Joanna Opalinska

Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis

Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.

p38 MAP Kinase Regulates Stem Cell Apoptosis in Human Hematopoietic Failure

Myelodysplastic syndromes (MDS) are clonal stem cell disorders that lead to ineffective hematopoiesis and are common causes of low blood counts in the elderly. The exact molecular mechanisms regulating increased stem apoptosis in these disorders are not well defined. p38 MAPK activation is important in regulating the growth inhibitory signals of TNF-α, TGF-β and Interferons on human hematopoiesis. Our findings show that p38 MAPK is overactivated in myelodysplasia bone marrows and regulates hematopoietic stem cell apoptosis. Inhibition of p38 MAPK by genetic or pharmacologic means decreases apoptosis and stimulates in vitro hematopoiesis from primary MDS hematopoietic progenitors. These studies point to the potential efficacy of selective p38α inhibitor, SCIO-469, in human bone marrow failure.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

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.

Nucleic acid drugs in the clinic.

As a number of diseases are caused, or accompanied, by abnormal gene expression an understandable temptation to modulate the expression of the abnormal gene’s mRNA or protein to restore proper functioning of the cellular machinery has arisen. In addition, as many traditional therapeutic interventions are accompanied by serious side effects (because the cell killing they cause is not specific to the tumors they are intended to treat) there is a natural desire to design drugs with a very targeted mode of action so as to minimize these side effects. The motivation for developing tumor-specific therapies has now become so strong and so pervasive that we are now truly entering an era of targeted therapeutics. One of the major breakthroughs in the field of targeted therapies, and an example that many are hoping to duplicate, has been the development and successful introduction into the clinic of the first small-molecule inhibitor of the bcr-abl tyrosine kinase – imatinib. Indeed, the spectacular success of imatinib has rapidly led to the development of second-generation inhibitors, which are now either approved themselves or are in advanced clinical trials. Monoclonal antibodies have also found their way to the bedside and are being used widely to treat malignant and non-malignant diseases. In the present treatment climate, dominated as it is by small-molecule drugs and antibodies, one could wonder whether alternative approaches, such as RNA or gene-targeted nucleic acid-based drugs, are still needed. For reasons discussed in the body of this review, many colleagues believe that there is still a place for nucleic acid-based therapeutics. Here, the reason for believing that this is true is reviewed in the context of nucleic acid drug development and the early clinical experience with these new medicines.

A new PML-RARs fusion transcript hints at the important role of PML dysregulation in the pathogenesis of APL.

Acute promyelocytic leukemia is associated with the 15:17 chromosomal translocation, which results in the formation of a fusion PML-RARa oncoprotein. This oncoprotein blocks the transcription of retinoic acid regulated genes and leads to maturation arrest at promyelocytic stage of differentiation [1]. Multiple studies have now shown that the length of PML in the fusion protein can vary while the RARa portion remains constant and contains the DNA binding and ligand binding motifs (B-F domains) of the retinoic acid receptor protein (Figure 1). Depending on the location of the breakpoint in the PML gene, 3 main types of fusion proteins have been described on the basis of their size [2]. The short (S) isoform is generated by fusion of PML exons 1 through 3 (encoding the RING, B boxes, and coiled-coil domain) to exon 3 of RARa. The longer (L) isoform contains exons 1 through 6 of PML and intermediate length variable (V) isoforms are formed after breakpoints around exon 6 of PML (Figure 1). Approximately 70% of APL patients express the L isoform, 20% the S isoform and the rest the V isoform. Even though these isoforms have differing sensitivities to ATRA, all respond to high doses given as a part of induction therapy. Other rare PML-RARa isoforms have also been noted that are formed because of alternative splicing of the PML transcripts. Interestingly each APL patient exhibits a unique isoform consistent with the clonal nature of this leukemia. In the study by Zayed et al. [3] the authors describe a new fusion PML-RARa transcript, which is formed by breakpoint between exons 5 and 6 in the PML gene. In contrast with the other known isoforms, this fusion oncoprotein leads to a frame shift in the RARa sequence, which then codes for a truncated version of RARa. This predicted short version of protein will not have the Retinoic acid binding regions and thus theoretically would be resistant to ATRA therapy. This hypothesis is validated by the continued presence of Promyelocytic blasts after ATRA treatment in this report. Though this study describes a novel breakpoint, there are some questions raised by these findings. Even though the authors have studied the sequence of the fusion transcript at the RNA level, a determination of the exact breakpoint at the DNA level would have confirmed that this new RNA transcript is not formed as a result of alternative splicing. Additionally, the authors could not detect the transcript by RT-PCR at day 25 of treatment even in the presence of blasts in the marrow. Though RNA degradation could be a possibility, the authors do not comment if this conjecture was borne out by RT PCR studies of other housekeeping genes. The second explanation could be the rare possibility of a second leukemic clone (without the PML-RARa fusion protein) that could have become dominant after ATRA treatment. Cytogenetic examination performed at Day 25 would have been helpful in ruling out this rare possibility. A major point this paper illustrates is the importance of PML in the pathogenesis of APL. Since the fusion protein described by the authors encodes a very short fragment of the RARa protein, the presence of a dominant negative truncated PML is the most likely cause of this patient’s malignancy. PML is a protein that is involved in multiple pathways regulating cell proliferation, apoptosis, and aging [4]. Notably, PML participates in p53 dependent tumor suppressor pathways by regulating the acetylation and transcriptional activation of this