Loss Of Bmi1 Research Articles

Abstract C072: Bmi1 expression is upregulated in response to injury/stress in the pancreas

Abstract Pancreatic homeostasis is regulated in part through epigenetic mechanisms that restrict or enable access of pancreatic lineage transcription factors to their target genes. These epigenetic protein cascades are also required for regeneration of the pancreas after injury. B-cell Moloney murine leukemia virus integration site 1 (Bmi1), a central protein in the Polycomb repressor complex 1 (PRC1), has been shown to be required for the regeneration of the pancreas after injury and its absence prevents pancreatic neoplasia in mice. Previous work also suggests that Bmi1 marks a subset of acinar cells contributing to pancreatic renewal and tissue maintenance. It remains unclear how Bmi1 expression levels are regulated in response to pancreatic stress and injury. Here we assess the baseline expression of Bmi1 in pancreatic tissue with a genetic reporter mouse model as well as changes in Bmi1 mRNA levels at different time points after the induction of pancreatitis in wild-type mice via RNA in situ hybridization (RNAscope). We also compare the ability of the murine pancreas to regenerate after acute pancreatitis in the presence and absence of Bmi1. Our data indicates that rather than a specific subpopulation, most, if not all, acinar cells in the pancreas express Bmi1 during their life and specific loss of Bmi1 in the pancreas impairs regeneration after acute pancreatitis. We also show an increase in Bmi1 mRNA levels within the pancreas shortly after the induction of acute pancreatitis which reduces as the pancreas resumes its normal histology. Altogether, our data indicate that Bmi1 expression is dynamic, responsive to injury and necessary for pancreatic homeostasis and regeneration. Citation Format: Nur Muhammad Renollet, Joyce K Thompson, Emily Wu, Osama Alkhalili, Nina Steele, Filip Bednar. Bmi1 expression is upregulated in response to injury/stress in the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C072.

Abstract 4441: The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells

Abstract Dysregulation of epigenetic factors is a key component of tumorigenesis. BMI1, a member of the polycomb repressor complex 1 (PRC1), is an oncogenic factor studied in many types of cancer, including pancreatic ductal adenocarcinoma (PDAC). PDAC is the third most common cause of cancer-related death in the United States and investigation of the biology involved in transformation is critical for improving outcomes in this devastating disease. In PDAC, BMI1 is required for initiation of pancreatic cancer in mice and enhances in vitro growth of human and murine tumor cell lines. CRISPR-mediated knock out of BMI1 results in reduced subcutaneous and orthotopic tumor growth in mice and decreased expression of genes related to metabolism, specifically glycolysis, and cell proliferation. BMI1 has also been implicated in regulation of metabolism in other epithelial tumor types, including ovarian cancer. Given these preliminary findings, we hypothesized that loss of BMI1 in multiple human PDAC cell lines would result in altered metabolic activity, reducing the growth and tumorigenic properties of these cells. We demonstrated that BMI1 expression is higher in PDAC cell lines compared to normal pancreatic epithelial cells and normal human donor pancreatic tissue. Following CRISPR-mediated BMI1 knock out in human PDAC cell lines, we investigated the alterations to metabolism using metabolic flux analysis. Loss of BMI1 resulted in lower basal and compensatory rates of glycolysis and increased oxygen consumption rates compared to wild-type cells. Metabolomic analysis demonstrated reduction in all the enzymes involved in the glycolysis pathway with loss of BMI1. Cell growth was reduced by loss of BMI1 in vitro, and orthotopic injection into mice of human cells with BMI1 knock out resulted in decreased tumor size compared to wild-type, recapitulating these findings in vivo. Together, these data identify a role of BMI1 in promotion of tumor growth through regulation of metabolism in pancreatic cancer. Experiments are ongoing to determine the underlying mechanism of this regulation and implications on therapeutic targeting with BMI1/PRC1 complex inhibitors in conjunction with traditional chemotherapy. Citation Format: Jamie N. Mills, Dominik Awad, Heather K. Schofield, Joyce K. Thompson, Hannah Watkoske, Damien Sutton, Nicholas Nedzesky, Donovan Drouillard, Zeribe Nwosu, Carlos Espinoza, Yaqing Zhang, Annachiara Del Vecchio, Christopher J. Halbrook, Marina Pasca di Magliano, Costas A. Lyssiotis, Filip Bednar. The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4441.

Abstract 17416: Epigenetic Regulation of Fibroblast Fate Transition in Cardiac Wound Healing

Background: Fibroblast transitioning between physiological and pathological states can define cardiac wound healing ability of the heart in response to stress. Preventing fibroblast activation and differentiation into myofibroblast formation can attenuate cardiac fibrosis and adverse cardiac remodeling. Changes in chromatin structure and accessibility are one of the essential drivers of gene expression/repression involved in myocardial injury. One such driver of chromatin remodeling is the group of polycomb proteins that alter expression of genes involved in cardiac wound healing including B cell specific Moloney murine leukemia virus integration site 1 (Bmi1), a component of the polycomb repressive complex 1 (PRC1). Bmi1 have been linked to self-renewal, proliferation, quiescence, and lineage commitment in many organs however, how Bmi1 can mediate fibroblasts states and mediate wound healing after cardiac injury is not understood. Hypothesis: Bmi1 mediates myofibroblast activation and transition, scar maturation, stability, and fibrotic responses by regulating histone marks after injury. Methods and Results: To establish the role of Bmi1 in cardiac wound healing, we developed an inducible knock out mouse model for Bmi1, which showed decrease in cardiac structure and function with maladaptive remodeling following myocardial injury. Bmi1 knockout (KO) mice showed significant decrease in ejection fraction and fractional shortening measured by echocardiography after cardiac injury. Concurrently, this significant decrease in heart function was associated with increased fibrosis and DNA damage analyzed using immunohistochemistry. Additionally, we found that the adult cardiac fibroblasts, isolated from the Bmi1 KO mice have increased expression of fibrotic genes including TGFβ, Acta2, and collagen using RTPCR analysis. Using single cell sequencing RNA and ATAC sequencing and cellular staining assays, we found significant changes in fibrotic signaling pathways of TGFβ. We found SMAD3 chromatin accessibility was increased with the loss of Bmi1 with the decrease in H2AK119Ub. Conclusion: Bmi1 mediates wound healing ability by regulating cardiac fibroblast transition after cardiac injury.

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.

Bmi1 Augments Proliferation and Survival of Cortical Bone-Derived Stem Cells after Injury through Novel Epigenetic Signaling via Histone 3 Regulation.

Ischemic heart disease can lead to myocardial infarction (MI), a major cause of morbidity and mortality worldwide. Multiple stem cell types have been safely transferred into failing human hearts, but the overall clinical cardiovascular benefits have been modest. Therefore, there is a dire need to understand the basic biology of stem cells to enhance therapeutic effects. Bmi1 is part of the polycomb repressive complex 1 (PRC1) that is involved in different processes including proliferation, survival and differentiation of stem cells. We isolated cortical bones stem cells (CBSCs) from bone stroma, and they express significantly high levels of Bmi1 compared to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs). Using lentiviral transduction, Bmi1 was knocked down in the CBSCs to determine the effect of loss of Bmi1 on proliferation and survival potential with or without Bmi1 in CBSCs. Our data show that with the loss of Bmi1, there is a decrease in CBSC ability to proliferate and survive during stress. This loss of functionality is attributed to changes in histone modification, specifically histone 3 lysine 27 (H3K27). Without the proper epigenetic regulation, due to the loss of the polycomb protein in CBSCs, there is a significant decrease in cell cycle proteins, including Cyclin B, E2F, and WEE as well as an increase in DNA damage genes, including ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). In conclusion, in the absence of Bmi1, CBSCs lose their proliferative potential, have increased DNA damage and apoptosis, and more cell cycle arrest due to changes in epigenetic modifications. Consequently, Bmi1 plays a critical role in stem cell proliferation and survival through cell cycle regulation, specifically in the CBSCs. This regulation is associated with the histone modification and regulation of Bmi1, therefore indicating a novel mechanism of Bmi1 and the epigenetic regulation of stem cells.

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.

Abstract A22: Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1

Abstract The initial stages of pancreatic ductal adenocarcinoma (PDA) involve an oncogenic Kras-driven metaplastic transition of acinar cells to a more duct-like phenotype referred to as acinar to ductal metaplasia (ADM). The exact mechanism by which Kras reprograms acinar cells to a more duct-like cell at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells in other contexts and rely on appropriate chromatin context for their activity. Deletion of Bmi1, a component of the Polycomb Repressor Complex 1, completely represses Kras-driven ADM and neoplasia in genetically engineered mouse models of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used a genetically engineered mouse model of early pancreatic neoplasia bearing an acinar cell-specific tamoxifen inducible Cre-recombinase under the elastase promoter (Ela-CreER) combined with the Kras G12D oncogenic allele (Kras−LSL-G12D/+). We added the conditional Bmi1 knockout allele (Bmi1−fl) and the Rosa26 tdTomato reporter to generate Ela-CreER, Kras−LSL-G12D/+, Bmi1−fl/fl, R26 tdTomato mice and their litter mate controls with and without KrasG12D and Bmi1. We activated the Cre-recombinase in 6- to 8-week-old mice with 5 daily tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (eight doses per day for two days) one week after the first tamoxifen dose. We used FACS to sort tdTomato+ cells one week after pancreatitis induction and analyzed RNA expression levels of 26 TFs, previously reported to play a role in pancreatic development, homeostasis and neoplasia. Expression levels of three lineage markers (amylase, elastase and cytokeratin 19) were also measured by quantitative RT-PCR. Tamoxifen gavage induced high tdTomato expression in the acinar compartment of Ela-CreER+ mice. RT-qPCR of tdTomato+ cells indicated a loss of acinar specific TFs in mice expressing mutant Kras one week after induction of pancreatitis. This is consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase expression at the mRNA level. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in expression of three key acinar-specific fate TFs, Mist1, Hnf1a and Nr5a2. The mRNA expression levels of other acinar TFs like Pdx1 and Ptf1a/p48 did not recover with loss of Bmi1 and mutant Kras expression. Together, our data suggest that oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Jack Blumberg, Osama Alkahili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A22.

Abstract C56: Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia

Abstract Rare acinar cells expressing Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), represent a reservoir of cells contributing to pancreatic repair during injury and stress. This is marked by acinar-ductal metaplasia (ADM), a transition of acinar cells to a more duct-like phenotype, which is seen in pancreatitis and early stages of Kras-driven pancreatic neoplasia. The mechanism behind Kras-driven ADM at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells and rely on appropriate chromatin context for their activity. Deletion of Bmi1 represses Kras-driven ADM and neoplasia in genetically engineered mouse models (GEMMs) of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used GEMMs to study the expression of Bmi1 in the acinar compartment and its role in early pancreatic neoplasia. We combined an acinar cell-specific tamoxifen-inducible Cre-recombinase under the elastase promoter (Ela-CreER) with the Kras G12D oncogenic allele and the conditional Bmi1 knockout allele and the Rosa26 tdTomato reporter to generate Ela-CreER, KrasLSL-G12D/+, Bmi1, R26 tdTomato mice and their littermate controls. We also generated Ela-CreER, Bmi1GFP, R26tdTomato and Bmi1-CreER, R26 tdTomato mice to quantify the fraction of acinar cells expressing Bmi1. We activated the Cre-recombinase in 6- to 8-week-old Ela-CreER mice with 5 tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (8 hourly injections x 2 days) one week after the first tamoxifen dose. We FACS sorted tdTomato+ cells from untreated mice (0 hr) and one week (168 hr) after pancreatitis induction and analyzed RNA levels of 8 TFs, reported to play a role in pancreatic development, homeostasis, and neoplasia. Expression levels of two lineage markers (amylase and elastase) were also measured by RT-qPCR. Lineage tracing in Bmi1-CreER and Ela-CreER, Bmi1GFP mice revealed expression of Bmi1 in vast majority of acinar cells. RT-qPCR of tdTomato+ cells in the Ela-CreER models indicated a loss of acinar-specific TFs in mice expressing mutant Kras one week after induction of pancreatitis consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase mRNA levels. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in RNA expression of three key acinar-specific fate TFs—Mist1, Hnf1a, and Nr5a2. The mRNA levels of other key acinar TFs—Pdx1 and Ptf1a/p48—did not recover with the loss of Bmi1 in the presence of KrasG12D. Together, our data suggest that Bmi1 is expressed in the majority of acinar cells and regulates oncogenic Kras-driven ADM by altering master TF gene regulatory networks. Its deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Emily Wu, Osama Alkhalili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C56.

Loss of BMI1 in mature olfactory sensory neurons leads to increased olfactory basal cell proliferation

Damage to olfactory sensory neurons (OSNs), situated within the neuroepithelium of the olfactory cleft, may be associated with anosmia. Although their direct contact with the nasal airspace make OSNs vulnerable to injury and death, multiple mechanisms maintain epithelium integrity and olfactory function. We hypothesized that BMI1, a polycomb protein found to be enriched in OSNs, may function in neuroprotection. Here, we explored BMI1 function in a mouse model. Utilizing a mouse genetic approach to delete Bmi1 selectively in mature OSNs, we investigated changes in OE homeostasis by performing immunohistochemical, biochemical, and functional assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunostaining, and electro-olfactograms were used to compare gene expression, cell composition, and olfactory function in OSN-specific BMI1 knockout mice (n = 3 to 5) and controls. Chromatin studies were also performed to identify protein-DNA interactions between BMI1 and its target genes (n = 3). OSN-specific BMI1 knockout led to increased neuron death and basal cell activation. Chromatin studies suggested a mechanism of increased neurodegeneration due to de-repression of a pro-apoptosis gene, p19ARF. Despite the increased turnover, we found that olfactory neuroepithelium thickness and olfactory function remained intact. Our studies also revealed the presence of additional polycomb group proteins that may compensate for the loss of BMI1 in mature OSNs. The olfactory neuroepithelium employs multiple mechanisms to maintain epithelial homeostasis. Our findings provide evidence that in a mouse model of BMI1 deletion, the overall integrity and function of the olfactory neuroepithelium are not compromised, despite increased neuronal turnover, reflecting a remarkable reparative capacity to sustain a critical sensory system.

Impaired Expression of Rearranged Immunoglobulin Genes and Premature p53 Activation Block B Cell Development in BMI1 Null Mice

SUMMARYB cell development is a highly regulated process that requires stepwise rearrangement of immunoglobulin genes to generate a functional B cell receptor (BCR). The polycomb group protein BMI1 is required for B cell development, but its function in developing B cells remains poorly defined. We demonstrate that BMI1 functions in a cell-autonomous manner at two stages during early B cell development. First, loss of BMI1 results in a differentiation block at the pro-B cell to pre-B cell transition due to the inability of BMI1-deficient cells to transcribe newly rearranged Igh genes. Accordingly, introduction of a pre-rearranged Igh allele partially restored B cell development in Bmi1−/− mice. In addition, BMI1 is required to prevent premature p53 signaling, and as a consequence, Bmi1−/− large pre-B cells fail to properly proliferate. Altogether, our results clarify the role of BMI1 in early B cell development and uncover an unexpected function of BMI1 during VDJ recombination.

Hoxc-Dependent Mesenchymal Niche Heterogeneity Drives Regional Hair Follicle Regeneration

Mesenchymal niche cells instruct activity of tissue-resident stem and progenitor cell populations. Epithelial stem cells in hair follicles (HFs) have region-specific activity, which may arise from intrinsic cellular heterogeneity within mesenchymal dermal papilla (DP) cells. Here we show that expression of Hoxc genes is sufficient to reprogram mesenchymal DP cells and alter the regenerative potential of epithelial stem cells. Hoxc gene expression in adult skin dermis closely correlates with regional HF regeneration patterns. Disrupting the region-specific expression patterns of Hoxc genes, by either decreasing their epigenetic repression via Bmi1 loss or inducing ectopic interactions of the Hoxc locus with an active epigenetic region, leads to precocious HF regeneration. We further show that a single Hoxc gene is sufficient to activate dormant DP niches and promote regional HF regeneration through canonical Wnt signaling. Altogether, these results reveal that Hoxc genes bestow mesenchymal niches with tissue-level heterogeneity and plasticity.

Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling.

Establishment of an inflammatory microenvironment. Suppression of BMI-1 reverses THP-1-CM-induced inflammatory cytokine production in CRC. Loss of BMI-1 suppressed TLR4/MD-2/MyD88 complex-mediated NF-κB signaling.

Abstract 4875: Identification of the cellular origin and "stemness" phenotype of Malignant Rhabdoid Tumors (MRT) may represent a new therapeutic approach in paediatric oncology

Abstract Malignant Rhabdoid Tumors (MRT) are highly aggressive childhood malignancies characterized by a single mutation; biallelic inactivation of SMARCB1, a component of the SWI/SNF chromatin remodeling complex. These tumors may occur anywhere, most frequently in the brain (Atypical Teratoid/Rhabdoid Tumor, ATRT) and in the kidneys and soft tissues (Extra Cranial Rhabdoid tumor, ECRT). MRT presents immune markers from multiple lineages exhibiting a teratoid phenotype, characterized by cells with heterogeneous morphology within the same tumor leading to speculation that the MRT cell of origin is a type of multipotent stem cell. Despite recent advances in treating other solid tumors, treatment for MRT still remains ineffective and we hypothesized that MRT “stemness” characteristics are key to its aggressive clinical course and resistance to treatment. Here we analyze the expression profiles of primary MRT (n= 134) and catalog the expression of a program of “stemness” genes capable of driving aberrant self-renewal. Further, we show by re-expression of SMARCB1 in MRT cells that several of these key “stemness” genes are aberrantly activated by SMARCB1 mutation. One such gene, BMI1 was further demonstrated to be critical to MRT tumorigenesis by shRNA knockout, and a novel anti-BMI1 drug, PTC209 was tested to show efficacy in MRT. Knockdown of BMI1 in MRT cells reduced the self-renewal capability of cells as seen from the number of tumorspheres formed in limiting dilution assay (LDA). Interestingly, BMI1 loss triggers upregulation of p16(INK4a) expression and this mimics the expression profile when SMARCB1 was re-expressed into MRT cells. To identify a putative MRT cell of origin we performed a meta-analysis cross-referencing expression profiles from primary MRT (n= 20), and functional models in which SMARCB1 was re-expressed (n=5 lines) with expression profiles from multiple stem cell types including epithelial, embryonic, neural mesenchymal and neural crest (n= 446). Analysis by t-SNE and NMF consensus clustering suggested that MRT bore the closest resemblance to either early neural progenitors (ATRT) or early neural crest cells (ECRT). In MRT cells in which SMARCB1 was forcibly re-expressed, cells became gradually more differentiated and their expression profile altered from that of an early neural crest to a more differentiated mesenchymal pattern. For the first time, we show evidence that ECRTs and ATRTs appear to have different cells of origin, despite having the same mutation and tumor appearance. We further demonstrate that SMARCB1 expression is necessary for MRT cells to maintain a de-differentiated “stem-like” state and finally that the SMARCB1-dependent stemness gene BMI1 shows potential as a novel therapeutic target in MRT. Citation Format: Ras A. Ramli, Martina A. Finetti, Matthew P. Selby, Yura Grabovska, Stephen Crosier, Amanda J. Smith, Steven C. Clifford, Daniel Williamson. Identification of the cellular origin and "stemness" phenotype of Malignant Rhabdoid Tumors (MRT) may represent a new therapeutic approach in paediatric oncology [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 4875. doi:10.1158/1538-7445.AM2017-4875

Bmi1 Loss in the Organ of Corti Results in p16ink4a Upregulation and Reduced Cell Proliferation of Otic Progenitors In Vitro.

The mature mammalian organ of Corti does not regenerate spontaneously after injury, mainly due to the absence of cell proliferation and the depletion of otic progenitors with age. The polycomb gene B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) promotes proliferation and cell cycle progression in several stem cell populations. The cell cycle inhibitor p16ink4a has been previously identified as a downstream target of Bmi1. In this study, we show that Bmi1 is expressed in the developing inner ear. In the organ of Corti, Bmi1 expression is temporally regulated during embryonic and postnatal development. In contrast, p16ink4a expression is not detectable during the same period. Bmi1-deficient mice were used to investigate the role of Bmi1 in cochlear development and otosphere generation. In the absence of Bmi1, the postnatal organ of Corti displayed normal morphology at least until the end of the first postnatal week, suggesting that Bmi1 is not required for the embryonic or early postnatal development of the organ of Corti. However, Bmi1 loss resulted in the reduced sphere-forming capacity of the organ of Corti, accompanied by the decreased cell proliferation of otic progenitors in otosphere cultures. This reduced proliferative capacity was associated with the upregulation of p16ink4a in vitro. Viral vector-mediated overexpression of p16ink4a in wildtype otosphere cultures significantly reduced the number of generated otospheres in vitro. The findings strongly suggest a role for Bmi1 as a promoter of cell proliferation in otic progenitor cells, potentially through the repression of p16ink4a.

Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

The polycomb complex proto-oncogene BMI1 [B lymphoma Mo-MLV insertion region 1 homolog (mouse)] is essential for self-renewal of normal and cancer stem cells. BMI1-null mice show severe defects in growth, development, and survival. Although BMI1 is known to exert its effect in the nucleus via repression of 2 potent cell-cycle regulators that are encoded by the Ink4a/Arf locus, deletion of this locus only partially rescues BMI1-null phenotypes, which is indicative of alternate mechanisms of action of BMI1. Here, we show that an extranuclear pool of BMI1 localizes to inner mitochondrial membrane and directly regulates mitochondrial RNA (mtRNA) homeostasis and bioenergetics. These mitochondrial functions of BMI1 are independent of its previously described nuclear functions because a nuclear localization-defective mutant BMI1 rescued several bioenergetic defects that we observed in BMI1-depleted cells, for example, mitochondrial respiration, cytochrome c oxidase activity, and ATP production. Mechanistically, BMI1 coprecipitated with polynucleotide phosphorylase, a ribonuclease that is responsible for decay of mtRNA transcripts. Loss of BMI1 enhanced ribonuclease activity of polynucleotide phosphorylase and reduced mtRNA stability. These findings not only establish a novel extranuclear role of BMI1 in the regulation of mitochondrial bioenergetics, but also provide new mechanistic insights into the role of this proto-oncogene in stem cell differentiation, neuronal aging, and cancer.-Banerjee Mustafi, S., Aznar, N., Dwivedi, S. K. D., Chakraborty, P. K., Basak, R., Mukherjee, P., Ghosh, P., Bhattacharya, R. Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

BMI1 Regulation of Self-Renewal and Multipotency in Human Mesenchymal Stem Cells.

We have previously described generation of mesenchymal stem cells (MSCs) from human embryonic and induced pluripotent stem cells. One of the central questions in stem cell biology is to understand how stem cells regulate the decision to self-renew vs. differentiate, at the molecular level. In the current studies we used loss-of-function and gain-of-function analyses in primary human MSCs to demonstrate that BMI1 is a critical regulator for self-renewal and multipotency in this interesting cell type. Knockdown of BMI1 in MSCs reduced self-renewal by upregulation of p16(INK4A) and increased apoptosis. Knockdown of p16(INK4A) partially rescued the self-renewal defect in MSCs with loss of BMI1. Overexpressed BMI1 reduced apoptosis and increased cell proliferation by repressing p16(INK4A). Loss of BMI1 resulted in deregulation of PPARγ, an adipogenic factor, and imprinted gene network (IGN), which blocks osteogenesis. Knockdown of PPARγ or IGN in BMI1 defect models restored osteogenesis. Overexpression of BMI1 repressed transcripts of RUNX2 and PPARγ, in osteogenesis and adipogenesis, respectively, which lead to decreased lineage specification potential in MSCs. These data show that BMI1 regulates cell proliferation, apoptosis, and differentiation of human MSCs.

Polycomb Repressive Complex 1 Enhances HSC Self-Renewal through Repressing the Canonical Wnt Signaling Pathway

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. While studies on individual PRC1 component suggest that PRC1 plays an important role in hematopoiesis, how PRC1 regulates transcriptional repression in hematopoietic stem cells (HSCs) is largely unknown. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the PRC1 complex. Bmi1 is a positive regulator of HSC self-renewal; however, the role of Mel18 in hematopoiesis has been controversial. To determine whether Bmi1 and Mel18 play redundant or distinct role in HSC self-renewal, we have generated Bmi1 and Mel18 conditional knockout mice. While acute deletion of Mel18 affects neither HSC frequency nor lineage commitment, we found that Mel18-deficent hematopoietic progenitor cells showed enhanced replating potential compared to wild type cells. To determine the role of Mel18 in HSC self-renewal, we performed serial HSC transplantation assays and found that the repopulating ability of Mel18-/- HSCs was significantly higher than WT HSCs in both primary and secondary transplantation assays, demonstrating that the loss of Mel18 enhances HSC self-renewal in vivo. We hypothesize that loss of Bmi1 and Mel18 in hematopoietic stem cells will disrupt PRC1 complex and impairs HSC self-renewal. To determine the role of PRC1 complex in HSCs, we analyzed the HSC behavior in Bmi1 and Mel18 double-deficient mice. While we found that Bmi1-deficient HSCs showed decreased repopulating potential compared to WT HSCs 16 weeks following transplantation, loss of both Bmi1 and Mel18 in HSCs resulted in even more severe self-renewal defects. In addition, loss of both Bmi1 and Mel18 resulted in decreased myeloid differentiation and increased B cell differentiation compared to WT, Mel18 KO, and Bmi1 KO mice. These data demonstrate that Bmi1 and Mel18 have non-overlapping role in HSC maintenance and lineage commitment.Given that Bmi1 plays a dominant role in the PRC1 complex, we decided to identify Bmi1 target genes in hematopoietic stem cells to understand how PRC1 complex regulates HSC self-renewal. We performed transcript profiling assays to compare gene expression in HSCs isolated from wild type and Bmi1-/- mice. The Ingenuity Pathways indicates that the canonical Wnt signaling is significantly elevated in Bmi1 null HSCs compared to WT HSCs. We confirmed the upregulation of several genes of the Wnt pathway in Bmi1 null HSCs by quantitative real-time PCR analysis. To determine whether Bmi1 can repress the activation of Wnt signaling in cells, we utilized the Top-Flash Wnt reporter system. Stimulation of 293T cells with Wnt3a activates the Wnt reporter and this activation can be efficiently repressed by Bmi1. Furthermore, we detected the association of Bmi1 with the Lef1, Tcf4, and Axin2 promoters in Baf3 cells by ChIP experiment. Thus, Bmi1 directly represses the expression of several Wnt genes in hematopoietic cells. To determine the functional significance of activation of Wnt signaling in Bmi1 null HSCs, we have generated R26StopFL Bmi1-Apcf/f-Mx1-Cre+ and Bmi1f/f-Ctnnb1f/f-Mx1-Cre+ mice. Loss of Apc in hematopoietic cells activates the Wnt signaling pathway and impairs HSC self-renewal. We found that expressing three-copies of Bmi1 from the Rosa26 locus enhanced the self-renewal capabilities of Apc deficient HSCs in transplantation assays. Ctnnb1 encodes b-catenin and loss of Ctnnb1 in HSCs diminishes Wnt signaling. Acute deletion of Bmi1 in hematopoietic compartments resulted in decreased bone marrow cellularity and enhanced apoptosis of hematopoietic stem and progenitor cells. Deletion of Ctnnb1 in Bmi1 null hematopoietic cells rescued these defects. Thus, impaired HSC self-renewal seen in Bmi1 null mice is, at least in part, due to activation of the canonical Wnt signaling pathway.Taken together, we demonstrate that PRC1 complex enhances HSC self-renewal through inhibiting the canonical Wnt signaling. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-061: Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Many characteristic mutations in PDAC are known, but this information has so far failed to produce the development of effective treatments, highlighting the need for deeper understanding of the processes that drive tumorigenesis. B-cell specific Moloney murine leukemia virus insertion site 1 (Bmi1), a Polycomb repressive group protein, is upregulated in PDAC and associated with poor prognosis. Our lab has shown that despite an oncogenic K-ras mutation, mice with pancreas specific loss of Bmi1 do not develop precancerous lesions, termed PanINs (Pancreatic Intraepithelial Neoplasia). This lack of PanIN development was also seen in the presence of pancreatitis, which is usually known to synergize with oncogenic K-ras to speed PanIN development. In other cancer types, Bmi1's effect on tumorigenesis was mechanistically linked to its regulation of the Ink4a/ARF genetic locus. However, we found that in PDAC, PanIN initiation was independent of Bmi1 control this locus. Further, impairment in the regulation of ROS generation was seen in vitro in pancreatic cancer cell lines lacking Bmi1. Regulating ROS generation is a vital step in the neoplastic process and has been shown in other systems to be controlled by Bmi1. Overall, in this work we have shown that expression of the Polycomb group protein Bmi1 is necessary for the initiation of pancreatic precancerous lesions, and that the mechanism of Bmi1 requirement is independent of its repression of the Ink4a/ARF genetic locus. Given the recent pre-clinical development of a Bmi1 inhibitor, this work could provide rationale for future treatment of pancreatic cancer, a truly devastating disease. Citation Format: Heather Schofield, Filip Bednar, Meredith Collins, Wei Yan, Yaqing Zhang, Nikhil Shyam, Jaime Eberle, Kenneth Olive, Nabeel Bardeesy, Daisuke Nakada, Diane Simeone, Sean Morrison, Marina Pasca di Magliano. Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-061. doi:10.1158/1538-7445.AM2015-LB-061

Bmi1 promotes erythroid development through regulating ribosome biogenesis.

While Polycomb group protein Bmi1 is important for stem cell maintenance, its role in lineage commitment is largely unknown. We have identified Bmi1 as a novel regulator of erythroid development. Bmi1 is highly expressed in mouse erythroid progenitor cells and its deficiency impairs erythroid differentiation. BMI1 is also important for human erythroid development. Furthermore, we discovered that loss of Bmi1 in erythroid progenitor cells results in decreased transcription of multiple ribosomal protein genes and impaired ribosome biogenesis. Bmi1 deficiency stabilizes p53 protein, leading to upregulation of p21 expression and subsequent G0/G1 cell cycle arrest. Genetic inhibition of p53 activity rescues the erythroid defects seen in the Bmi1 null mice, demonstrating that a p53-dependent mechanism underlies the pathophysiology of the anemia. Mechanistically, Bmi1 is associated with multiple ribosomal protein genes and may positively regulate their expression in erythroid progenitor cells. Thus, Bmi1 promotes erythroid development, at least in part through regulating ribosome biogenesis. Ribosomopathies are human disorders of ribosome dysfunction, including Diamond-Blackfan anemia (DBA) and 5q- syndrome, in which genetic abnormalities cause impaired ribosome biogenesis, resulting in specific clinical phenotypes. We observed that BMI1 expression in human hematopoietic stem and progenitor cells from patients with DBA is correlated with the expression of some ribosomal protein genes, suggesting that BMI1 deficiency may play a pathological role in DBA and other ribosomopathies.

Bmi1 regulates auditory hair cell survival by maintaining redox balance.

Reactive oxygen species (ROS) accumulation are involved in noise- and ototoxic drug-induced hair cell loss, which is the major cause of hearing loss. Bmi1 is a member of the Polycomb protein family and has been reported to regulate mitochondrial function and ROS level in thymocytes and neurons. In this study, we reported the expression of Bmi1 in mouse cochlea and investigated the role of Bmi1 in hair cell survival. Bmi1 expressed in hair cells and supporting cells in mouse cochlea. Bmi1−/− mice displayed severe hearing loss and patched outer hair cell loss from postnatal day 22. Ototoxic drug-induced hair cells loss dramatically increased in Bmi1−/− mice compared with that in wild-type controls both in vivo and in vitro, indicating Bmi1−/− hair cells were significantly more sensitive to ototoxic drug-induced damage. Cleaved caspase-3 and TUNEL staining demonstrated that apoptosis was involved in the increased hair cell loss of Bmi1−/− mice. Aminophenyl fluorescein and MitoSOX Red staining showed the level of free radicals and mitochondrial ROS increased in Bmi1−/− hair cells due to the aggravated disequilibrium of antioxidant–prooxidant balance. Furthermore, the antioxidant N-acetylcysteine rescued Bmi1−/− hair cells from neomycin injury both in vitro and in vivo, suggesting that ROS accumulation was mainly responsible for the increased aminoglycosides sensitivity in Bmi1−/− hair cells. Our findings demonstrate that Bmi1 has an important role in hair cell survival by controlling redox balance and ROS level, thus suggesting that Bmi1 may work as a new therapeutic target for the prevention of hair cell death.