H3K27 Methylation: A Focal Point of Epigenetic Deregulation in Cancer.

Epigenetics, the modification of chromatin without changing the DNA sequence itself, determines whether a gene is expressed, and how much of a gene is expressed. Methylation of lysine 27 on histone 3 (H3K27me), a modification usually associated with gene repression, has established roles in regulating the expression of genes involved in lineage commitment and differentiation. Not surprisingly, alterations in the homeostasis of this critical mark have emerged as a recurrent theme in the pathogenesis of many cancers. Perturbations in the distribution or levels of H3K27me occur due to deregulation at all levels of the process, either by mutation in the histone itself, or changes in the activity of the writers, erasers, or readers of this mark. Additionally, as no single histone mark alone determines the overall transcriptional readiness of a chromatin region, deregulation of other chromatin marks can also have dramatic consequences. Finally, the significance of mutations altering H3K27me is highlighted by the poor clinical outcome of patients whose tumors harbor such lesions. Current therapeutic approaches targeting aberrant H3K27 methylation remain to be proven useful in the clinic. Understanding the biological consequences and gene expression pathways affected by aberrant H3K27 methylation may lead to identification of new therapeutic targets and strategies.

MMSET/WHSC1 enhances DNA damage repair leading to an increase in resistance to chemotherapeutic agents.

MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double-strand breaks (DSBs). By using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET-high cells had increased damage at baseline. Upon addition of a DNA-damaging agent, MMSET-high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET-low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC.

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3'-untranslated region (3'-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

miR-433 is aberrantly expressed in myeloproliferative neoplasms and suppresses hematopoietic cell growth and differentiation.

BCR-ABL-negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation, but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and microRNA (miRNA) deregulation may have an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared with controls (P<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2.

The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal transition and invasive properties of prostate cancer.

Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial-mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

Functional characterization of Wilms tumor-suppressor WTX and tumor-associated mutants.

The WTX, Wilms tumor-associated tumor-suppressor gene, is present on the X chromosome and a single WTX mutation may be sufficient to promote carcinogenesis. Unlike the WT1 tumor suppressor, a transcription factor, WTX lacks conserved functional protein domains. To study the function of WTX, we constructed inducible cell lines expressing WTX and tumor-associated WTX mutants. Induction of WTX inhibited cell growth and caused G(1)/G(0) arrest. In contrast, a short, tumor-associated truncation mutant of WTX358 only slightly inhibited cell growth without a significant cell-cycle arrest, although expression of a longer truncation mutant WTX565 led to the growth inhibition and cell-cycle arrest to a similar extent as wild-type WTX. Like WT1, WTX slowed growth and caused cell-cycle arrest through p21 induction. Gene expression profiling showed that these two tumor-suppressors regulated genes in similar pathways, including those implicated in control of the cellular growth, cell cycle, cell death, cancer and cardiovascular system development. When gene expression changes mediated by wild-type WTX were compared with those affected by mutant forms, WTX565 showed a 55% overlap (228 genes) in differentially regulated genes, whereas WTX358 regulated only two genes affected by wild-type WTX, implying that amino-acid residues 358-561 are critical for WTX function.

Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms.

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3-23.3 (n=1), 9q33.1-34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31-36.33 (n=6), 17q21.2-q21.31 (n=5) and 17q25.1-25.3 (n=5) and deletions affecting 18p11.31-11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a 'HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal.

Mutations with epigenetic effects in myeloproliferative neoplasms and recent progress in treatment: Proceedings from the 5th International Post-ASH Symposium.

Immediately following the 2010 annual American Society of Hematology (ASH) meeting, the 5th International Post-ASH Symposium on Chronic Myelogenous Leukemia and BCR-ABL1-Negative Myeloproliferative Neoplasms (MPNs) took place on 7-8 December 2010 in Orlando, Florida, USA. During this meeting, the most recent advances in laboratory research and clinical practice, including those that were presented at the 2010 ASH meeting, were discussed among recognized authorities in the field. The current paper summarizes the proceedings of this meeting in BCR-ABL1-negative MPN. We provide a detailed overview of new mutations with putative epigenetic effects (TET oncogene family member 2 (TET2), additional sex comb-like 1 (ASXL1), isocitrate dehydrogenase (IDH) and enhancer of zeste homolog 2 (EZH2)) and an update on treatment with Janus kinase (JAK) inhibitors, pomalidomide, everolimus, interferon-α, midostaurin and cladribine. In addition, the new 'Dynamic International Prognostic Scoring System (DIPSS)-plus' prognostic model for primary myelofibrosis (PMF) and the clinical relevance of distinguishing essential thrombocythemia from prefibrotic PMF are discussed.

Retinoblastoma protein and the leukemia-associated PLZF transcription factor interact to repress target gene promoters.

Translocations of the retinoic acid receptor-alpha (RARalpha) locus with the promyelocytic leukemia zinc-finger (PLZF) or PML genes lead to expression of oncogenic PLZF-RARalpha or PML-RARalpha fusion proteins, respectively. These fusion oncoproteins constitutively repress RARalpha target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARalpha have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here, we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (poxvirus and zinc-finger, POZ) and DNA-binding (zinc-finger) domains. In addition, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction of Rb with PML or E2F1, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. These data are supported by chromatin immunoprecipitation analysis, which indicates that PLZF associates with the promoter region of CDC6, a known E2F/Rb target gene. Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F/Rb target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to function in stem cells and taken together these data suggest that interactions between PLZF and Rb could be important in stem cell biology.

ATF-2 controls transcription of Maspin and GADD45 alpha genes independently from p53 to suppress mammary tumors.

The activating transcription factor, ATF-2, is a target of p38 and JNK that are involved in stress-induced apoptosis. Heterozygous Atf-2 mutant (Atf-2+/-) mice are highly prone to mammary tumors. The apoptosis-regulated gene GADD45alpha and the breast cancer suppressor gene Maspin, both of which are known to be p53 target genes, are downregulated in the mammary tumors arisen in Atf-2+/- mice. Here, we have analysed how ATF-2 controls the transcription of GADD45alpha and Maspin. ATF-2 and p53 independently activate the GADD45alpha transcription. ATF-2 does not directly bind to the GADD45alpha promoter; instead, it is recruited via Oct-1 and NF-I. ATF-2 simultaneously binds to Oct-1, NF-I and breast cancer suppressor BRCA1 to activate transcription. With regard to Maspin, ATF-2 and p53 directly bind to different sites in the Maspin promoter to independently activate its transcription. Consistent with the observation that ATF-2 and p53 independently activate the transcription of Maspin and GADD45alpha is that the loss of one copy of p53 shortened the period required for mammary tumor development in Atf-2+/- mice. These studies suggest the functional link between the ATF-2 and the two tumor suppressors BRCA1 and p53.

Epigenetic regulation of normal and malignant hematopoiesis.

The molecular processes governing hematopoiesis involve the interplay between lineage-specific transcription factors and a series of epigenetic tags, including DNA methylation and covalent histone tail modifications, such as acetylation, methylation, phosphorylation, SUMOylation and ubiquitylation. These post-translational modifications, which collectively constitute the 'histone code', are capable of affecting chromatin structure and gene transcription and are catalysed by opposing families of enzymes, allowing the developmental potential of hematopoietic stem cells to be dynamically regulated. The essential role of these enzymes in regulating normal blood development is highlighted by the finding that members from all families of chromatin regulators are targets for dysregulation in many hematological malignancies, and that patterns of histone modification are globally affected in cancer as well as the regulatory regions of specific oncogenes and tumor suppressors. The discovery that these epigenetic marks can be reversed by compounds targeting aberrant transcription factor/co-activator/co-repressor interactions and histone-modifying activities, provides the basis for an exciting field in which the epigenome of cancer cells may be manipulated with potential therapeutic benefits.

Sprouty2 inhibits BDNF-induced signaling and modulates neuronal differentiation and survival.

Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.

The PLZF gene of t (11;17)-associated APL.

The PLZF gene is one of five partners fused to the retinoic acid receptor alpha in acute promyelocytic leukemia. PLZF encodes a DNA-binding transcriptional repressor and the PLZF-RARalpha fusion protein like other RARalpha fusions can inhibit the genetic program mediated by the wild tpe retinoic acid receptor. However an increasing body of literature indicates an important role for the PLZF gene in growth control and development. This information suggests that loss of PLZF function might also contribute to leukemogenesis.

Minimally Invasive Limited Ligation Endoluminal-assisted Revision (MILLER) for treatment of dialysis access-associated steal syndrome.

Dialysis-associated steal syndrome (DASS) is defined as a clinical condition caused by arterial insufficiency distal to the dialysis access owing to diversion of blood into the fistula or graft. The incidence of symptomatic DASS requiring treatment is 1-8%. The etiology is iatrogenic and symptoms are quite debilitating. Banding of the access inflow has largely been abandoned because of the inherent problem with balancing fistula flow with distal flow complicated by a high incidence of subsequent access thrombosis. In this study, we are reporting a modification to the traditional banding procedure, which markedly improves banding outcomes. We are reporting 16 patients who underwent a new standardized minimally invasive banding procedure performed in an outpatient setting with minimal morbidity. This modified banding procedure requires a small (1-2 cm) skin incision for the placement of a ligature and utilizes a 4 or 5 mm diameter endoluminal balloon to achieve and standardize the desired reduction of inflow size. All 16 patients had immediate symptomatic and angiographic improvement after the procedure. Follow-up showed none of the patients had recurrence of symptoms or thrombosis of the access. In our experience, this procedure is an excellent treatment option because of its simplicity and should be considered as a first-line treatment for patients with DASS.

Canalicular immunostaining of aminopeptidase N (CD13) as a diagnostic marker for hepatocellular carcinoma.

BACKGROUND: Aminopeptidase N (CD13) is expressed in normal and neoplastic liver tissue, where it exhibits a characteristic canalicular pattern (CD13(can)), similar to that seen for CD10 and when antibodies crossreact with biliary glycoprotein I (p-CEA). AIM: To compare the putative diagnostic use of CD13(can) in differentiating between hepatocellular (HCC) and non-hepatocellular carcinomas metastatic to the liver (non-HCC). METHODS: A retrospective study comparing 53 HCC specimens with 32 non-HCC specimens. Immunostaining was performed with HepPar1 and antibodies directed against CD10, CD13, p-CEA, and alpha fetoprotein (AFP). RESULTS: In the HCC group, a canalicular staining pattern was found for CD13, p-CEA, and CD10 in 51, 43, and 33 specimens, respectively. HepPar1 was positive in 29 and AFP in 17 HCC specimens. In the non-HCC group, canalicular immunostaining for CD10 and p-CEA was confined to non-neoplastic liver tissue. One poorly differentiated cholangiocarcinoma showed apical expression of CD13, resembling to some extent CD13(can). Sensitivity and specificity were 96.2% and 97.0%, respectively, for CD13(can), 81.1% and 100% for p-CEA(can), 62.3% and 100%, for CD10(can), 54.7% and 99.9% for HepPar1, and 32.1% and 100% for AFP. CONCLUSIONS: These results show that CD13(can) is more sensitive in differentiating between HCC and non-HCC than CD10(can), p-CEA(can), HepPar1, and AFP.

The human promyelocytic leukemia zinc finger gene is regulated by the Evi-1 oncoprotein and a novel guanine-rich site binding protein.

PLZF (promyelocytic leukemia zinc finger ) is a transcription factor disrupted in t(11;17)-associated acute promyelocytic leukemia which is highly expressed in undifferentiated myeloid cells. To address the tissue-specific regulation of the promoter, we isolated sequences 1.2-kb 5' to the transcriptional start site. Sequence analysis demonstrated that this region contains one TATA box and several putative transcription factor binding sites including four G/C-rich sites and one Evi-1-like site. A fragment of the promoter spanning 158-bp upstream of the transcription start site displayed relative specificity for PLZF-expressing myeloid cells. Functional promoter assays revealed that an Evi-1-like site at -140/-130 was essential for full promoter activity in every cell line tested while a G-rich site at -15/-7 was important for tissue specificity. Electrophoretic mobility shift assays showed that Evi-1 binds specifically to -140/-130 Evi-1-like site and overexpression of Evi-1 in K562 cells activated the PLZF promoter. UV cross-linking assays showed that the proximal, tissue specific element at -15/-7 bound a novel 28 kDa protein. These results indicate as with other myeloid genes, a relatively small segment of DNA can direct tissue-specific expression, but unlike other myeloid promoters, no critical PU.1 or C/EBP sites were found.

Early detection and longitudinal changes in amyotrophic lateral sclerosis by (1)H MRSI.

OBJECTIVE: To determine 1) the reproducibility of metabolite measurements by (1)H MRS in the motor cortex; 2) the extent to which (1)H MRS imaging (MRSI) detects abnormal concentrations of N-acetylaspartate (NAA)-, choline (Cho)-, and creatine (Cre)-containing compounds in early stages of ALS; and 3) the metabolite changes over time in ALS. METHODS: Sixteen patients with definite or probable ALS, 12 with possible or suspected ALS, and 12 healthy controls underwent structural MRI and multislice (1)H MRSI. (1)H MRSI data were coregistered with tissue-segmented MRI data to obtain concentrations of NAA, Cre, and Cho in the left and right motor cortex and in gray matter and white matter of nonmotor regions in the brain. RESULTS: The interclass correlation coefficient of NAA was 0.53 in the motor cortex tissue and 0.83 in nonmotor cortex tissue. When cross-sectional data for patients were compared with those for controls, the NAA/(Cre + Cho) ratio in the motor cortex region was significantly reduced, primarily due to increases in Cre and Cho and a decrease in NAA concentrations. A similar, although not significant, trend of increased Cho and Cre and reduced NAA levels was also observed for patients with possible or suspected ALS. Furthermore, in longitudinal studies, decreases in NAA, Cre, and Cho concentrations were detected in motor cortex but not in nonmotor regions in ALS. CONCLUSION: Metabolite changes measured by (1)H MRSI may provide a surrogate marker of ALS that can aid detection of early disease and monitor progression and treatment response.