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The bacteriophage Mu Mor activator protein is absolutely required for transcription from the Mu middle promoter P(m). However, when RNA polymerase (RNAP) was incubated with P(m) DNA in the absence of Mor, a band at promoter position -51 was hypersensitive to DNase I cleavage, demonstrating an interaction of RNAP with the promoter DNA. The hypersensitivity was similar at four different lengths of P(m) DNA assayed from -62 to +10, -62 to +46, -96 to +10, and -96 to +46. The hypersensitivity occurred equally well at 5 °C, 15 °C, and 30 °C, indicating that it did not require open complex formation, which only occurred at 30 °C. The -51 hypersensitivity at 5 °C and 15 °C was eliminated by the addition of heparin, consistent with the possibility that it arose by formation of unstable closed complexes of RNAP bound to P(m) DNA. Generation of the hypersensitive band required the complete RNAP with its αCTDs, but neither the αCTD nor intact α were sufficient for the interaction and resulting hypersensitivity. There was no correlation between the level of hypersensitivity observed in vitro and the level of Pm activity in vivo, as assayed by the Mor-dependent production of β-galactosidase from a P(m)-lacZ fusion. In an "order of addition" experiment, preincubation of P(m) DNA with Mor followed by addition of RNAP led to the fastest open complex formation, whereas preincubation of P(m) DNA with RNAP gave the slowest. These results support the conclusion that Mor recruits RNAP to P(m) rather than reposition a prebound RNAP, as occurs for C-dependent repositioning of RNAP at the Mu late promoter Pmom .

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines byMycobacterium smegmatisorM. bovisBCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer usingM. smegmatisor BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected withM. smegmatishyperconjugation mutants.M. smegmatisstrains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation inoriMresulted in a threefold increase in gene transfer to J774 cells. Moreover,M. smegmatisstrains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120hE) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P< 0.05) and threefold (P< 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120hE.M. smegmatisand other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. To study this at a genome-wide level in melanoma we analyzed the methylome of 44 cases of malignant melanoma. We saw widespread demethylation in melanoma occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions showed demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. The colony stimulating factor-1 receptor was aberrantly expressed and hypomethylated in nearly all cases. The expression of CSF1R was validated by IHC on primary tumors and by qPCR and Western blotting in BRAF mutant and WT cell lines. CSF1R can be aberrantly expressed via an upstream LTR element in Hodgkin’s lymphoma. After analyzing our patient samples and the cell lines, we have found this aberrant transcript may be the dominant form in melanoma as well. Expression of one of its ligands IL34 was also shown in the cell lines by both ELISA and qPCR pointing to a potential autocrine regulatory loop. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in 2D and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness - suggesting a functional role for CSF1R in melanoma. Treatment of melanoma with small molecule inhibitors of BRAF V600E is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and the rebound activation of the MAPK-pathway. We are suggesting a role for CSF1R in this process. In Western experiments, the rebound of phospho-ERK after BRAF inhibitor treatment was accelerated with the addition of CSF1R ligands, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing shRNA against CSF1R recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the receptor, as well as its maturation, evidenced by the appearance of the high MW form. Utilizing shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression in resistant tumors. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo. The CSF1R inhibitor, PLX3397 is currently in clinical trials for glioblastoma, prostate, breast cancers and other cancers. These data present a preclinical rationale for its study in malignant melanoma. Citation Format: Orsolya Giricz, Yongkai Mo, Caroline Y. Hu, Yiting Yu, Kith Pradhan, Matthias Bartenstein, Nandini Ramachandra, Veronika Polishchuck, Kimberly B. Dahlman, Tushar Bhagat, Hoa Nguyen, Bernice Matusow, Rafe Shellooe, Elizabeth Burton, Paraic Kenny, John Greally, Jeffrey Sosman, Gideon Bollag, Brian West, Amit Verma. Aberrant expression of CSF1R in melanoma is driven through an endogenous viral promoter and it contributes to malignant growth and the acquisition of resistance against BRAF inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3332. doi:10.1158/1538-7445.AM2017-3332

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions, and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA, and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, whereas epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors, and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.

Epigenetic changes in cancer are thought to contribute to regulation of invasion and metastasis. To study this at a genome-wide level in melanoma we analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it to melanocyte controls. We saw widespread demethylation in melanoma occurring preferentially outside of CpG islands. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways aberrantly expressed and regulated by loss of DNA methylation. The colony stimulating factor-1 receptor (CSF1R) was aberrantly expressed and hypomethylated in nearly all cases. The expression of CSF1R was validated by immunohistochemistry on primary tumors and by Western blotting in BRAF V600E mutant and WT melanoma cell lines. Expression of its ligand IL34, but not of CSF1 was also shown in the melanoma cells by both ELISA and qPCR. The effects of a small molecule inhibitor, PLX3397 as well as shRNA-mediated knockdown of the receptor were investigated in traditional and 3D cell culture. We saw inhibition of cell growth, smaller colony size, increased apoptosis and decreased invasiveness - suggesting a functional role for CSF1R in melanoma. Treatment of melanoma with small molecule inhibitors of BRAF V600E is effective for a time, but resistance invariably develops. The feedback activation of EGFR, BRAF amplification, BRAF splice variants and others are known to aid in the acquisition of resistance and lead to rebound activation of the MAPK-pathway. In Western blotting experiments, the rebound of ERK phosphorylation after BRAF inhibitor treatment was accelerated with the addition of the CSF1R ligands CSF1 and IL34, or delayed with PLX3397, also attenuating AKT phosphorylation. Melanoma cells stably expressing CSF1R shRNA recapitulated the effects of the inhibitor. Assaying the cells at different time points during a long-term V600E inhibitory experiment, we saw increasing levels of the transcription factor RUNX1, followed by increasing levels of IL34 and of the CSF1R protein, as well as its maturation, evidenced by the appearance of the high MW form. Utilizing shRNA-mediated knockdown of RUNX1 resulted in lower levels of the CSF1R and IL34 transcripts and delayed the rebound. Analysis of primary RNA-Seq data showed an increase in RUNX1, CSF1R and IL34 expression as resistance was acquired. Co-inhibition of CSF1R and BRAF was also tested and resulted in synergistic blockade of cell growth in vitro and xenograft growth in vivo. The CSF1R inhibitor, PLX3397, is in clinical trials for breast and other cancers, and these data present a preclinical rationale for its study in malignant melanoma. Citation Format: Orsolya Giricz, Yongkai Mo, Caroline H. Hu, Kimberly Dahlman, Nandini Ramachandra, Matthias Bartenstein, Kith Pradhan, Tushar Bhagat, Yiting Yu, Hoa Nguyen, Elizabeth Burton, Bernice Matusow, Gaston Habets, Rafe Shellooe, Gideon Bollag, Brian West, John Greally, Jeffrey Sosman, Paraic Kenny, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma and points to the role of CSF1R-RUNX1 axis in resistance against BRAF inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1885.

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

Epigenetic alterations can direct carcinogenesis by leading to transcriptional changes and inducing genomic instability. We analyzed the methylome of malignant melanoma and observed widespread loss of DNA methylation that was found to preferentially occur outside of CpG islands. Demethylation was seen to occur early during carcinogenesis, was independent of mutational status and correlated with genomic instability. Parallel transcriptomic analyses revealed that various immune and cancer associated pathways were overexpressed and were associated with promoter demethylation. The CSF1-receptor (CSF1R) was aberrantly overexpressed and hypomethylated in nearly all cases and was strikingly expressed via an aberrant upstream promoter in 10% of melanomas. shRNA mediated knockdown and inhibition of CSF1R kinase via a clinically relevant inhibitor, PLX3397, led to decreased 3D growth and invasiveness. Co-inhibition of CSF1R and BRAF resulted in synergistic blockade of BRAF-mutant melanoma xenograft growth. Thus, widespread epigenetic changes are seen in melanoma and CSF1R is a potential therapeutic target in this disease. Citation Format: Yongkai Mo, Orsolya Giricz, Caroline H. Hu, Kimberly B. Dahlman, Sanchari Bhattacharyya, Hoa Nguyen, Bernice Matusow, Tushar Bhagat, Rafe Shellooe, Elizabeth Burton, James Tsai, Chao Zhang, Gaston Habets, Yu Shyr, John Greally, Yiting Yu, Gideon E. Bollag, Richard Stanley, Jeffrey Trent, Paraic A. Kenny, Brian L. West, Jeffrey Sosman, Amit K. Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma and reveals CSF-1 receptor as an aberrant regulator of malignant growth and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4781. doi:10.1158/1538-7445.AM2014-4781

Epigenetic changes in cancer are thought to contribute to regulation of tumor invasion and metastasis, but this previously has not been studied at a genome wide level in melanoma. We analyzed the methylome of 44 cases of malignant melanoma with the HELP (HpaII tiny fragment enriched by LM-PCR) assay and compared it with healthy melanocyte controls. We observed widespread demethylation in malignant melanoma, preferentially outside of CpG islands. The epigenomic loss of methylation was independent of mutational status of BRAF, RAS and Kit. Comparison of primary and metastatic lesions demonstrated that demethylation occurs early during carcinogenesis with very few additional alterations in advanced tumors. Parallel transcriptomic analysis revealed many known and novel oncogenic pathways that were aberrantly expressed and regulated by loss of DNA methylation. Strikingly, the colony stimulating factor-1 receptor (CSF1R, c-fms) was aberrantly expressed and hypomethylated in nearly all cases. CSF1R is a transmembrane tyrosine kinase receptor that predominantly regulates macrophages, osteoclasts, and microglia, but is known to sometimes be aberrantly expressed by malignant cells in Hodgkins lymphoma. The expression of CSF1R on malignant melanocytes was validated by immunohistochemical analysis of primary tumors. In a melanoma cell line (A2058) we found through PCR sequencing of the cDNA 5' untranslated region that the CSF1R can be expressed through an aberrant promoter, as has been described for Hodgkin lymphoma. A custom Taqman assay was developed for this unique transcript, and then used to detect the transcript in 4 of 40 samples in a panel of melanoma biopsies, suggesting that aberrant CSF1R expression in melanoma is not uncommon. Expression of CSF1R protein in A2058 cells was confirmed by FACS using anti-CD115 antibodies, and by Western blot using antibodies directed to the C-terminus. Expression of the ligand CSF-1 was also found in A2058 cells by both ELISA and Taqman assays. Inhibition of A2058 cell growth by PLX3397, a clinically relevant small molecule inhibitor of CSF1R kinase, could be observed in 3D cell culture, indicating that under some conditions an autocrine stimulation of growth occurs. shRNA mediated knockdown of CSF1R also demonstrated decreased colony size and increased apoptosis in 3D culture conditions. The invasiveness of A2058 cells was decreased after treatment with PLX3397 or anti-CSF1 antibodies, suggesting a role for melanoma cancer cell expression of CSF1R in metastasis. Since A2058 cells possess an oncogenic BRAF mutation, co-inhibition of CSF1R and BRAF was tested and resulted in synergistic blockade of xenograft growth. The CSF1R inhibitor, PLX3397, is under investigation in clinical trials for breast, glioma, and other cancers, and these data present a preclinical rationale for its study in malignant melanoma. Citation Format: Yongkai Mo, Orsolya Giricz, Caroline Hu, Kimberly Dahlman, Sanchari Bhattacharyya, Hoa Nguyen, Bernice Matusow, Tushar Bhagat, Yiting Yu, Rafe Shellooe, Elizabeth Burton, Gaston Habets, John Greally, Paraic Kenny, Jeffrey Sosman, Gideon Bollag, Brian L. West, Amit Verma. Integrated epigenomic profiling reveals widespread demethylation in melanoma, and reveals aberrant CSF-1 receptor expression as a regulator of malignant growth and invasion inhibited by PLX3397. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A26.

Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though global epigenetic alterations in MDS have not been well described. The transmission of these epigenetic marks during hematopoietic differentiation and their role in disease pathophysiology is also unknown. We first compared global methylation profiles of 8 bone marrow samples with peripheral leucocytes by using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. We observed a high correlation (r=0.89–0.96) of epigenetic marks between bone marrow and peripheral blood samples suggesting that a majority of epigenetic marks can be also be seen in differentiated cells. We subsequently compared peripheral blood leucocytes from 20 patients with MDS with 10 age-matched normal and anemic controls. Parallel gene expression analysis was performed using 37K oligo maskless arrays on cDNA from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Epigenetic profiling demonstrated two clusters of MDS based on similarity of aberrant epigenetic changes. Overall, there was a trend towards hypermethylation in MDS, albeit not statistically significant given the large number of relatively unchanged genes. Detailed analysis revealed several novel differentially methylated genes that had corresponding changes in gene expression, when MDS samples were compared to the controls with a low false discovery rate of analysis. Interesting genes getting hypomethylated and overexpressed included TNF superfamily member 9, granulocyte pep A, microsomal glutathione S-transferase, homeo box B4, mitochondrial RPL11, and others. Similarly, the set of genes that were getting hypermethylated with associated decrease in gene expression included Evi-1, DAPK, HOXB3, Protein Phosphatase 1, CEBPB, mutated in colorectal cancer (MCC), myeloid-lymphoid or mixed-lineage leukemia 5 (MLL5), plasminogen-related protein B, ovarian cancer related protein 1 (ORP1), and others. In addition, we did array-based comparative genome hybridization (aCGH) to look at exact genome copy number changes in these samples. We found changes that were not detectable by conventional karyotyping in all samples. Commonly seen alterations were del(14q11), del(20q11), del(5q13), del(8p23), amp(1q42), amp(5q11), amp(17q12), amp(19q13) and amp(7q22). Integrative analysis revealed sets of genes that were either silenced by methylation or deletion in different patients. Thus, our data demonstrates that promoter DNA methylation changes are an important phenomenon in MDS evolution, and are associated with changes in expression of genes playing important roles in cancer development and/or progression. We also show that previously unrecognizable changes in copy number exist in most patients with MDS. In addition, our work shows that whole genome methylation assays, even when done on peripheral blood leukocytes, can be used for potential biomarker studies in the diagnosis of MDS.

The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of bone marrow stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of bone marrow stroma in this disease. To accomplish this aim, bone marrow mononuclear cells from MDS patients and controls were grown to form adherent cell layers and then depleted for hematopoietic elements by immunomagnetic CD45 negative selection. CD45 negative adherent cells were subsequently expanded and then used for whole genome methylation studies using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) which uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantify individual promoter CpG island methylation. A custom whole genome human promoter array (Roche-Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Global epigenetic profiling revealed that MDS stroma (n=6) was epigenetically distinct from normal bone marrow stroma (n=4) (ANOVA, P In subsequent studies, we profiled stroma from another set of MDS patients who had been treated with the DNMT inhibitor, 5-Azacytidine (n=4). In contrast to untreated MDS patients, there were no significant epigenetic differences between these 5-Azacytidine treated MDS patients and healthy controls (p = NS). These 5-Azacytidine exposed stroma cells did not demonstrate global hypomethylation (as hypothesized after DNMT inhibitor treatment) and were characterized by both hyper- and hypo-methylated loci similar to healthy controls. Thus our results reveal that MDS is characterized by widespread aberrant epigenetic changes in the bone marrow microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, and we hypothesize that those stroma effects contribute in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire bone marrow, including the microenvironment, if we are to improve our understanding of the pathophysiology of MDS and further improve therapy.