Martin Corcoran

A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells

PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and β. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA methyltransferase 3a and Enhancer of Zeste. In contrast, the β isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.

Cloning of two candidate tumor suppressor genes within a 10 kb region on chromosome 13q14, frequently deleted in chronic lymphocytic leukemia

Previous studies have indicated the presence of a putative tumor suppressor gene on chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have previously defined a minimally deleted region of 130 kb centromeric to the marker D13S272, and constructed a PAC and cosmid contig encompassing this area. In the present study we have made a detailed restriction and transcriptional map of the region of interest. Using these tools we have screened a panel of 206 primary CLL clones and three cell lines. In five CLL cases we found limited deletions defining the region of interest to an area of no more than 10 kb. Two adjacent genes, termed Leu1 and Leu2 (leukemia-associated gene 1 and 2), were mapped to the minimally deleted region, with several patients showing deletion borders within these genes. The Leu1 and Leu2 genes show little homology to previously published genes at the nucleotide and expected translated amino acid sequence level. Mutational analysis of the Leu1 and 2 genes in 170 CLL samples revealed no small intragenic mutations or point mutations. However, in all cases of 13q14 loss examined, the first exon of both genes, which are only 300 bp apart, were deleted. We conclude that the Leu1 and Leu2 genes are strong candidates as tumor suppressor gene(s) involved in B-CLL leukemogenesis.

Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.

Dysregulation of cyclin dependent kinase 6 expression in splenic marginal zone lymphoma through chromosome 7q translocations.

The increased or inappropriate expression of genes with oncogenic properties through specific chromosome translocations is an important event in the pathogenesis of B-cell lymphoproliferative diseases. Recent studies have found deletions or translocations of chromosome 7q to be the most common cytogenetic abnormality observed in SLVL, a leukemic variant of SMZL, with the q21 – q22 region being most frequently affected. In three patients with translocations between chromosomes 2 and 7, the cloning of the breakpoints at 7q21 revealed that each was located within a small region of DNA 3.6 kb upstream of the transcription start site of cyclin dependent kinase 6 (CDK6). In each case the translocation event was consistent with aberrant VJ recombination between the immunoglobulin light chain region (Ig kappa) on chromosome 2p12 and DNA sequences at 7q21, resembling the heptamer recombination site. The t(7;21) breakpoint in an additional patient with splenic marginal zone lymphoma (SMZL), resided 66 kb telomeric to the t(2;7) breakpoints juxtaposing CDK6 to an uncharacterized transcript. In two of the SLVL patient samples, the CDK6 protein was found to be markedly over expressed. These results suggest that dysregulation of CDK6 gene expression contributes to the pathogenesis of SLVL and SMZL.

DLEU2, frequently deleted in malignancy, functions as a critical host gene of the cell cycle inhibitory microRNAs miR-15a and miR-16-1

The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.

Identification of a novel gene, FGFR1OP2, fused to FGFR1 in 8p11 myeloproliferative syndrome

The 8p11 myeloproliferative syndrome (EMS) is an aggressive hematological malignancy caused by the fusion of diverse partner genes to fibroblast growth factor receptor 1 (FGFR1). The partner proteins promote dimerization and ligand‐independent activation of FGFR1‐encoded tyrosine kinase, deregulating hemopoiesis in a manner analogous to BCR‐ABL in chronic myeloid leukemia. Here, we describe the identification of a new FGFR1 fusion gene in a patient who presented with T‐cell lymphoblastic lymphoma in conjunction with an acquired ins(12;8)(p11;p11p22). Initial FISH analysis and Southern blotting confirmed that FGFR1 was disrupted. Using 5′‐RACE PCR, we identified part of a novel gene, FGFR1OP, at chromosome band 12p11 that was fused to exon 9 of FGFR1.FGFR1OP2 is predicted to be translated into an evolutionarily conserved protein containing coiled‐coil domains but no other recognizable motifs. The presence of the chimeric gene was confirmed by RT‐PCR, genomic DNA PCR, and FISH. These data further support the central role of deregulated FGFR1 in the pathogenesis of EMS.

Identification of four new translocations involving FGFR1 in myeloid disorders

The 8p11 myeloproliferative syndrome (EMS) is associated with three translocations, t(8;13)(p11;q12), t(8;9)(p11;q33), and t(6;8)(q27;p11), that fuse unrelated genes (ZNF198, CEP110, and FOP, respectively) to the entire tyrosine kinase domain of FGFR1. In all cases thus far examined (n = 10), the t(8;13) results in an identical mRNA fusion between ZNF198 exon 17 and FGFR1 exon 9. To determine if consistent fusions are also seen in the variant translocations, we performed RT‐PCR on four cases and sequenced the products. For two patients with a t(8;9), we found that CEP110 exon 15 was fused to FGFR1 exon 9. For two patients with a t(6;8), we found that FOP exon 5 (n = 1) or exon 7 (n = 1) was fused to FGFR1 exon 9. To determine if FGFR1 might be involved in other myeloid disorders with translocations of 8p, we developed a two‐color FISH assay using two differentially labeled PAC clones that flank FGFR1. Disruption of this gene was indicated in a patient with a t(8;17)(p11;q25) and Ph‐negative chronic myeloid leukemia in association with systemic malignant mast cell disease, a patient with acute myeloid leukemia with a t(8;11)(p11;p15), and two cases with T‐cell lymphoma, myeloproliferative disorder, and marrow eosinophilia with a t(8;12)(p11;q15) and ins(12;8)(p11;p11p21), respectively. For the patient with the t(8;11), the chromosome 11 breakpoint was determined to be in the vicinity of NUP98. We conclude that 1) all mRNA fusions in EMS result in splicing to FGFR1 exon 9 but breakpoints in FOP are variable, 2) two‐color FISH can identify patients with EMS, and 3) the t(8;17)(p11;q25), t(8;11)(p11;p15), t(8;12)(p11;q15), and ins(12;8)(p11;p11p21) are novel karyotypic changes that most likely involve FGFR1.

The p53 target Wig-1 regulates p53 mRNA stability through an AU-rich element

The p53 target gene Wig-1 encodes a double-stranded-RNA-binding zinc finger protein. We show here that Wig-1 binds to p53 mRNA and stabilizes it through an AU-rich element (ARE) in the 3′ UTR of the p53 mRNA. This effect is mirrored by enhanced p53 protein levels in both unstressed cells and cells exposed to p53-activating stress agents. Thus, the p53 target Wig-1 is a previously undescribed ARE-regulating protein that acts as a positive feedback regulator of p53, with implications both for the steady-state levels of p53 and for the p53 stress response. Our data reveal a previously undescribed link between the tumor suppressor p53 and posttranscriptional gene regulation via AREs in mRNA.

A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B-cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5

B‐cell chronic lymphocytic leukemia (B‐CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2‐42), which corresponds to the minimal region shared by B‐CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc‐finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B‐CLL rearrangements.

Characterisation of dic(9;20)(p11–13;q11) in childhood B-cell precursor acute lymphoblastic leukaemia by tiling resolution array-based comparative genomic hybridisation reveals clustered breakpoints at 9p13.2 and 20q11.2

Although the dic(9;20)(p11–13;q11) is a recurrent chromosomal abnormality in paediatric B‐cell precursor acute lymphoblastic leukaemia (BCP ALL), occurring in approximately 2% of the cases, its molecular genetic consequences have not been elucidated. In the present study, high‐resolution genome‐wide array‐based comparative genomic hybridisation (array‐CGH) and fluorescence in situ hybridisation (FISH) were used to characterise the 9p and 20q breakpoints (BPs) in seven childhood BCP ALLs with dic(9;20), which was shown to be unbalanced in all of them, resulting in loss of 9p13.2‐pter. Five of the cases had loss of 20q11.2‐qter, whereas two displayed gain of 20cen‐pter. All BPs on 9p clustered in a 1.5 Mb segment of the sub‐band 9p13.2; in three of the cases, the 20q BPs mapped to three adjacent clones covering a distance of 350 kb at 20q11.2. Thus, the aberration should be designated dic(9;20)(p13.2;q11.2). One of the ALLs, shown to have a complex dic(9;20), was further investigated by FISH, revealing a rearrangement of the haemapoietic cell kinase isoform p61 (HCK) gene at 20q11. The disruption of HCK may result in a fusion gene or in loss of function. Unfortunately, lack of material precluded further analyses of HCK. Thus, it remains to be elucidated whether dic(9;20)(p13.2;q11.2) leads to a chimaeric gene or whether the functionally important outcome is loss of 9p and 20q material.