High Levels 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-KLF4 axis deficiency improves responses to neoadjuvant concurrent chemoradiotherapy in patients with rectal cancer

PurposeNeoadjuvant concurrent chemoradiotherapy (CCRT) is a gold standard treatment for patients with stage II/III rectal cancer. B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) is a member of the polycomb group of proteins that are involved in regulating gene expression. High levels of BMI1 have been demonstrated to contribute to the malignant phenotypes of several cancers; however, its relevance in rectal cancer treated with CCRT is largely unknown. Methods and materialsWe used two patient cohorts to address the clinical relevance of BMI1 in human cancers. In addition, HT-29 and HCT-116 cells were chosen as our in vitro models to verify the role of BMI1 in cell response to ionizing radiation. Stemness-related proteins were analyzed by western blotting and cell survival was determined using clonogenic assays. ResultsBMI1 overexpression was found to significantly correlate with advanced pre-treatment nodal status (N1-N2; p < 0.001), post-treatment tumor stage (T1-T2; p = 0.015), inferior tumor regression grade (p = 0.001), and also an independent prognosis factor in 172 rectal cancer patients receiving CCRT. Serial cell-based functional examination indicated that BMI1 deficiency sensitized cells to radiation treatment by modulating the gene expression of Kruppel-like factor 4 (KLF4) and enhanced radiosensitivity in microsatellite stable (MSS) colorectal cancers. Overexpression of KLF4 partially overcame BMI1-deficiency-mediated γ-H2AX expression after ionizing radiation exposure. Consistent with in vitro data, an analysis of an additional 30 rectal cancer tissue specimens revealed a positive correlation between BMI1 and KLF4 (p = 0.02). ConclusionHigher levels of BMI1 are associated with poor therapeutic response and adverse outcomes in rectal cancer patients receiving CCRT.

Reduction of gastric cancer proliferation and invasion by miR-15a mediated suppression of Bmi-1 translation.

B-cell specific moloney leukemia virus insertion site 1 (Bmi-1) gene plays important roles in gastric cancer, but the epigenetic regulatory mechanism by microRNA (miRNA) and the functional significance of Bmi-1 inhibition in gastric cancer remains elusive. In this study, we systematically investigated the functional roles of miRNA mediated Bmi-1 suppression in gastric cancer. Our results show that the expression of miR-15a is significantly reduced in gastric cancer and the protein expression levels of Bmi-1 are inversely correlated with miR-15a (P = 0.034) in gastric cancer patient samples. Functional studies revealed that ectopic expression of miR-15a decreased Bmi-1 in gastric cancer cell lines with reduced proliferation and tumor invasion. High levels of Bmi-1 in gastric cancer patients are significantly associated with better overall survival (P = 0.024) based on the Kaplan-Meier survival analysis.

Abstract A130: Targeting BMI-1 by the novel BMI-1 inhibitor PTC596 in acute leukemia

Abstract Leukemia stem cells are resistant to chemotherapeutic agents and contribute to disease relapse. BMI-1, a part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and leukemia stem cells. We have reported that acute myeloid leukemia (AML) patients with higher levels of BMI-1 have worse overall survival and that the translational BMI-1 inhibitor PTC-209 induces apoptosis in patient-derived CD34+CD38low/- AML cells. PTC596 is a second-generation BMI-1 inhibitor that accelerates BMI-1 degradation. Based on preclinical data showing excellent anti-tumor activities against solid cancers, PTC596 is currently in PH1 clinical development. We here investigated preclinical activities of PTC596 against acute leukemias in vitro and in vivo. A total of 6 AML (MOLM-13, OCI-AML3, U-937, MV4-11, MOLM-14 and HL60) and 3 acute lymphoblastic leukemia (ALL) (Reh, NALM6 and MOLT-4) cell lines were exposed to PTC596 for 48 hours. PTC596 exhibited dose- and time-dependent anti-proliferative and cytotoxic (apoptotic) activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) were 30.7 ± 4.1 nM (mean ± SEM) for AML and 37.7 ± 8.0 nM for ALL. The ED50 values (effective concentration inducing 50% killing as measured by Annexin V/PI induction) were 60.3 ± 6.7 nM for AML and 77.8 ± 11.5 nM for ALL. PTC596 induced apoptosis in AML cells irrespective of their p53 status. 72-hour treatment of MOLM-13 and OCI-AML3 cells with 100nM PTC596 reduced total BMI-1 protein levels by 87% and 61%, respectively. Forced overexpression of BMI-1 in K562 cells abrogated PTC596-induced apoptosis (∼ 80% at 50 nM PTC596; P &amp;lt; 0.05). 16-hour treatment of MOLM-13 cells with 200nM PTC596 resulted in reduced levels of non-phosphorylated BMI-1 by 85%, along with increased levels of phosphorylated BMI-1. PTC596 reduced the levels of ubiquitylated histone H2A, which indicates a deactivation of the PRC1 complex function. The anti-leukemia activity of PTC596 was tested in xenograft (K562) and syngeneic (L210) mouse models. PTC596 at a daily dose of 20 mg/kg significantly delayed an enlargement of the implanted tumor of K562 by ∼55%. PTC596 significantly prolonged survival of mice injected with syngeneic L210 leukemia cells (27 versus 37 days; 76% extension of lifespan with a median survival time; P &amp;lt; 0.05). Collectively, BMI-1 inhibition by PTC596 offers a novel therapeutic strategy for acute leukemias. Citation Format: Yuki Nishida, Aya Maeda, Liangxian Cao, Melissa Dumble, Shinya Kimura, Thomas W. Davis, Kensuke Kojima. Targeting BMI-1 by the novel BMI-1 inhibitor PTC596 in acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A130.

Predicting clinical outcome in feline oral squamous cell carcinoma: tumour initiating cells, telomeres and telomerase.

Feline oral squamous cell carcinoma (SCC) has very poor prognosis. Here, a retrospective pilot study was conducted on 20 feline oral SCC patients who underwent stereotactic radiation therapy (SRT), to evaluate: (1) the value of putative tumour initiating cell (TIC) markers of human head and neck SCC (CD44, Bmi-1); (2) telomere length (TL) specifically in putative TICs; and (3) tumour relative telomerase activity (TA). Significant inverse correlations were found between treatment outcomes and Bmi-1 expression, supporting the predictive value of Bmi-1 as a negative prognostic indicator. While TL exhibited a wide range of variability, particularly in very short fractions, many tumours possessed high levels of TA, which correlated with high levels of Bmi-1, Ki67 and EGFR. Taken together, our results imply that Bmi-1 and telomerase may represent novel therapeutic targets in feline oral SCC, as their inhibition-in combination with SRT-would be expected to have beneficial treatment outcome.

Adult Thymic Epithelium Contains Nonsenescent Label-Retaining Cells

Progress in our understanding of thymic epithelial cell (TEC) renewal and homeostasis is hindered by the lack of markers for TEC progenitors. Stem and progenitor cell populations display remarkable diversity in their proliferative behavior. In some but not all tissues, stemness is associated with quiescence. The primary goal of our study was to discover whether quiescent cells were present in neonatal and adult TECs. To this end, we used a transgenic label-retaining cell (LRC) assay in which a histone H2B-GFP fusion protein is expressed under the control of the reverse tetracycline-controlled transactivator and the tetracycline operator minimal promoter. In adult mice, we found that both cortical and medullary TECs (cTECs and mTECs) proliferated more actively in females than males. Moreover, we observed three main differences between neonatal and adult TECs: 1) neonatal TECs proliferated more actively than adult TECs; 2) whereas cTECs and mTECs had similar turnover rates in young mice, the turnover of mTECs was more rapid than that of cTECs in adults; and 3) although no LRCs could be detected in young mice, LRCs were detectable after a 16-wk chase in adults. In female mice, LRCs were found almost exclusively among cTECs and expressed relatively low levels of p16INK4a, p19ARF, and Serpine1, and high levels of Bmi1, Foxn1, Trp63, and Wnt4. We conclude that LRCs in adult TECs are not senescent postmitotic cells and may represent the elusive progenitors responsible for TEC maintenance in the adult thymus.

B-Cell Lymphoma Cells Overexpressing BMI-1 Are Correlated with Drug Resistance through Enhanced Expression of Survivin and Are Effectively Eliminated by T Cells with Anti-CD38 Chimeric Receptor

AbstractAbstract 3613BMI-1 is essential for the self-renewal and proliferation of leukemic and hematopoietic stem and progenitor cells. Increased expression of BMI-1 is known to be an indicator for a poor prognosis in cancer patients. Analysis of the expression of BMI-1 and survivin in 6 patients with B-cell lymphoma (3 drug-resistant and 3 sensitive cases) showed that in the drug-resistant patients, high levels of BMI-1 and survivin were maintained even after drug administration in vitro. However, there observed was a down-regulation of both BMI-1 and survivin expression in the drug-sensitive patients. BMI-1 transduction induced the drug-resistance of two B-cell lymphoma cell lines, HT and RL, to the anti-cancer drugs etoposide and oxaliplatin, but not to irinotecan. The expression of survivin was clearly augmented in the cells transduced with BMI-1. Moreover, we detected sustained expression of survivin level in the presence of etoposide in the BMI-1-overexpressing cells. By contrast, the mock-transduced cells succumbed in the medium with anti-cancer drugs with an accompanying decrease in the expression of survivin as well as BMI-1. Survivin has been reportedly implicated in resistance to chemotherapeutic agents. Intriguingly, survivin mRNA levels in BMI-1-overexpressing cells were consistent with those in controls. Also, the level of survivin was enhanced by treatment with a proteasomal inhibitor, MG132, suggesting that overexpression of BMI-1 stabilized survivin expression post-translationally. We further showed that sh RNA-mediated knockdown of BMI-1 or survivin restored sensitivity to etoposide in the HT cells overexpressing BMI-1. Our findings suggest survivin as a potential target for BMI-1. Thus BMI-1, by acting as an upstream regulator, may control the expression of survivin, facilitating drug resistance in B-cell lymphoma. Next, we examined whether B-cell lymphoma cells overexpressing BMI-1 are abrogated by immunotherapy with T cells containing anti-CD38 chimeric receptor in vitro. Interestingly, these B-cell lymphoma cells were effectively eliminated by specific T cells against B-cell lymphoma cells bearing CD38. These results suggest that the immunotherapy is useful for treatment of patients with B-cell lymphoma cells overexpressing BMI-1, which are refractory to chemotherapeutic reagents. BMI-1 may be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas. Disclosures:No relevant conflicts of interest to declare.

Abstract 4261: Inhibition of Bmi-1 suppressed the tumorigenicity of oral cancer-derived ALDH1-positive cells

Abstract Bmi-1, a member of the Polycomb family of transcriptional repressors, is essential for maintaining the self-renewal abilities of adult stem cells. Bmi-1 has been demonstrated to play a role in tumorigenesis in head and neck squamous cell carcinomas (HNSCCs). A recent study has further suggested that ALDH1 may be considered to be a putative marker for HNSCC-derived cancer stem cells. However, the role that Bmi-1 plays in HNSCC-derived ALDH1-positive cells (HNSCC- ALDH1+) has yet to be determined. In this study, we demonstrated that HNSCC- ALDH1+ cells possess tumor initiating properties, are capable of self-renewal, and express higher levels of Bmi-1 as compared to HNSCC-ALDH1− cells. To further explore the functional role of Bmi-1 in HNSCC-ALDH1+ cells, we used a lentiviral vector expressing shRNA to knock down Bmi-1 expression (sh-Bmi-1) in HNSCC- ALDH1+ cells. Silencing of Bmi-1 significantly enhanced the sensitivity of HNSCC- ALDH1+ cells to chemoradiation and increased the degree of chemoradiation- mediated apoptosis that occurred. Importantly, knockdown of Bmi-1 increased the effectiveness of radiotherapy and led to the inhibition of tumor growth in nude mice transplanted with HNSCC-ALDH1+ cells. Kaplan-Meier survival analysis indicated that the mean survival rate of HNSCC-ALDH1+ tumor-bearing immunocompromised mice treated with radiotherapy was significantly improved by treatment with sh-Bmi-1 as well. In summary, these results suggest that Bmi-1 is a potential target for increasing the sensitivity of HNSCC cancer stem cells to chemoradiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4261.

Abstract 3353: The CD44 hi cell population from malignant pleural effusion derived lung tumors displays increased tumorigenic potential

Abstract We have developed a new model system (http://dx.plos.org/10.1371/journal.pone.0005884) to test whether we can isolate candidate Cancer Stem Cell (CSC) populations from clinical specimens and validate that they possess increased tumorigenic and/or metastogenic potential. The model utilizes primary tumor cell populations derived from human malignant pleural effusions (MPE), which are cultured in an autologous tumor microenvironment (TME). This model system maintains intratumoral heterogeneity, and contains molecular signatures, surface phenotypes, and metabolic biomarkers that are associated with candidate CSC. The current study compares isogenic primary tumor subpopulations, separated on the basis of a surface biomarker expression, in terms of differences in molecular marker expression, and bioassays that measure clonogenic efficiency and tumorigenic potential. CD44, a surface CSC-biomarker, is expressed on most of the primary tumor cells (75-95%) derived from MPE. The CD44 expressing tumor cells can be divided into two groups (CD44hi) and (CD44lo) on the basis of CD44 surface labeling intensity. The CD44hi and CD44lo tumor cells were sorted (10% of each) from total tumor cell populations by FACS and evaluated for their clonogenicity on plastic (colony forming efficiency) and for colony forming units in soft agar (anchorage independent growth). Studies show that the CD44hi tumor cells are 40-60% more efficient in forming colonies, and give rise to larger colonies than CD44lo tumor cells. Expression of other candidate CSC biomarkers was also compared in the two cell subsets to determine the molecular correlates of CD44hi versus CD44lo cells. CD44hi cells coexpress higher levels of BMI-1, hTERT, EZH2, and OCT-4 by RT-PCR than CD44lo tumor cells, but there is no apparent difference in the expression of SUZ12 between these two populations. CD44hi cells also have increased ALDH expression based on the Aldefluor assay. To determine whether these subpopulations differed in terms of tumorigenic potential, limiting dilutions of cells were heterotopically (subcutaneous flank region) injected in NOD/SCID (IL-2gRnull) mice in comparison to CD44lo tumor cells. Low numbers of CD44hi cells (down to 300 cells) efficiently formed tumors, whereas isogenic CD44lo tumor cells failed to form tumors at the same cell doses. Thus, CD44hi cells have higher tumorigenic potential than CD44lo cells, and these studies suggest that these primary tumor populations are preferentially enriched for the candidate CSC phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3353.

Abstract 5169: The polycomb group protein BMI-1 cooperates with Yes-Associated Protein, YAP, to suppress cell contact inhibition in Ewing sarcoma cells

Abstract The polycomb group family protein BMI-1 is frequently deregulated in cancer. BMI-1 has been shown to promote stemness and tumorigenicity largely through epigenetic repression of the CDKN2A locus, inhibiting the expression of cell-cycle inhibitors p16INK4A and p14ARF. We have previously shown that BMI-1 functions as an oncoprotein in Ewing sarcoma, promoting anchorage independent growth in vitro and tumorigenicity in vivo independently of CDKN2A repression. In the current study we have investigated the potential contribution of BMI-1 to loss of cell contact inhibition, a fundamental transforming property of cancer cells. Using cells that stably express an shRNA against BMI-1, we discovered that loss of BMI-1 expression restores contact inhibition to CDKN2A-null Ewing sarcoma cells. Significantly, although proliferation of cells in log phase growth is unaffected by loss of BMI-1, at high cell density BMI-1 knockdown cells undergo cell cycle arrest and death. In contrast, control vector-transduced cells continue to enter cell cycle and proliferate, avoiding contact inhibition. Although many signaling pathways are involved in mediating the cell contact inhibition response, inactivation of the Yes-Associated Protein (YAP), a key downstream target of the Hippo pathway, has been implicated in both Drosophila and mammalian cells. Intriguingly, our data show that, although the Hippo pathway is activated in response to increasing cell density, as evidenced by increasing YAP phosphorylation, YAP is not degraded in control cells that express high levels of BMI-1. In contrast, silencing BMI-1 expression results in loss of YAP protein at high cell density coincident with induction of contact inhibition. Together, these findings suggest that YAP is a novel downstream target of BMI-1 and that BMI-1-mediated stabilization of YAP renders cancer cells impervious to cell contact inhibition. Experiments are currently ongoing to elucidate the precise molecular mechanism linking BMI-1 to YAP; however, preliminary studies show that knockdown of BMI-1 has no effect on YAP1 transcript expression at either low or high cell density indicating that the effects of BMI-1 on YAP stabilization are most likely to be post-translational and indirect. Cancer cells escape cell contact inhibition to achieve unrestrained proliferation and enhanced invasive and metastatic properties. Our findings suggest that BMI-1 may be a key upstream mediator of this critical cancer-associated phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5169.

Expression of Proteins P16INK 4a, P53 and Bmi-1 in Hematopoietic Stem Cells of Patients with MDS. the Role of Cellular Senescence.

Abstract 5049Expression of Proteins P16INK 4a, P53 and Bmi-1 in Hematopoietic Stem Cells of Patients with MDS. The Role of Cellular Senescence.Irene Constandinidou1, Eleni Kalivioti1,Vasilios Fertakis1, Costas Dallas1, Polyxeni Lampropoulou1, Evangelia Tzouvara1, Panagiotis Zoumboulis2, Nicholas Zoumbos.11Hematology Division, University of Patras, Medical School, Patras, Greece2Orthopaedic Division, University Hospital of Patras, Patras, Greece IntroductionThe myelodysplastic syndromes (MDS) comprise a spectrum of heterogeneous clonal stem/progenitor cell disorders characterized by marrow failure. One potential reason, apart from apoptosis, for hematopoietic stem cells (HSCs) functional failure is cellular senescence, which is believed to have evolved as a tumor suppressor mechanism capable of arresting growth to reduce risk of malignancy. It can be activated by both telomere-dependent (telomere shortening) and telomere-independent pathways (DNA damage, oncogenic or oxidative stress). There are two widely recognized major tumor suppressor pathways p16/pRb and p53-p14/ARF that regulate cellular senescence. Activation of either pathway is profoundly associated with cellular senescence. Bmi-1, on the other hand, is a transcriptional repressor that plays an essential role in the self-renewal of HSCs and leukemic stem cells as well. One of its major targets is the INK/ARF locus, which encodes p16 and p53 independently. AimTo study the role of cellular senescence in the pathogenesis of MDS through the expression of p16, p53, phospho p53 (activated form of p53, phosphorylated at Ser-15) and bmi-1. Patients and methodsWe examined the expression level of p16INK4a, Bmi-1, p53 and phospho p53 by flow cytometry (direct and indirect staining) in CD34+ bone marrow mononuclear cells (BMMNCs). Furthermore, we analyzed the relative telomere length (RTL) in BMMNCs by quantitative fluorescence in situ hybridization assay using flow cytometry (flow-FISH).We verified the presence of p16 by SDS PAGE western blotting. We studied 36 samples of MDS patients (11 RA, 6 RARS, 3 RCMD, 4 5q- and 12 RAEB I-II), 17 samples of age-matched healthy controls after informed consent and 8 cord blood samples. All MDS diagnoses were histologically confirmed by bone marrow examination and categorized into low and high risk according to the international prognostic scoring system (IPSS). ResultsExpression of p16 is significantly increased in low risk MDS (mean± SD=8,7±12) when compared to healthy controls (p=0,02) and high risk patients(p=0,004). Patients with 5q- syndrome express lower level of p16 in comparison with the other low risk group patients. Increased level of bmi-1 expression is noticed in high risk group (mean± SD=30,3 ±33,9) when compared to low risk group (mean± SD=25±24,6) and healthy controls (mean± SD=18 ±16,4), while 5q- syndrome patients appear to express higher level of bmi-1 than any other risk group. Significantly increased expression of p53 (p=0,03) is noticed in high risk group (mean ±SD=64,3±27) when compared to low risk (mean±SD=34,2±28,8). Patients with 5q- syndrome express increased level of p53 (mean± SD=57,4±26,3) similar to that of high risk group. Expression of phospho p53 seams increased (p=0,055) in low risk group(mean± SD=7,1± 13,9) in comparison to high risk group (mean± SD=0,09± 0,18) and healthy controls (mean± SD=0,19± 0,63). Phospho p53 is expressed in lower level in 5q- syndrome patients. Regarding the RTL there is a significant difference (p=0,0018) between MDS (mean± SD=9,7± 2,7) and cord blood samples (mean± SD=13,8± 1,9). ConclusionsIncreased levels of p16 and phospho p53 in low risk MDS suggest that cellular senescence may contribute to the ineffective hematopoiesis of MDS and probably in a telomere independent way. Low expression level of of phospho p53 in high risk MDS raises the question of genetic integrity of p53 in this group of patients and when combined with high levels of bmi-1 and low expression of p16 may play a role in the disease progression to AML. Further investigation of bmi-1, especially in the 5q- group - and other markers of cellular senescence (SA- b galactosidase, SAHF) is needed in order to clarify the impact of cellular senescence in different risk groups of MDS. DisclosuresNo relevant conflicts of interest to declare.

BMI1, the Polycomb-Group Gene, Is Recurrently Targeted by Genomic Rearrangements in Progressive B-Cell Leukemia/Lymphoma.

Abstract 1099Poster Board I-121The 10p11 chromosomal site is recurrently affected by genomic abnormalities in B cell leukemia/lymphoma, but the molecular consequences of these aberrations remain unknown. We have collected three cases (2 female, 1 male; 67-69 years) of chronic lymphocytic leukemia (CLL) with a novel IGH-mediated t(10;14)(p11;q32) (#1 and 2) or its IGL variant t(10;22)(p11;q11) (#3), and one case of blastic mantle cell lymphoma (MCL) with a complex t(11;14)-positive karyotype including add(10)(p12) (#4). Case 1 showed unmutated VH genes and usage of VH3-21/D1-26/JH6. Cases 2 and 3 revealed mutated VH genes and usage of VH3-21/D1-14/JH4 and VH3-11/D4-4/JH6, respectively. In cases 1 and 2 with a monoallelic loss of RB1, the t(10;14)(p11,q32) was found as a secondary change at time of CLL progression/Richter transformation observed 148 and 50 months after diagnosis, respectively. In case 3, the sole t(10;22)(p11;q11) was identified at time of CLL diagnosis. All three patients died from disease progression 37-149 months after diagnosis. Of note, both patients with t(10;14) died from refractory disease 1 and 3 months after CLL progression/transformation and genetic detection f t(10;14). The patient #3 did not require therapy, but died from septic shock 37 months after diagnosis, and autopsy revealed diffuse and massive lymph node and spleen involvement. The patient with MCL (#4), 67 years old man with a previous history of prostatic adenocarcinoma and CLL, developed secondary central nervous system liesions and died 40 months after diagnosis of MCL. Extensive FISH studies of t(10;14) and t(10;22) mapped the 10p11 breakpoint in the region of the BMI1 locus. SNP array analysis of case 4 complemented by FISH identified amplification of the 10p11 region covering BMI1. Immunostaining with anti-BMI1 serum performed in 2 available cases (#2 and 3) showed expression of the BMI1 protein by neoplastic cells. This approach, however, did not allow to estimate whether the level of BMI1 expression in these cases was higher than in control CLL cases. BMI1 (B-cell-specific Moloney murine leukemia virus integration site 1) is a member of the Polycomb group of genes that are important epigenetic chromatin modifiers with an essential role in embryogenesis and the maintenance of adult stem cells. BMI1 was first identified as a protooncogene that cooperates with c-myc in the generation and development of mouse pre B-cell lymphomas. The BMI1 protein is a component of Polycomb repressive complex1 (PRC1), one of two multiprotein PRCs that maintain cell identity by gene suppression. The gene plays an important role in self-renewal of hematopoietic and neural stem cells. Multiple lines of evidence implicate BMI1 and other Polycomb genes in tumorigenesis. Among others, an aberrant expression of BMI1 has been observed in a wide spectrum of cancers and usually correlated with unfavourable clinical outcome. BMI1 is one of the genes marking the ABC-type signature of DLBCL, associated with a poor prognosis. High levels of BMI1 mRNA/protein were also detected in CLL, MCL, MM and HL. In some MCLs, overexpression of BMI1 correlated with genomic amplification of the BMI1 gene detected by aCGH in 12-24% of analysed cases (Jares and Campo, 2008). BMI1 has been shown to contribute to cell cycle regulation and senescence by acting as transcriptional repressor of the INK4a-ARF tumor suppressors. Despite the wealthy information on BMI1 contributing to a variety of tumors, molecular mechanisms underlying deregulation of BMI1 in neoplasms and functional consequences of these events remain elusive.In summary, we show for the first time that BMI1, a postulated lymphoma-related oncogene, is recurrently targeted by IG-mediated chromosomal translocations in CLL and confirm its amplification in MCL. We found that in both cases with t(10;14)/IGH-BMI1, the translocation was acquired during clinical transformation of CLL initially harboring the del(13q)/RB1. The adverse prognostic impact of BMI1 aberrations in all four collected cases is reflected by a constantly aggressive clinical course of disease and short survival of the affected patients (1-50 months). In this regard, further molecular studies of the BMI1 pathway and its downstream partners are necessary to develop novel therapeutic approaches targeting this oncogene in human cancers. DisclosuresNo relevant conflicts of interest to declare.

Age-Dependent Decline in β-Cell Proliferation Restricts the Capacity of β-Cell Regeneration in Mice

OBJECTIVEThe aim of this study was to elucidate whether age plays a role in the expansion or regeneration of β-cell mass.RESEARCH DESIGN AND METHODSWe analyzed the capacity of β-cell expansion in 1.5- and 8-month-old mice in response to a high-fat diet, after short-term treatment with the glucagon-like peptide 1 (GLP-1) analog exendin-4, or after streptozotocin (STZ) administration.RESULTSYoung mice responded to high-fat diet by increasing β-cell mass and β-cell proliferation and maintaining normoglycemia. Old mice, by contrast, did not display any increases in β-cell mass or β-cell proliferation in response to high-fat diet and became diabetic. To further assess the plasticity of β-cell mass with respect to age, young and old mice were injected with a single dose of STZ, and β-cell proliferation was analyzed to assess the regeneration of β-cells. We observed a fourfold increase in β-cell proliferation in young mice after STZ administration, whereas no changes in β-cell proliferation were observed in older mice. The capacity to expand β-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age. The ability of β-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16Ink4aexpression in the β-cells. Young Bmi1−/− mice that prematurely upregulate p16Ink4afailed to expand β-cell mass in response to exendin-4, indicating that p16Ink4alevels are a critical determinant of β-cell mass expansion.CONCLUSIONSβ-Cell proliferation and the capacity of β-cells to regenerate declines with age and is regulated by the Bmi1/p16Ink4apathway.

Essential role for activation of the Polycomb group (PcG) protein chromatin silencing pathway in metastatic prostate cancer: Berezovska OP, Glinskii AB, Yang Z, Li XM, Hoffman RM, Glinsky GV, Translational and Functional Genomics Laboratory, Ordway Cancer Center, Ordway Research Institute, Inc., Center for Medical Sciences, Albany, NY

The Polycomb group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of Bmi1 oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA-gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from blood and shown to overexpress both BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.

Essential Role for Activation of the Polycomb Group (PcG) Protein Chromatin Silencing Pathway in Metastatic Prostate Cancer

The Polycomb-group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of the BMI1 oncogene causes neoplastic transformation of lymphocytes and plays an essential role in the pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both the BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from the circulation which overexpress both the BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased the BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate important biological role of PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.