Ink4a Deletion Research Articles

EGFRvIII uses intrinsic and extrinsic mechanisms to reduce glioma adhesion and increase migration.

A lack of biological markers has limited our ability to identify the invasive cells responsible for glioblastoma multiforme (GBM). To become migratory and invasive, cells must downregulate matrix adhesions, which could be a physical marker of invasive potential. We engineered murine astrocytes with common GBM mutations, e.g. Ink4a (Ink) or PTEN deletion and expressing a constitutively active EGF receptor truncation (EGFRvIII), to elucidate their effect on adhesion. While loss of Ink or PTEN did not affect adhesion, counterparts expressing EGFRvIII were significantly less adhesive. EGFRvIII reduced focal adhesion size and number, and these cells - with more labile adhesions - displayed enhanced migration. Regulation appears to depend not on physical receptor association to integrins but, rather, on the activity of the receptor kinase, resulting in transcriptional integrin repression. Interestingly, EGFRvIII intrinsic signals can be propagated by cytokine crosstalk to cells expressing wild-type EGFR, resulting in reduced adhesion and enhanced migration. These data identify potential intrinsic and extrinsic mechanisms that gliomas use to invade surrounding parenchyma.

Intersection of retinoblastoma tumor suppressor function, stem cells, metabolism, and inflammation.

The Retinoblastoma (RB) tumor suppressor regulates G1/S transition during cell cycle progression by modulating the activity of E2F transcription factors. The RB pathway plays a central role in the suppression of most cancers, and RB mutation was initially discovered by virtue of its role in tumor initiation. However, as cancer genome sequencing has evolved to profile more advanced and treatment‐resistant cancers, it has become increasingly clear that, in the majority of cancers, somatic RB inactivation occurs during tumor progression. Furthermore, despite the presence of deregulation of cell cycle control due to an INK4A deletion, additional CCND amplification and/or other mutations in the RB pathway, mutation or deletion of the RB gene is often observed during cancer progression. Of note, RB inactivation during cancer progression not only facilitates G1/S transition but also enhances some characteristics of malignancy, including altered drug sensitivity and a return to the undifferentiated state. Recently, we reported that RB inactivation enhances pro‐inflammatory signaling through stimulation of the interleukin‐6/STAT3 pathway, which directly promotes various malignant features of cancer cells. In this review, we highlight the consequences of RB inactivation during cancer progression, and discuss the biological and pathological significance of the interaction between RB and pro‐inflammatory signaling.

LKB1 suppresses melanoma metastasis: the answer is YES

<scp>LKB</scp>1 suppresses melanoma metastasis: the answer is <scp>YES</scp>

Ionizing Radiation Induces Hematopoietic Stem Cell Senescence and Long-Term Bone Marrow Suppression in a p16Ink4a/Arf-Independent manner

Abstract 1345Many patients receiving chemotherapy and/or ionizing radiation (IR) develop residual (or long-term) bone marrow (BM) injury that can not only limit the success of cancer treatment but also adversely affect their quality of life. Although residual BM injury has been largely attributed to the induction of hematopoietic stem cell (HSC) senescence, neither the molecular mechanisms by which chemotherapy and/or IR induce HSC senescence have been clearly defined, nor has an effective treatment been developed to ameliorate the injury. The Ink4a-Arf locus encodes two important tumor suppressors, p16Ink4a (p16) and Arf. Both of them have been implicated in mediating the induction of cellular senscence in a variety of cells including HSCs. Therefore, we examined the role of p16 and/or Arf in IR-induced HSC senescence and long-term BM suppression using a total body irradiation (TBI) mouse model. The results from our studies show that exposure of wild-type (WT) mice to a sublethal dose (6 Gy) of TBI induces HSC senescence and long-term BM suppression. The induction of HSC senescence is not associated with a reduction in telemore length in HSCs and their progeny, but is associated with significant increases in the production of reactive oxygen species (ROS), the expression of p16 and Arf mRNA, and the activity of senescence-associated β-galacotosidase (SA-β-gal) in HSCs. However, genetical deletion of Ink4a and/or Arf has no effect on TBI-induced HSC senescence, as HSCs from the Ink4a and/or Arf knockout mice after exposure to TBI exhibit similar changes as those seen in the cells from irradiated WT mice in comparison with the cells from un-irradiated mice with correspondent genotypes. In addition, TBI-induced long-term BM suppression is also not attenuated by the deletion of the Ink4a and/or Arf genes. These findings suggest that IR induces HSC senescence and long-term BM suppression in a p16Ink4a/Arf-independent manner. Disclosures:No relevant conflicts of interest to declare.

Lethal Myelofibrosis Induced by Bmi1-Deficient Hematopoietic Cells Unveils a Tumor Suppressor Function of the Polycomb Group Genes

Abstract 390Polycomb-group (PcG) proteins form polycomb repressive complex (PRC) 1 and PRC2, and function as transcriptional repressors through histone modifications. They maintain proliferative capacity of hematopoietic stem and progenitor cells by repressing transcription of tumor suppressor genes, thus have been characterized as oncogenes. However, identification of inactivating mutations of a PcG gene, EZH2, unveiled its tumor suppressor function in myeloid malignancies including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis reminiscent of PMF in mice. We previously reported that deletion of both Ink4a and Arf in Bmi1-deficient mice substantially restores the defective self-renewal capacity of HSCs. To evaluate the repopulating capacity of Bmi1−/−Ink4a-Arf−/− BM cells precisely, we transplanted wild-type, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− BM cells into lethally irradiated mice without competitor cells. The recipients repopulated with Bmi1−/−Ink4a-Arf−/− donor cells had 2-fold more megakaryocyte/erythroid progenitors (MEPs) and extramedullary hematopoiesis as evident from a significant increase in the number of LSK HSCs/MPPs in spleen. All the control recipient mice repopulated with Ink4a-Arf−/− BM cells eventually developed sarcoma or lymphoma and succumbed to die by 11 months post-transplant as previously reported with the Ink4a-Arf−/− mice. On the other hand, the recipient mice repopulated with Bmi1−/−Ink4a-Arf−/− BM cells died much earlier than the Ink4a-Arf−/− controls, displayed more progressive pancytopenia, and showed marked hepatosplenomegaly and hypoplastic BM with massive fibrosis at their terminal stage. Although abnormal megakaryocytosis was not obvious in BM and spleen because of severe fibrosis at the terminal stage of the disease, the mice at earlier time point after transplantation showed marked megakaryocytosis in both BM and spleen, implicating pathological megakaryocytosis in the development of lethal myelofibrosis. Together, lethal myelofibrosis induced by Bmi1−/−Ink4a-Arf−/− hematopoietic cells follows the natural course of human PMF.To identify the responsible genes for PMF-like disease in the absence of Bmi1, we compared gene expression profiles of LSK HSCs/MPPs and common myeloid progenitors (CMPs) from wild-type, Ink4a-Arf −/−, and Bmi1−/−Ink4a-Arf −/− BM cells. Absence of Bmi1 caused de-repression of a cohort of genes. We then compared the list of de-repressed genes with that of PMF-associated genes identified by a gene expression profiling of CD34+ cells from human PMF patients (Guglielmelli et al., Stem Cells 25:165–173, 2007). Hmga2, a well-known oncogene, appeared to be commonly upregulated in Bmi1−/−Ink4a-Arf −/− CMPs and PMF CD34+ cells. Chromatin immunoprecipitation assays demonstrated that Bmi1 directly represses the expression of Hmga2 by marking its promoter with a repressive histone mark. To test contribution of Hmga2 to the development of PMF-like disease in mice, we examined the effects of Hmga2 overexpression on hematopoiesis. Forced expression of Hmga2 in HSCs promoted expansion of progenitor cells and significantly facilitated megakaryocytopoiesis in vitro. Hmga2-overexpressing HSCs also induced a mild myeloproliferative state with enhanced megakaryocytopoiesis in recipient mice, although fibrosis was not obviously induced. These results implicated Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1.Collectively, our findings unveiled the tumor suppressor function of Bmi1. Corresponding to our findings, mice with hypomorphic mutations of Eed and Suz12 reportedly showed enhanced hematopoiesis. All these findings might be in line with tumor suppressor function of EZH2 observed in MDS and MPN patients, and suggestive of a broad range of target genes of the PcG proteins, which include both oncogenes and tumor suppressor genes. Although the tumor suppressor genes have been stresses as PcG targets, our findings shed a light on the role of PcG proteins in gene silencing of oncogenes. Thus, the PcG proteins fine-tune the growth of hematopoietic cells in both a positive and a negative manner to maintain hematopoietic homeostasis. Disclosures:No relevant conflicts of interest to declare.

INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.

SCCRO Promotes Glioma Formation and Malignant Progression in Mice

Originally identified as an oncogene activated by amplification in squamous cell carcinomas, several lines of evidence now suggest that squamous cell carcinoma-related oncogene (SCCRO; aka DCUN1D1) may play a role in the pathogenesis of a wide range of human cancers including gliomas. SCCRO's oncogenic function is substantiated by its ectopic expression, resulting in transformation of cells in culture and xenograft formation in nude mice. The aim of this study was to assess the in vivo oncogenicity of SCCRO in a murine model. Ubiquitous expression of SCCRO resulted in early embryonic lethality. Because SCCRO overexpression was detected in human gliomas, its in vivo oncogenic activity was assessed in an established murine glioma model. Conditional expression of SCCRO using a replication-competent ASLV long terminal repeat with splice acceptor/nestin-(tumor virus-A) tv-a model system was not sufficient to induce tumor formation in a wild-type genetic background, but tumors formed with increasing frequency and decreasing latency in facilitated background containing Ink4a deletion alone or in combination with PTEN loss. Ectopic expression of SCCRO in glial progenitor cells resulted in lower-grade gliomas in Ink4a-/- mice, whereas its expression in Ink4a-/-/PTEN-/- background produced high-grade glioblastoma-like lesions that were indistinguishable from human tumors. Expression of SCCRO with platelet-derived growth factor-beta (PDGF-β) resulted in an increased proportion of mice forming glioblastoma-like tumors compared with those induced by PDGF-β alone. This work substantiates SCCRO's function as an oncogene by showing its ability to facilitate malignant transformation and carcinogenic progression in vivo and supports a role for SCCRO in the pathogenesis of gliomas and other human cancers.

The polycomb group gene product Ezh2 regulates proliferation and differentiation of murine hepatic stem/progenitor cells

Polycomb group proteins initiate and maintain gene silencing through chromatin modifications and contribute to the maintenance of self-renewal in a variety of stem cells. Among polycomb repressive complexes (PRCs), PRC2 initiates gene silencing by methylating histone H3 lysine 27, and PRC1 maintains gene silencing through mono-ubiquitination of histone H2A lysine 119. We have previously shown that Bmi1, a core component of PRC1, tightly regulates the self-renewal of hepatic stem/progenitor cells. In this study, we conducted lentivirus-mediated knockdown of Ezh2 to characterise the function of Ezh2, a major component of PRC2, in hepatic stem/progenitor cells. Loss of Ezh2 function in embryonic murine hepatic stem/progenitor cells severely impaired proliferation and self-renewal capability. This effect was more prominent than that of Bmi1-knockdown and was partially abrogated by the deletion of both Ink4a and Arf, major targets of PRC1 and PRC2. Importantly, Ezh2-knockdown but not Bmi1-knockdown promoted the differentiation and terminal maturation of hepatocytes, followed by the up-regulation of several transcriptional regulators of hepatocyte differentiation. Our findings indicate that Ezh2 plays an essential role in the maintenance of both the proliferative and self-renewal capacity of hepatic stem/progenitor cells and the full execution of their differentiation.

Bmi1 Is Essential for Leukemic Reprogramming of Myeloid Progenitor Cells.

Abstract 1423Poster Board I-446The polycomb group (PcG) protein Bmi1 plays an essential role in the maintenance of self-renewing hematopoietic stem cells (HSCs). Derepressed p16Ink4a and p19Arf are tightly associated with a loss of self-renewing capacity of HSCs in Bmi1-deficient mice. Deletion of both Ink4a and Arf genes substantially restores the self-renewal capacity of Bmi1−/− HSCs. Thus, Bmi1 maintains HSCs by acting as a critical failsafe against the p16Ink4a- and p19Arf-dependent senescence pathway. Meanwhile, Bmi1 was originally identified as a collaborating oncogene in the induction of lymphoma and was subsequently reported to be overexpressed in various human cancers including leukemia. Recent studies have demonstrated that PcG proteins bind to multiple regions of the genome and regulate a bunch of target genes. Therefore, we asked whether Bmi1 is essential for leukemic stem cells (LSCs) and tried to identify critical target genes for Bmi1 other than Ink4a and Arf in leukemia. We expressed the MLL-AF9 leukemic fusion gene in purified Lin−Sca-1−c-Kit+CD34+FcγRII/ IIIhi granulocyte/macrophage progenitors (GMPs) from wild-type, Bmi1−/−, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− mice and performed in vitro myeloid progenitor replating assay. GMPs from 4 different genetic backgrounds were all immortalized in vitro, although Bmi1-deficient cells showed a slightly decreased replating efficiency. We then infused the immortalized cells into lethally irradiated recipient mice. Mice infused with wild-type and Ink4a-Arf−/− cells developed acute myelogenous leukemia (AML) at 30 to 60 days after infusion. Mice infused with Bmi1−/− cells did not develop leukemia at all. While a significant portion of mice infused with Bmi1−/−Ink4a-Arf−/− cells developed AML, although they took much longer time compared to those mice infused with wild-type and Ink4a-Arf−/− cells. These results indicate that as in HSCs, the Ink4a /Arf locus is one of the major targets for Bmi1 in leukemogenesis. In order to find unknown targets of Bmi1 in LSCs, we compared gene expression profiles of purified c-KithiFcRγII/IIIhiCD34+ cells from Ink4a-Arf−/− and Bmi1−/−Ink4a-Arf−/− immortalized cells. We found that the loss of Bmi1 did not affect the induction of MLL-AF9 target gene expression. By contrast, a number of genes were derepressed in the absence of Bmi1. Among these, Tbx15, a transcriptional co-repressor gene, appeared to be regulated by Bmi1 and a potential tumor suppressor gene in the development of leukemia. Of interest, the majority of derepressed target genes in transformed Bmi1−/−Ink4a-Arf−/− cells, including Tbx15, remained unchanged by re-expression of Bmi1. Correspondingly, re-introduction of Bmi1 to transformed Bmi1−/−Ink4a-Arf−/− cells failed to rescue their compromised leukemogenic activity in vivo. Our findings suggest that Bmi1 is required for faithful epigenetic reprogramming of myeloid progenitors into LSCs by leukemic fusions and contributes to establish LSC-specific transcriptional profiles to confer full leukemogenic activity on LSCs. Disclosures:No relevant conflicts of interest to declare.

The Inhibitor of Growth 1 (ING1) Is Involved in Trichostatin A-Induced Apoptosis and Caspase 3 Signaling in p53-Deficient Glioblastoma Cells

Prognosis for patients with glioblastoma multiforme (GBM) is poor. Inhibitors of histone deacetylases (HDACi) like trichostatin A (TSA) are promising alternatives to conventional treatment. Deficient tumor suppressor functions, such as TP53 mutations and p14(ARF)/p16(INK4a) deletions, are characteristic for GBM and can cause resistance to DNA damaging agents such as cisplatin and to HDACi like TSA. The type II tumor suppressor Inhibitor of growth 1 (ING1) is involved in DNA damage response and histone modification. We have previously shown that ING1 is downregulated in GBM and involved in glioma-induced angiogenesis and in cisplatin-induced apoptosis in malignant glioma cells. Hence, the goal of our present study was to investigate whether TSA affects ING1 protein expression and also whether modulating ING1 levels affects TSA-induced apoptosis in malignant glioma cells that contain deficient p53 function and inactive pl4(ARF)/p16(INK4a) signaling. If so, we asked, which apoptotic pathway might be the major mediator beyond this interaction. To test whether ING1 proteins function in TSA-induced apoptosis in GBM, we analyzed TSA effects in LN229 GBM cells, which harbor TP53 mutations and INK4a deletion, following ING1 knockdown by siRNA. Expression of ING1, acetylated core histones H3 and H4, and the proapoptotic proteins caspase 3 and Fas-associated death domain (FADD) was determined by Western blotting. Percentages of apoptotic cells were obtained by flow cytometry. TSA induced the major ING1 isoform p33(ING1b) and increased levels of both histone acetylation and apoptosis in LN229 cells. ING1 knockdown cells revealed marked resistance to TSA-induced apoptosis, impairment of caspase 3 activation, and suppression of FADD. The data suggest that ING1 contributes to TSA-induced apoptosis in GBM cells with deficient p53 and p14(ARF)/p16(INK4a) functions, possibly by regulating FADD/caspase 3 signaling.

Activated TNF-α/NF-κB signaling via down-regulation of Fas-associated factor 1 in asbestos-induced mesotheliomas from Arf knockout mice

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of cyclin dependent kinase 4) and ARF (alternative reading frame) here, that are translated from alternatively spliced mRNAs. Human ARF is implicated as a tumor suppressor gene, mainly in association with the simultaneous deletion of INK4A. However, questions remain as to whether loss of ARF alone is sufficient to drive tumorigenesis. Here, we report that mice deficient for Arf are susceptible to accelerated asbestos-induced malignant mesothelioma (MM). MMs arising in Arf (+/-) mice consistently exhibit biallelic inactivation of Arf, but, unexpectedly, do not acquire additional recurrent genetic alterations that we previously identified in asbestos-induced MMs arising in Nf2 (+/-) mice. Array CGH analysis was used to detect a recurrent deletion at chromosome 4C6 in MMs from Arf (+/-) mice. A candidate gene in this region, Faf1 (FAS-associated factor 1), was further explored, because it encodes a protein implicated in tumor cell survival and in the pathogenesis of some human tumor types. We confirmed hemizygous loss of Faf1 and down-regulation of Faf1 protein in a series of MMs from Arf (+/-) mice, and we then showed that Faf1 regulates TNF-alpha-mediated NF-kappaB signaling, a pathway previously implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a significant role in driving MM pathogenesis, and implicate Faf1 as a key component in the TNF-alpha/NF-kappaB signaling node that has now been independently implicated in asbestos-induced oncogenesis.

Ultra-High Resolution Analysis of Genomic Alterations in High-Risk Acute Lymphoblastic Leukemia.

Previous genomic analyses identified a high frequency of genetic alterations targeting B-lymphoid development (>40%) and cell cycle/tumor suppression (CDKN2A/B, 35%) in pediatric B-progenitor acute lymphoblastic leukemia (B-ALL). The frequency and nature of copy number alterations (CNAs) differs significantly across ALL subtype with less than one CNA/case in MLL ALL and over 8 lesions/case in BCR-ABL1 and ETV6-RUNX1 ALL; and the near obligate presence of IKZF1 (Ikaros) deletion in BCR-ABL1 ALL, but never in ETV6-RUNX1 ALL. Although important insights have been obtained from these genome-wide CNAs, it is important to realize that the SNP array platforms that have been used are limited by uneven probe spacing and poor coverage of some genes (including IKZF1 and CDKN2A/B). Moreover, additional CNAs are detected as array resolution increases (e.g. 6.5 lesions per BCR-ABL1 case using 315,000 markers, and 8.8 for 615,000 marker data). Thus, the true frequency of CNAs in ALL remains unknown. To address this question, we now report ultra-high resolution CNA analysis using 2.17 million feature oligonucleotide arrays (Roche Nimblegen) of 20 MLL, 20 BCR-ABL1, and 4 miscellaneous karyotype B-ALL cases. All had previously been examined using Affymetrix 500K SNP arrays. We identified a mean of 6.2 deletions (range 0–38) and 1.4 gains (0–13) per case. There were more lesions in BCR-ABL1 than MLL ALL cases (mean 11.2 v 1.15 deletions, P<0.0001; 2.3 v 0.45 amplifications, P=0.01). Notably, more lesions were detected by this 2.17 million feature array in comparison to previous Affymetrix SNP array data in BCRABL1 ALL (13.5 v 8.8 lesions/case, P=0.001) but not MLL ALL (1.6 v 1.3 lesions/case, P=NS). Moreover, the Nimblegen platform robustly identified focal deletions in genes poorly covered by SNP arrays (IKZF1, CDKN2A/B) and detected additional deletions not previously identified (e.g. deletion of HBS1L immediately adjacent to MYB). The most common lesions in ALL were deletions of IKZF1 (80% BCR-ABL1, none in MLL), CDKN2A/B (60% BCR-ABL1, 20% MLL) and CNAs of PAX5 (50% BCR-ABL1, 20% MLL). High array resolution enabled PCR-based mapping and sequencing of the genomic breakpoints of multiple recurring deletions (e.g. ADD3, BTLA, CDKN2A/B, C20orf94, HBS1L, IKZF1 and PAX5), each of which bore hallmarks of aberrant RAG-mediated recombination.Due to the small size of the CDKN2A/B genomic locus (41.5 Kb), previous genomic analyses have been unable to accurately determine the frequency of deletion of each of the three tumor suppressors (INK4A, ARF, and INK4B) encoded by this locus. We performed genomic quantitative PCR (gqPCR) across the locus (CDKN2A exons 1b, 1a and 2; CDKN2B exons 1 and 2), which revealed complete concordance between deletions identified by the Nimblegen array and confirmatory gqPCR. However, gqPCR was required to precisely delineate the extent of deletion in each case. All deletions involved both CDKN2A and CDKN2B loci except one MLL case (CDKN2A only), and the same degree of deletion (mono- or bi-allelic) was usually uniform across the locus for each case. Exceptions included two cases harboring homozygous deletions of INK4B and ARF but hemizygous deletion of INK4A, and a case with deletion of ARF-INK4A but not INK4B. To extend this analysis to epigenetic silencing of CDKN2A/B, we performed quantitative methylation analysis of six CDKN2A and two CDKN2B CpG islands by MALDI-TOF mass spectrometry for all BCR-ABL1 cases and 18 CDKN2A/B non-deleted MLL, ETV6-RUNX, and T-ALL cases. Strikingly, whereas high level CpG methylation of CDKN2B and CDKN2A exons 2 and 3 was observed in half the MLL and T-ALL cases, no high level methylation was observed in BCR-ABL1 ALL.These data demonstrate that identification of all CNAs in BCR-ABL1 ALL is critically dependent on array resolution, whereas MLL -rearranged leukemias harbor very few CNAs. Importantly, the use of high density arrays with even probe distribution improves the ability to robustly identify focal CNAs in genes poorly covered by SNP arrays, and to further characterize the mechanism of deletion by sequencing. Furthermore, even with the highest resolution arrays, complementary quantitative PCR across CDKN2A/B is required to precisely define the extent of deletion of this compact gene cluster. Thus, oligonucleotide array or sequencing-based platforms allowing true tiling across the genome will be required to determine the full complement of CNAs in ALL.

The ARFul truth about melanoma susceptibility genes

The ARFul truth about melanoma susceptibility genes

BCR–ABL and CDKN2A: a dropped connection

In their recent Review of chronic myeloid leukamia (CML) stem cell biology (Savona , M. & Talpaz , M.Getting to the stem of chronic myeloid leukemia. Nature Rev. Cancer 8, 341–350)1, Savona and Talpaz focus their attention exclusively on deregulated self-renewal mechanisms in Philadelphia chromosome (Ph)+ hematopoietic stem cells (HSCs) in chronic-phase CML and on the role of -catenin abnormalities in granulocyte–macrophage progenitors in myeloid blast crisis. However, a review of genomic abnormalities in lymphoid blast crisis provides insights into alternative mechanisms of progenitor self-renewal in BCR–ABL-driven diseases. Deletion of the CDKN2A locus (encoding the INK4A and ARF tumour suppressor genes) frequently occurs in the progression of CML from chronic-phase to lymphoid, but not myeloid, blast crisis2, and a recent genome-wide analysis3 (published concurrently with this CML review) has reinforced the conclusions of earlier studies. INK4A and ARF share common second and third exons, are translated in alternate reading frames, and exhibit no sequence similarity4. Whereas p16INK4A is a bona fide cyclin-dependent kinase inhibitor, p14ARF (p19Arf in mice) is not, but instead regulates p53 tumour suppressor function through its interaction with MDM2 (Ref. 5). BCR–ABL induces p19Arf in vivo to oppose tumour development, but the combination of BCR–ABL expression and Arf inactivation is sufficient to generate leukaemia-initiating cells in a murine model of Ph+ acute lymphocytic leukaemia6. Therefore, in BCR–ABL-driven lymphoid leukaemias — specifically, CML lymphoid blast crisis and Ph+ acute lymphocytic leukaemia — deletion of INK4A and ARF in committed Ph+ lymphoid blasts is expected to simultaneously disrupt both the RB and p53 tumour suppressor networks, thereby facilitating leukaemia-initiating cell self-renewal and enhancing targeted therapeutic resistance in vivo.

The regulation of stem cell self‐renewal

The mechanisms that regulate stem cell self‐renewal are fundamental to the formation and maintenance of normal hematopoiesis as well as hematopoietic malignancies. Fetal stem cells differ phenotypically and functionally from adult stem cells in diverse tissues. However, little is known about how these differences are regulated. To address this we compared the gene expression profiles of fetal versus adult hematopoietic stem cells (HSCs) and discovered that the Sox17 transcription factor is specifically and preferentially expressed in fetal and neonatal but not adult HSCs (Cell 130:470). Germline deletion of Sox17 led to severe fetal hematopoietic defects, including a lack of detectable definitive HSCs. Conditional deletion of Sox17 led to the loss of fetal and neonatal but not adult HSCs. Sox17 expression by HSCs was extinguished three to four weeks after birth as HSCs acquired adult properties. During this transition, loss of Sox17 expression by individual cells correlated with slower proliferation and the acquisition of an adult phenotype. Sox17 thus distinguishes the transcriptional regulation of fetal and neonatal HSCs from that of adult HSCs.As HSCs enter the postnatal period, they become dependent upon the polycomb family transcriptional repressor, Bmi‐1. Bmi‐1 is required for the postnatal maintenance of every type of stem cell yet examined including HSCs (Nature 423:302, 2003) and neural stem cells (Nature 425:962, 2003). Bmi‐1‐deficient mice die by early adulthood with stem cell depletion, growth retardation, ataxia, and seizures. These phenotypes correlate with increased expression of the cyclin‐dependent kinase inhibitor p16Ink4a and the p53 agonist p19Arf, which promote cellular senescence. Deletion of Ink4a and/or Arf from Bmi‐1−/− mice partially rescues stem cell self‐renewal and stem cell frequency (G&amp;D 19:1432, 2005). Despite ongoing Bmi‐1 expression, Ink4a expression increases with age, reducing stem cell frequency and function (Nature 443:448). The regulation of stem cell self‐renewal throughout life by networks of proto‐oncogenes and tumor suppressors emphasizes the mechanistic links between stem cell self‐renewal and cancer cell proliferation.

Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice

The polycomb group (PcG) protein Bmi1 plays an essential role in the self-renewal of hematopoietic and neural stem cells. Derepression of the Ink4a/Arf gene locus has been largely attributed to Bmi1-deficient phenotypes in the nervous system. However, its role in hematopoietic stem cell (HSC) self-renewal remained undetermined. In this study, we show that derepressed p16Ink4a and p19Arf in Bmi1-deficient mice were tightly associated with a loss of self-renewing HSCs. The deletion of both Ink4a and Arf genes substantially restored the self-renewal capacity of Bmi1−/− HSCs. Thus, Bmi1 regulates HSCs by acting as a critical failsafe against the p16Ink4a- and p19Arf-dependent premature loss of HSCs. We further identified a novel role for Bmi1 in the organization of a functional bone marrow (BM) microenvironment. The BM microenvironment in Bmi1−/− mice appeared severely defective in supporting hematopoiesis. The deletion of both Ink4a and Arf genes did not considerably restore the impaired BM microenvironment, leading to a sustained postnatal HSC depletion in Bmi1−/−Ink4a-Arf−/− mice. Our findings unveil a differential role of derepressed Ink4a and Arf on HSCs and their BM microenvironment in Bmi1-deficient mice. Collectively, Bmi1 regulates self-renewing HSCs in both cell-autonomous and nonautonomous manners.

Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways

Bmi-1 is required for the post-natal maintenance of stem cells in multiple tissues including the central nervous system (CNS) and peripheral nervous system (PNS). Deletion of Ink4a or Arf from Bmi-1(-/-) mice partially rescued stem cell self-renewal and stem cell frequency in the CNS and PNS, as well as forebrain proliferation and gut neurogenesis. Arf deficiency, but not Ink4a deficiency, partially rescued cerebellum development, demonstrating regional differences in the sensitivity of progenitors to p16Ink4a and p19Arf. Deletion of both Ink4a and Arf did not affect the growth or survival of Bmi-1(-/-) mice or completely rescue neural development. Bmi-1 thus prevents the premature senescence of neural stem cells by repressing Ink4a and Arf, but additional pathways must also function downstream of Bmi-1.

Coexistence of Copy Number Changes of Different Genes (INK4A, erbB-1, erbB-2, CMYC, CCND1 and ZNF217) in Urothelial Tumors

The aim of this study was to establish the frequency of combinatorial and separate copy number changes of INK4A (9p21), erbB-1 (7p11), erbB-2 (17q17–21), CMYC (8q24), CCND1 (11q13) and ZNF217 (20q13) in urothelial tumors; a tissue microarray of 159 urothelial bladder tumors was analyzed by fluorescence in situ hybridization. A total of 38 invasive tumors were successfully analyzed for all 6 loci. Normal gene copy numbers of all loci were established in 13 tumors (34.2%). In 25 tumors (65.8%), at least one aberration was found. Single abnormalities were detected in 16 tumors (64%), while double or higher abnormalities were found in 9 tumors (39%). The most frequent genetic change was deletion of INK4A (60% of aberrant tumors), followed by increased copy number changes of ZNF217 (36%), CCND1 (28%), CMYC (12%) and erbB-1 (4%). It was significantly more frequent in pT1 than in pT2–4 tumors and was predominantly found separately, while oncogene copy number increases were usually combined with another aberration and were not associated with the tumor stage. We concluded that INK4A loss is usually found as a single aberration in bladder cancer, which is more frequent in pT1 than in pT2–4 tumors. Overrepresentations of putative oncogenes are present in these two groups with similar frequency and are rarely found as single abnormality.

Deletion of one copy of the p16INK4A tumor suppressor gene is implicated as a predisposing factor in pediatric leukemia

The p16 INK4A tumor suppressor gene is frequently disrupted by mutation or deletion in a wide range of cancer types, ranging from leukemia to cancers of the bladder, skin, lung, liver, and spleen. We have previously shown that deletion of at least one copy of the p16 INK4A gene is associated with an increased risk of relapse in pediatric leukemia. Our data suggest that hemizygous p16 INK4A deletion may be constitutional, conferring susceptibility to leukemia. Confirmation of this association is worthy of a larger study. Data from primary leukemia specimens are also presented here which examined the possibility that the remaining allele of the gene was inactivated by another mechanism such as mutation or was silenced by methylation. These possibilities were formally excluded in a case of hemizygous loss of the p16 INK4A gene in leukemia, establishing that in this case the p16 INK4A deletion was either semidominant or fully haploinsufficient for relapse susceptibility in this disease. Implementation of high throughput methods such as those used here for detecting hemizygous loss of tumor suppressor genes will become increasingly important for molecular diagnosis of cancer. This is particularly true for the emerging class of tumor suppressor genes where deletion of one allele is sufficient to confer cancer susceptibility or poor prognosis with standard treatment.

Prognostic impact of INK4A deletion in Ewing sarcoma.

The primary genetic alteration in > 95% of Ewing sarcomas (ES) is a specific fusion of EWS with FLI1 or ERG. Secondary genetic alterations possibly involved in progression of ES are not well understood. A recent study found loss of the negative cell cycle regulator gene INK4A in 8 of 27 ES samples (30%). To confirm these findings and evaluate their prognostic significance, the authors studied INK4A deletion in 41 ES samples from 39 patients. Using Southern blot analysis with an INK4A p16 cDNA probe, the intensity of the INK4A bands in ES DNA samples was normalized to that of a control probe and compared with nondeleted control DNA; > 50% signal reduction was scored as evidence of deletion. All ES tumor DNA samples previously were confirmed to have EWS rearrangements on the same Southern blots, using a cDNA probe spanning the EWS breakpoint region. Tumors from 7 patients (18%) showed INK4A deletion independent of disease stage (localized or metastatic) or sample source (primary tumor or metastasis). INK4A was a strong negative factor for disease specific survival in univariate analysis (P = 0.001) and in multivariate analysis including stage (relative risk = 6; P = 0.001). INK4A deletions appear to be the most frequent secondary molecular genetic alteration found to date in ES. Their possible clinical usefulness in identifying a subset of ES patients with poor prognosis merits systematic prospective analysis. [See related article on pages 783-92.]