Abstract Background: 5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome wide assays for 5-hmC determination are needed as many of the techniques commonly used to assay 5-methylcytosine (5-mC), including conventional methyl-sensitive restriction digest and bisulfite sequencing, are incapable of distinguishing between 5-mC and 5-hmC. Results: Glycosylation of 5-hmC residues by beta-Glucosyl Transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. We used this premise to modify the HELP-tagging assay to identify both 5-mC and 5-hmC loci in the genome. The HELP tagging assay uses massive parallel sequencing to analyze the cytosine methylation status of 2.1 million CpGs in the genome. Libraries were generated from genomic DNA digested with HpaII, MspI and β-GT +MspI. Comparison of HpaII and MspI digested samples led to determination of 5-mC loci, while comparison of β-GT +MspI with MspI digested samples identified 5-hmC residues. This modified “HELP-GT” assay allowed multiplexing of 8 libraries per sequencing lane to generate an average of 6-10 million HpaII/MspI reads per sample with an average depth of coverage between 6-11x for each CCGG site. A custom bioinformatics pipeline was created to identify 5-hmC sites that were validated at global level by LS-MS and at the locus specific level by qRT-PCR of 5-hmC pulldown DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. This assay was then used to analyze 5-hmC profiles of two pancreatic cancer cell lines (Pa03C and Pa04C) that were compared with pancreatic control cells (HPNE). Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells with significantly increased 5-hmC at Promoters, Gene bodies and Transcription factor binding sites. 5-hmC was also increased at many oncogenic promoters such as GATA6 in pancreatic cancer and correlated with its overexpression. 5-hmC profiles were also able to distinguish between cancer and control cells with greater discrimination (unsupervised hierarchical clustering) when compared to 5-mC patterns. Conclusions: The HELP-GT assay allows a high resolution, simultaneous determination of 5-hmC and 5-mC loci from small amounts of DNA with the utilisation of modest sequencing resources. This assay is able to provide single base pair resolution analysis of over 1 million sites in the human genome with the use of 1μg of genomic DNA. Redistribution of 5-hmC seen in cancer highlights the importance of examining this modification in conjugation with conventional methylome analysis. Citation Format: Sanchari Bhattacharyya, Yiting Yu, Masako Suzuki, Nathaniel Cambpell, Jozef Mazdo, Aparna Vasantkumar, Tushar D. Bhagat, Sangeeta Nischal, Ulrich Steidl, Lucy Godley, Anirban Maitra, John M. Greally, Amit Verma. Genome wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4231. doi:10.1158/1538-7445.AM2013-4231
Abstract Ribosomes are essential components of the protein translation machinery and are composed of more than 80 unique large and small ribosomal proteins. Recent studies show that in addition to their roles in protein translation, ribosomal proteins are also involved in extra-ribosomal functions of DNA repair, apoptosis and cellular homeostasis. Consequently, alterations in the synthesis or functioning of ribosomal proteins can lead to various hematologic disorders. These include congenital anemias such as Diamond Blackfan anemia and Shwachman Diamond syndrome; both of which are associated with mutations in various ribosomal genes. Acquired uniallelic deletion of RPS14 gene has also been shown to lead to the 5q syndrome, a distinct subset of MDS associated with macrocytic anemia. Recent evidence shows that specific ribosomal proteins are overexpressed in liver, colon, prostate and other tumors. Ribosomal protein overexpression can promote tumorigenesis by interactions with the p53 tumor suppressor pathway and also by direct effects on various oncogenes. These data point to a broad role of ribosome protein alterations in hematologic and oncologic diseases.
Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS.
Abstract 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.
Abstract The microenvironment where a tumor originates plays an important role in its initiation, growth, progression and metastatic capability. Since in most cancers the microenvironment is not derived from the malignant clone and does not contain oncogenic mutations, it is likely that the tumor microenvironment is reprogrammed epigenetically to support the growth of the tumor. To test this hypothesis, Fibroblasts associated with primary pancreatic adenocarcinomas (N=7) & healthy fibroblast controls (N=4) were analyzed for genome wide alterations in DNA cytosine methylation by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR). Methylome profiling revealed widespread aberrant cytosine methylation in Pancreatic cancer associated fibroblasts (CAFs) with demethylation of many important gene promoters as the predominant epigenetic event. In addition to loss of methylation, aberrant hypermethylation of GALNTS, JAZF1, MTCH1, SP8, GRB14 was also seen in CAFs. In addition to epigenetic differences, pancreatic CAFs were also found to have widespread transcriptmoic differences as seen by parallel gene expression profiling. Next, we analyzed tumor mediated reprogramming of microenvironment at a higher resolution by a Massive parallel sequencing based methylation analysis (HELP-Tagging) on CAFs differentiated from mesenchymal stem cells (MSCs) in the presence of pancreatic cancer cell conditioned medium. HELP-tagging showed widespread epigenetic reprogramming with 11,100 hypomethylated and 1709 hypermethylated loci. Comparison of in-vitro reprogramed loci with the aberrantly methylated loci from primary CAFs showed a core set of 140 loci that were commonly differentially methylated in both samples. The chemokine receptor CXCR4 was observed to overexpressed & demethylated in both cohorts & was found to be expressed on the surface of primary pancreatic CAFs by immunohistochemistry. Functional studies demonstrated that co-culture of pancreatic cancer cells with CAFs (from MSCs) led to significant increase in malignant cell invasion when compared to co-culture with naïve MSCs. This increased invasion was abrogated by blockade of CXCR4 by AMD-3100 and by knockdown of CXCR4 by siRNAs in orthotopically derived CAFs; demonstrating a critical role for this receptor in regulating tumor promoting abilities of the microenvironment. Thus our results reveal for the first time that pancreatic CAFs are characterized by widespread epigenomic reprogramming that includes loss of methylation at many important loci. Validation of an aberrantly demethylated target, CXCR4, shows that inhibition of this receptor can abrogate the ability of CAFs in promoting cancer cell invasion. These results also provide a preclinical rationale for the use of clinically relevant CXCR4 antagonist AMD-3100 (plerixafor) in pancreatic cancer. Citation Format: Tushar D. Bhagat, Yanique Rattigan, Brijesh Patel, Strepell Mirte, Yiting Yu, Davendra Sohal, Matthias Bartenstein, Orsolya Giricz, Shanisha AK Gordon, Nishanth Vallumsetla, Rahul Polineni, Meher Walia, Paraic Kenny, John Greally, Debabrata Banerjee, Anirban Maitra, Amit Verma. Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2013-1504
