaired the PFS, especially for patients defined at "high risk" based on the cell of origin (no germinal center [GCB]), high BCL2 expression, and IPI 3-5. In this subgroup, the probability of relapse/progression was twice higher than that of patients carrying low BMI1 and USP22 levels. High expression of BMI1 and of USP22 might be a poor prognostic factor in DLBCL, and might represent the target for novel inhibitors.

BMI1 is required for melanocyte stem cell maintenance and hair pigmentation.

The epigenetic repressor BMI1 plays an integral role in promoting the self-renewal and proliferation of many adult stem cell populations, and also tumor types, primarily through silencing the Cdkn2a locus, which encodes the tumor suppressors p16Ink4a and p19Arf . However, in cutaneous melanoma, BMI1 drives epithelial-mesenchymal transition programs, and thus metastasis, while having little impact on proliferation or primary tumor growth. This raised questions about the requirement and role for BMI1 in melanocyte stem cell (McSC) biology. Here, we demonstrate that murine melanocyte-specific Bmi1 deletion causes premature hair greying and gradual loss of melanocyte lineage cells. Depilation enhances this hair greying defect, accelerating depletion of McSCs in early hair cycles, suggesting that BMI1 acts to protect McSCs against stress. RNA-seq of McSCs, harvested before onset of detectable phenotypic defects, revealed that Bmi1 deletion derepresses p16Ink4a and p19Arf , as observed in many other stem cell contexts. Additionally, BMI1 loss downregulated the glutathione S-transferase enzymes, Gsta1 and Gsta2, which can suppress oxidative stress. Accordingly, treatment with the antioxidant N-acetyl cysteine (NAC) partially rescued melanocyte expansion. Together, our data establish a critical function for BMI1 in McSC maintenance that reflects a partial role for suppression of oxidative stress, and likely transcriptional repression of Cdkn2a.

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.

A protein synthesis brake for hematopoietic stem cell maintenance.

Bmi1 is essential for normal and leukemic hematopoiesis, but its target genes in hematopoietic stem cells (HSCs) are incompletely understood. In this issue of Genes & Development, Burgess et al. (pp. 887-900) demonstrate a novel role of Bmi1 in regulating ribosome biogenesis and protein synthesis. Bmi1-deficient HSCs exhibited reduced transplantability, with the up-regulation of ARX and genes involved in ribosome biogenesis. However, depletion of ARX or its known targets, p16 Ink4a /p19 Arf , only partially rescues Bmi1 loss-induced hematopoietic defects. They further demonstrate an increased protein synthesis rate and resultant proteostatic stress in Bmi1 -/- HSCs, indicating a novel mechanism by which Bmi1 controls HSC maintenance.

BMI1 Silencing Induces Mitochondrial Dysfunction in Lung Epithelial Cells Exposed to Hyperoxia.

Acute Lung Injury (ALI), characterized by bilateral pulmonary infiltrates that restrict gas exchange, leads to respiratory failure. It is caused by an innate immune response with white blood cell infiltration of the lungs, release of cytokines, an increase in reactive oxygen species (ROS), oxidative stress, and changes in mitochondrial function. Mitochondrial alterations, changes in respiration, ATP production and the unbalancing fusion and fission processes are key events in ALI pathogenesis and increase mitophagy. Research indicates that BMI1 (B cell-specific Moloney murine leukemia virus integration site 1), a protein of the Polycomb repressive complex 1, is a cell cycle and survival regulator that plays a role in mitochondrial function. BMI1-silenced cultured lung epithelial cells were exposed to hyperoxia to determine the role of BMI1 in mitochondrial metabolism. Its expression significantly decreases in human lung epithelial cells (H441) following hyperoxic insult, as determined by western blot, Qrt-PCR, and functional analysis. This decrease correlates with an increase in mitophagy proteins, PINK1, Parkin, and DJ1; an increase in the expression of tumor suppressor PTEN; changes in the expression of mitochondrial biomarkers; and decreases in the oxygen consumption rate (OCR) and tricarboxylic acid enzyme activity. Our bioinformatics analysis suggested that the BMI1 multifunctionality is determined by its high level of intrinsic disorder that defines the ability of this protein to bind to numerous cellular partners. These results demonstrate a close relationship between BMI1 expression and mitochondrial health in hyperoxia-induced acute lung injury (HALI) and indicate that BMI1 is a potential therapeutic target to treat ALI and Acute Respiratory Distress Syndrome.

BMI-1 regulates DNA end resection and homologous recombination repair.

BMI-1 is an essential regulator of transcriptional silencing during development. Recently, the role of BMI-1 in the DNA damage response has gained much attention, but the exact mechanism of how BMI-1 participates in the process is unclear. Here, we establish a role for BMI-1 in the repair of DNA double-strand breaks by homologous recombination (HR), where it promotes DNA end resection. Mechanistically, BMI-1 mediates DNA end resection by facilitating the recruitment of CtIP, thus allowing RPA and RAD51 accumulation at DNA damage sites. Interestingly, treatment with transcription inhibitors rescues the DNA end resection defects of BMI-1-depleted cells, suggesting BMI-1-dependent transcriptional silencing mediates DNA end resection. Moreover, we find that H2A ubiquitylation at K119 (H2AK119ub) promotes end resection. Taken together, our results identify BMI-1-mediated transcriptional silencing and promotion of H2AK119ub deposition as essential regulators of DNA end resection and thus the progression of HR.

Resveratrol Attenuates High-Fat Diet-Induced Hepatic Lipotoxicity by Upregulating Bmi-1 Expression.

Resveratrol (RES), a natural polyphenol phytoalexin, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD). However, its roles on protection of liver from lipotoxicity and underlying mechanism are not fully understood. In this study, we investigated the impacts of RES on alleviating hepatic lipotoxicity and corresponding molecular mechanism. Impacts of RES on oleic acid (OA)-induced lipotoxicity were assessed in L02 cells and C57BL/6J mice, respectively. In L02 cells, lipotoxicity was assessed by detection of apoptosis, mitochondrial function, oxidative stress and ROS-related signaling. In mice, lipotoxicity was evaluated by detecting hepatic function, serum enzyme activity, and reactive oxygen species (ROS) levels. We found that RES reduced OA-induced apoptosis, mitochondrial dysfunction, ROS generation, and DNA damage in L02 cells. RES also decreased expression of cleaved caspase-3 and p53 and increased expression of B-cell lymphoma 2 (Bcl-2). Importantly, RES protected mice from high-fat diet-induced hepatic lipotoxicity, demonstrated by reduced ROS levels and lipid peroxidation. Mechanically, B lymphoma Mo-MLV insertion region 1 (Bmi-1) expression and antioxidative superoxide dismutase were increased after RES treatment. Further mechanistic analysis indicated that protection effects of RES against OA-induced lipotoxicity were abrogated by Bmi-1 small interference RNA (siRNA) in L02 cells. SIGNIFICANCE STATEMENT: Results from clinical studies about the effect of RES on NAFLD are inconsistent and inconclusive. This study confirms the protective role of RES as an anti-ROS agent and its ability to alleviate DNA damage through a pathway involving p53/p21 signaling. Further mechanistic analysis indicated that protection effects of RES were relative with Bmi-1. This is the first study on the role of Bmi-1 in the pathogenesis of NAFLD and the target of resveratrol against NAFLD.

Repression of the Antioxidant Pyrroloquinoline Quinone in Skin Aging Induced by Bmi-1 Deficiency.

It is uncertain whether Bmi-1 deficiency could lead to skin aging by redox imbalance and DNA damage. In this study, we first confirmed that Bmi-1 had a relatively high expression level in the skin and Bmi-1 expression levels gradually decreased with age. Then, we studied the role of Bmi-1 in the skin using a Bmi-1−/− mouse model. Bmi-1−/− mice were supplemented with or without pyrroloquinoline quinone (PQQ) for 5 weeks, and their skin phenotypes were compared with Bmi1−/− and wild-type littermates. Our results showed that Bmi-1−/− mice displayed decreased vertical thickness of skin, sparse hair follicles, and thinner and more irregular collagen bundles. Mechanistically, increased oxidative stress with reducing antioxidant capacity and induced DNA damage occurred in Bmi-1−/− mice. Subsequently, this would lead to reduced cell proliferation, increased cell senescence and matrix metalloproteinases (MMPs), and the degradation of fibroblast function and further reduce collagen synthesis. All pathological alterations in the skin of Bmi-1−/− mice were alleviated by PQQ supplementation. These results demonstrated that Bmi-1 might play a key role in protection from skin aging by maintaining redox balance and inhibiting DNA damage response and will be a novel and potential target for preventing skin aging.

Abstract 125: First report of a full spectrum of de novo induced human breast cancer subtypes generated from oncogene-transduced freshly isolated normal human mammary cells

Abstract Human breast cancers are genetically and biologically heterogeneous with variable responses to current treatments. As an alternative strategy to identify causal and shared mechanisms, we have been exploring the utility of creating de novo models of human breast cancers from freshly isolated, purified subsets of normal human mammary cells that are lentivirally transduced with defined genes of interest and then transplanted subcutaneously in Matrigel into highly immunodeficient adult female Nonobese-diabetic/Rag1-/--IL2Rγc-/- (NRG) mice. In an initial study using this approach, we discovered that forced expression of KRASG12D alone rapidly generates small (growth-arrested), but polyclonal and serially transplantable invasive ductal carcinomas (IDCs) from both normal basal and luminal progenitor subsets of normal human mammary cells. This outcome is neither replaced nor influenced by transduction of the cells with mutant forms of TP53 or PI3KCA (Nguyen et al Nature 2015). More recently, we have found that transduction of these normal human mammary cells with a cDNA encoding a myristylated form of AKT1 (myrAKT1) produces a model of ductal carcinoma in situ (DCIS), whereas forced expression of BMI1, MYC and TP53R273C with KRASG12D (KBMT) rapidly produces large and aggressive tumors within 6 weeks post-transplant, with intermediate outcomes obtained when either MYC or TP53R273C is omitted. Immunohistochemical staining for estrogen, progesterone and epidermal growth factor receptor 2 suggests that the KBMT tumors are triple negative, and that the proportion of proliferating cells in the KBMT tumors is higher than in the tumors induced by just KRASG12D. The latter finding is consistent with the known role of BMI1 as a positive upstream regulator of proliferation via its ability to inhibit expression of p16INK4a, although the relevance of this activity in the KBMT model has yet to be established. At the opposite end of the spectrum, we have also shown that co-induced suppression of Y-Box binding protein-1 (YBX1) expression blocks the in vivo growth of KRASG12D-induced IDCs and its forced increased expression enhances the growth of myrAKT-induced DCIS cells. Taken together, these findings illustrate the advantages of creating and characterizing de novo models of human breast cancers to identify and manipulate pathways that are required for the acquisition of malignant properties. Citation Format: Susanna Tan, Sylvain Lefort, Amal El-Naggar, Davide Pellacani, Poul H. Sorensen, Connie Eaves. First report of a full spectrum of de novo induced human breast cancer subtypes generated from oncogene-transduced freshly isolated normal human mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 125.

Exosome-Mediated Bmi1 Promotes Cholangiocarcinoma Growth and Metastasis Through the Differential Regulation of miR-320b and miR-27b-3p

Background: Bmi1 is up-regulated in many human malignancies. However, the role of Bmi1 in the progression of cholangiocarcinoma (CCA) remains largely undetermined. In the present study, we investigated the biological function and potential mechanisms of Bmi1 in the progression of CCA. Methods: The expression of Bmi1 was detected in 2 cohorts of human CCA tissues using immunohistochemistry. Function of Bmi1 or exosomal Bmi1 was investigated using CCK8, colony formation, transwell, vascular tube formation assays and tumour xenograft models. RNA-seq and miRNA-seq were used for identification of downstream targets and upstream regulation miRNAs. Findings: Bmi1 was significantly up-regulated in CCA tissues, and associated with tumour size and lymph nodes metastasis, which is an independent prognostic marker for CCA. Overexpresson of Bmi1 promoted CCA cells proliferation, migration, and invasion. And, depletion of Bmi1 could inhibit growth and metastases of CCA cells in vitro and in vivo. QBC-939-derived exosomes transferred Bmi1 to RBE cells and human umbilical vein endothelial cells, thus promoting proliferation, metastasis, and angiogenesis of CCA. Bmi1 is involved in regulation of p53 pathway and EMT in CCA. Based on the next-generation sequencing, we identified and verified Bmi1 was downregulated by miR-320b while upregulated by miR-27b-3p in CCA. Interpretation: Bmi1, or exosomal Bmi1, playes a critical role in tumourgenosis and metastasis of CCA, and can be served as a potential predictive biomarker for CAA prognosis. Funding: National Natural Science Foundation of China (No. 81900728); Shandong Province Natural Science Foundation (ZR2020MH238); Shandong Medical and Health Technology Development Project (2018WSB20002). Declaration of Interest: None to declare. Ethical Approval: The study was approved by the Ethics Committee of Qilu Hospital of Shandong University, and written informed consent was obtained from each patient.

Abstract PO-025: BMI1 is required for human and murine pancreatic cancer progression and controls metabolism and cell proliferation

Abstract Pancreatic cancer is the third leading cause of cancer-related death in the United States, and deeper understanding into factors that control its progression will be key to developing future treatments. Previously, we showed that Bmi1 is required for the initiation of pancreatic cancer in mice, and enhances growth of pancreatic cancer cells in vitro. However, the role of Bmi1 in established tumors and the mechanisms underlying its requirement have so far been elusive. To investigate this, we used a CRISPR/Cas9 strategy to delete BMI1 in primary cell lines derived from human pancreatic tumors as well as murine pancreatic cancer cell lines, and created clonal lines lacking BMI1 expression. We then used subcutaneous tumor growth models as well as transcriptomic and metabolomic profiling to define the role of BMI1 expression in tumor growth. BMI1 knockdown in human and mouse pancreatic cancer cells resulted in slower growth in vitro and in vivo compared to controls. Histologically, control tumors were moderately differentiated, while BMI1-/- tumors were small or histologically undetectable. BMI1-/- tumors with sufficient tumor tissue displayed prominent extracellular mucin pools, with few cancer cell clusters interspersed within mucin and dead cell debris. Interestingly, the remaining cancer cells in the BMI1-/- tumors, identified by CK19 immunostaining, still expressed phosphorylated ERK, indicating MAPK pathway activation, as expected in Kras mutant cells. Thus, BMI1 expression was required for the growth of both human and murine pancreatic cancer cells. Given this, we sought to determine the functional processes regulated by BMI1 to facilitate pancreatic cancer growth by using both transcriptomic and metabolomic comparison of WT or BMI1-/- human pancreatic cancer cells. Further analysis revealed changes in pathways with known importance in pancreatic cancer, including many related to metabolism and cell proliferation. Strikingly, the expression of every enzyme in the glycolytic pathway was downregulated in BMI1-/- cells. Loss of BMI1 also resulted in changes in pathways that control cell proliferation, and specifically the G2/M checkpoint, possibly explaining the growth defect observed in vitro and in vivo. Together, our data show that BMI1 is a key regulator of essential cellular processes for human and mouse pancreatic cancer growth, including metabolism and cell cycle progression. Further study will be required to determine the exact mechanism of BMI1 regulation of these pathways, as well as its potential as a therapeutic target in pancreatic cancer. Citation Format: Heather K. Schofield, Christopher J. Halbrook, Annachiara Del Vecchio, Donovan Drouillard, Zeribe C Nwosu, Joyce K. Thompson, Carlos Espinoza, Yaqing Zhang, Costas A Lyssiotis, Marina Pasca di Magliano, Filip Bednar. BMI1 is required for human and murine pancreatic cancer progression and controls metabolism and cell proliferation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-025.

Fluorescence imaging of biochemical relationship between ubiquitinated histone 2A and Polycomb complex protein BMI1

Several in vitro experiments have highlighted that the Polycomb group protein BMI1 plays a pivotal role in determining the biological functions of the Polycomb Repressor Complex 1 (PRC1), including its E3-ligase activity towards the Lys119 of histone H2A to yield ubiquitinated uH2A. The role of BMI1 in the epigenetic activity of PRC1 is particularly relevant in several cancers, particularly Non-Small Cell Lung Cancer (NSCLC). In this study, using indirect immunofluorescence protocols implemented on a confocal microscopy apparatus, we investigated the relationship between BMI1 and uH2A at different resolutions, in cultured (A549) and clinical NSCLC tissues, at the single cell level. In both cases, we observed a linear dependence of uH2A concentration upon BMI1 expression at the single nucleus level, indicating that the association of BMI1 to PRC1, which is needed for E3-ligase activity, occurs linearly in the physiological BMI1 concentration range. Additionally, in the NSCLC cell line model, a minor pool of uH2A may exist in absence of concurrent BMI1 expression, indicating non-exclusive, although predominant, role of BMI1 in the amplification of the E3-ligase activity of PRC1. A pharmacological downregulator of BMI1, PTC-209, was also tested in this context. Finally, the absence of significant colocalization (as measured by the Pearson's coefficient) between BMI1 and uH2A submicron clusters hints to a dynamic model where PRC1 resides transiently at ubiquitination sites. Beside unveiling subtle functional relationships between BMI1 and uH2A, these results also validate the use of uH2A as downstream “reporter” for BMI1 activity at the nuclear level in NSCLC contexts.

Co-inhibition of BMI1 and Mel18 enhances chemosensitivity of esophageal squamous cell carcinoma in vitro and in vivo.

Esophageal squamous cell carcinoma (ESCC) accounts for almost 90% of esophageal cancer cases and is the sixth most common cause of cancer-associated mortality worldwide. Cisplatin is the standard therapeutic reagent for ESCC; however, chemoresistance frequently occurs after a few weeks, which leads to ESCC recurrence. Aberrant expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) has been reported to activate multiple growth-regulatory pathways, induce antiapoptotic abilities in numerous types of cancer cells and promote chemoresistance. However, to the best of our knowledge, the role of BMI1 in cisplatin-resistant ESCC, and the interaction between BMI1 and its homologue melanoma nuclear protein 18 (Mel18) remain unknown. The present study identified that knockdown of BMI1 promoted cytotoxic effects of cisplatin, and co-inhibition of Mel18 and BMI1 enhanced cisplatin-induced apoptosis and cytotoxicity. Inhibition of BMI1 and Mel18 also suppressed the expression of c-Myc. Furthermore, this combined inhibition sensitized esophageal xenograft tumors to cisplatin to a greater extent compared with BMI1 inhibition alone. In summary, the current study demonstrated that inhibition of BMI1 and Mel18 could increase the sensitivity of esophageal cancer cells to cisplatin via inhibition of c-Myc. Therefore, combined targeting of BMI1 and Mel18 may serve as a promising therapeutic strategy for sensitizing ESCC to chemotherapy.

Abstract LB-207: A novel role for polycomb group protein BMI1 in prostate cancer

Abstract BMI1, a Polycomb Group (PcG) protein, plays a critical role in epigenetic regulation of cancer stem cell self-renewal, cell differentiation and proliferation. BMI1 is upregulated in multiple types of cancer, including prostate cancer. As a key component of Polycomb Repressive Complex 1 (PRC1), BMI1 can exert its oncogenic functions by enhancing the enzymatic activities of RING1B to ubiquitinate histone H2A at lysine 119 and repress gene transcription. Here we report a PRC1-independent role of BMI1 that is critical for castrate-resistant prostate cancer (CRPC) progression. BMI1 binds the androgen receptor (AR) and prevents MDM2-mediated AR protein degradation, resulting in sustained AR signaling in prostate cancer cells. More importantly, we demonstrate that targeting BMI1 effectively inhibits tumor growth of xenografts that have developed resistance to surgical castration and enzalutamide treatment. These results suggest that blocking BMI1 alone or in combination with anti-AR therapy can be more efficient to suppress prostate tumor growth. Citation Format: Sen Zhu, Dongyu Zhang, Lin Yan, Weihua Jiang, Qipeng Liu, Rui Wang, Bo Xia, Jonathan C. Zhao, Rong-Fu Wang, Xiaobing Shi, Hung-ming Lam, Xuesen Dong, Jindan Yu, Kaifu Chen, Qi Cao. A novel role for polycomb group protein BMI1 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-207.

Polycomb complex protein BMI1 confers resistance to tamoxifen in estrogen receptor positive breast cancer

We report that BMI1 promotes tamoxifen resistance in estrogen receptor (ER)-positive breast cancer (BC). BMI1 overexpression conferred MCF7 and TD47 cells resistance to tamoxifen; BMI1 knockdown sensitized the process. In MCF7-derived tamoxifen resistant cells, BMI1 expression was upregulated and BMI1 knockdown reduced the resistance. BMI1 is an oncogene; its oncogenic activity is attributed to BMI1-stimulated E3 ubiquitin ligase activity, a process that requires BMI1's ring finger (RF) domain. However, a BMI1 mutant without RF conferred tamoxifen resistance. Tamoxifen significantly reduced the growth of xenografts derived from MCF7 cells, but accelerated the growth of tumors produced by BMI1 overexpressing MCF7 cells. BMI1 enhances the pathways that promote resistance to endocrine therapy, including ER, androgen receptor, and MUC1. In patients with ER + BCs (n = 177), BMI1 expression was associated with BC recurrence. In the Curtis dataset consisting of ER + BCs (n = 1506) and ER- BCs (n = 474; cBioPortal), upregulations in BMI1 mRNA expression were correlated with ER + BCs; the upregulation was associated with a set of differentially expressed genes (DEGs). These DEGs were enriched with reductions in immunological processes, indicating a role of BMI1 in downregulation of the immune surveillance.

Polycomb subunit BMI1 determines uterine progesterone responsiveness essential for normal embryo implantation.

Natural and synthetic progestogens have been commonly used to prevent recurrent pregnancy loss in women with inadequate progesterone secretion or reduced progesterone sensitivity. However, the clinical efficacy of progesterone and its analogs for maintaining pregnancy is variable. Additionally, the underlying cause of impaired endometrial progesterone responsiveness during early pregnancy remains unknown. Here, we demonstrated that uterine-selective depletion of BMI1, a key component of the polycomb repressive complex-1 (PRC1), hampers uterine progesterone responsiveness and derails normal uterine receptivity, resulting in implantation failure in mice. We further uncovered genetic and biochemical evidence that BMI1 interacts with the progesterone receptor (PR) and the E3 ligase E6AP in a polycomb complex-independent manner and regulates the PR ubiquitination that is essential for normal progesterone responsiveness. A close association of aberrantly low endometrial BMI1 expression with restrained PR responsiveness in women who had previously had a miscarriage indicated that the role of BMI1 in endometrial PR function is conserved in mice and in humans. In addition to uncovering a potential regulatory mechanism of BMI1 that ensures normal endometrial progesterone responsiveness during early pregnancy, our findings have the potential to help clarify the underlying causes of spontaneous pregnancy loss in women.

Participation of BMI-1 protein in cancer

BMI-1 (B-lymphoma Mo-MLV insertion region 1) protein is a constituent of Polycomb Repressive Complex 1 (PRC1) that via ubiquitination of histone H2A affects expression of many genes. BMI-1 is involved in cellular processes such as DNA repair, proliferation, growth, senescence and apoptosis. BMI-1 plays a key role in biology of stem cells including cancer stem cells by regulation of their self-renewal and differentiation. Accumulating evidence has revealed that overexpression of BMI-1 in many human cancers correlates with disease progression and therapy failure. The results of in vitro and in vivo studies confirm the involvement of BMI-1 in tumor initiation as well as invasion, metastasis and chemoresistance. Taking into account significant role of BMI1 in tumorigenesis, especially associated with cancer stem cells, it seems that this gene may be a promising target of anticancer therapies.

Abstract 5526: Tumor suppressive role of BMI-1 through inhibition of JAK-STAT signaling in leukemia

Abstract BMI-1, which is one of the core components of polycomb repressive complex 1, is frequently found deregulated in patients with hematological disorders. In last decades, researchers concordantly agree that BMI-1 mediates tumorigenesis of leukemia stem cells through p16INK4A leukemogenic pathway. However, accumulating evidences contradict the idea that BMI-1 solely plays an oncogenic role in tumorigenesis. It has been shown BMI-1 depletion favors the development of myelofibrosis in mice; whereas high BMI-1 expression suppressed colony forming ability of MLL-ENL-transformed bone marrow and correlated with higher survival in some cancers. In this study, we hypothesized that BMI-1 plays a tumor suppressive role, which is independent of the regulation of INK4A-ARF locus, in human leukemia. BMI-1 was over-expressed in a panel of myeloid and lymphoid lineage leukemia cells, including HL-60, MV4-11, MonoMac-6, SEM, Nalm-20 and RS4;11. We observed no deregulation of p16INK4A and p14ARF genes by BMI-1, suggesting the regulation of INK4A-ARF locus is independent of BMI-1 modulation in leukemia cells. Nevertheless, over-expression of BMI-1 resulted in significant reduction of leukemia cell proliferation. It is noted that constitutively active JAK-STAT signaling pathway is crucial to leukemia cell survival. By modulation of BMI-1 level, we demonstrated suppression of the activated JAK-STAT signaling pathway in most of the leukemia cell lines with the exception of MonoMac-6 and RS4;11. This is in agreement with the high sensitivity to ruxolitinib, a JAK-STAT inhibitor, in all the tested leukemia cell lines except RS4;11. Importantly, we showed that BMI-1 over-expression could sensitize RS4;11 cells to reduce cell proliferation under ruxolitinib treatment. These results suggest that higher efficacy of ruxolitinib treatment could be achieved under a condition of high cellular level of BMI-1. We further demonstrated that ruxolitinib treatment was more effective in a cohort of AML patient samples (n = 25) with a context of higher BMI-1 expression (p < 0.05). Altogether, our results suggest that BMI-1 functions as a tumor suppressor gene via inhibition of the JAK-STAT signaling pathway. The endogenous level of BMI-1 could be served as an indicator for the effective treatment of JAK-STAT-dependent leukemia cells using ruxolitinib. Citation Format: Yuk Man Lam, Stephen Sze Yuen Lam, Anskar Yu Hung Leung, Ray Kit Ng. Tumor suppressive role of BMI-1 through inhibition of JAK-STAT signaling in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5526. doi:10.1158/1538-7445.AM2017-5526

MiR-203 Interplays with Polycomb Repressive Complexes to Regulate the Proliferation of Neural Stem/Progenitor Cells.

SummaryThe polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are two distinct polycomb group (PcG) proteins that maintain the stable silencing of specific sets of genes through chromatin modifications. Although the PRC2 component EZH2 has been known as an epigenetic regulator in promoting the proliferation of neural stem/progenitor cells (NSPCs), the regulatory network that controls this process remains largely unknown. Here we show that miR-203 is repressed by EZH2 in both embryonic and adult NSPCs. MiR-203 negatively regulates the proliferation of NSPCs. One of PRC1 components, Bmi1, is a downstream target of miR-203 in NSPCs. Conditional knockout of Ezh2 results in decreased proliferation ability of both embryonic and adult NSPCs. Meanwhile, ectopic overexpression of BMI1 rescues the proliferation defects exhibited by miR-203 overexpression or EZH2 deficiency in NSPCs. Therefore, this study provides evidence for coordinated function of the EZH2-miR-203-BMI1 regulatory axis that regulates the proliferation of NSPCs.

DIPG-18. VALIDATION OF BMI-1 AS A THERAPEUTIC TARGET IN DIFFUSE INTRINSIC PONTINE GLIOMA

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor. No effective curative therapy is currently available and no therapeutic advances have been made in several decades. BMI-1 is a member of the multimeric protein complex Polycomb repressor complex 1. It is overexpressed in a number of diseases and malignancies and has been implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. The role of BMI-1 in DIPG is largely unknown. Here, we show that BMI-1 is overexpressed in DIPG patient tissue samples and in patient-derived cancer stem cells independent of H3K27 mutation status. BMI-1 downregulation leads to the inhibition of DIPG patient-derived stem cell proliferation, cell cycle signaling, self-renewal, telomerase expression and activity, causes increased apoptosis, and suppresses DIPG cell invasion. Moreover, targeted inhibition of BMI-1 sensitizes DIPG cells to radiomimetic drug-induced DNA damage. Together, our data validate BMI-1 as a potential therapeutic target to treat children with DIPG regardless of H3 mutation status. *S. S Kumar, S. Sengupta and K. Lee contributed equally to this work.