MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation.

Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia.

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias.

High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.

Histone deacetylase 5 blocks neuroblastoma cell differentiation by interacting with N-Myc.

The N-Myc oncoprotein induces neuroblastoma, which arises from undifferentiated neuroblasts in the sympathetic nervous system, by modulating gene and protein expression and consequently causing cell differentiation block and cell proliferation. The class IIa histone deacetylase 5 (HDAC5) represses gene transcription, and blocks myoblast, osteoblast and leukemia cell differentiation. Here we showed that N-Myc upregulated HDAC5 expression in neuroblastoma cells. Conversely, HDAC5 repressed the ubiquitin-protein ligase NEDD4 gene expression, increased Aurora A gene expression and consequently upregulated N-Myc protein expression. Genome-wide gene expression analysis and protein co-immunoprecipitation assays revealed that HDAC5 and N-Myc repressed the expression of a common subset of genes by forming a protein complex, whereas HDAC5 and the class III HDAC SIRT2 independently repressed the expression of another common subset of genes without forming a protein complex. Moreover, HDAC5 blocked differentiation and induced proliferation in neuroblastoma cells. Taken together, our data identify HDAC5 as a novel co-factor in N-Myc oncogenesis, and provide the evidence for the potential application of HDAC5 inhibitors in the therapy of N-Myc-induced neuroblastoma and potentially other c-Myc-induced malignancies.

HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses.

The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds.

Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16(INK4a) during immortalization.

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16(INK4a) and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16(INK4a) on EC function and to identify molecular changes associated with repression of p16(INK4a). In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16(INK4a) showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16(INK4a)-negative and a p16(INK4a)-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16(INK4a) expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16(INK4a) expression. This corresponded with aberrant cytoskeletal architectures among p16(INK4a)-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16(INK4a)-positive cells. A direct link between p16(INK4a) repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16(INK4a). siRNA-mediated repression of p16(INK4a) significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16(INK4a) are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies.

Upregulation of survivin during immortalization of nontransformed human fibroblasts transduced with telomerase reverse transcriptase.

These investigations demonstrate that expression of the inhibitor of apoptosis family member, survivin, is dramatically increased during immortalization of nontransformed human fibroblasts that were transduced with telomerase reverse transcriptase (hTERT). Expression of survivin in immortalized fibroblasts peaked during G(2)/M phase of the cell cycle. However, the upregulation of survivin was dissociated from the rate of proliferation and proportion of G(2)/M cells. Depletion of survivin from immortal fibroblasts increased sensitivity to stress-induced apoptosis and resulted in an accumulation of cells with 4N DNA content. Conversely, overexpression of survivin in mortal fibroblasts conferred resistance to apoptosis. In contrast, very low levels of survivin in proliferating parental fibroblasts had no bearing on sensitivity to apoptosis. The upregulation of survivin did not appear to be a direct consequence of hTERT transduction. However, repression of hTERT resulted in the rapid downregulation of survivin in telomerase-immortalized fibroblasts and tumor cell lines, but not in cells immortalized via an Alternative Lengthening of Telomeres mechanism. These results have important therapeutic implications, as telomerase and survivin are both broadly expressed in human cancers. Selection during the immortalization process for cells expressing high levels of survivin may account for the abundance of survivin in diverse tumor types.

Telomere length of cord blood-derived CD34(+) progenitors predicts erythroid proliferative potential.

Excessive telomere shortening has been demonstrated in inherited and acquired blood disorders, including aplastic anemia and myelodysplastic syndromes. It is possible that replicative exhaustion, owing to critical telomere shortening in hematopoietic progenitor cells (HPCs), contributes to the development of cytopenias in these disorders. However to date, a direct link between the telomere length (TL) of human HPCs and their proliferative potential has not been demonstrated. In the present investigation, the TL and level of telomerase enzyme activity (TA) detected in cord blood (CB)-derived HPCs was found to predict erythroid expansion (P<0.01 and P=0.01 respectively). These results were corroborated by a correlation between proliferation of erythroid cells and telomere loss (P=0.01). In contrast, no correlations were found between initial TL, telomere loss or TA and the expansion of other myeloid lineage-committed cells. There was also no correlation between TL or TA and the number of clonogenic progenitors, including primitive progenitors derived from long-term culture. Our investigations revealed upregulation of telomerase to tumor cell levels in CD34- cells undergoing erythroid differentiation. Together, these results provide new insight into the regulation of TL and TA during myeloid cell expansion and demonstrate that TL is an important determinant of CB-derived erythroid cell proliferation.

Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo.

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation.

Clonal variation in phenotype and life span of human embryonic fibroblasts (MRC-5) transduced with the catalytic component of telomerase (hTERT).

Expression of telomerase (hTERT) in certain cell types has been shown to extend cellular life span without malignant transformation. We studied the phenotype of 26 telomerase-transduced fibroblast clones (TTFC) generated from a mass culture of hTERT retrovirally transduced MRC-5 cells. About two-thirds of the transduced clones senesced at the expected time or shortly thereafter, despite high levels of expression of telomerase and telomere length maintenance. The remaining one-third of the clones were "immortalized" (followed for over 200 cumulative population doublings). All clones maintained a nontransformed phenotype: contact inhibition, anchorage dependency, lack of tumor formation in nude mice, dose dependency to serum and growth factors, low expression of a matrix metalloproteinase associated with metastatic invasion (MMP-9) and high expression of its inhibitor TIMP-1, and no cytogenetic abnormalities by G-banding. In addition, fibroblast-specific biological parameters, such as colony size, production of collagenase, and response to MMC and gamma radiation were tightly regulated at the clonal and subclonal levels.

Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation.

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts.

Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture.

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34(+) cells and primary colony forming unit-cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34(+) cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P <.01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice.

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-ras reconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-ras mutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.

Retroviral transduction of hematopoietic progenitor cells with mutant p53 promotes survival and proliferation, modifies differentiation potential and inhibits apoptosis.

Mutations in the p53 tumor suppressor gene have been shown to be associated with many human tumors and various leukemias and lymphomas. To examine whether constitutive overexpression of mutant p53 can effect transformation of normal hematopoietic cells, a mutant p53 gene was introduced into normal murine bone marrow hematopoietic cells by retroviral gene transfer. Compared to vector alone-infected cells, hematopoietic progenitor cells transduced with mutant p53 showed increased proliferative potential, enhanced cloning efficiencies and a modified differentiation pattern in vitro. In addition, mutant p53-transduced hematopoietic cells were more resistant to loss of viability and/or induction of apoptosis when cultured in a low concentration of serum or in the absence of both growth factors and serum. These effects occurred rapidly with no apparent contributory secondary events. No permanent cell lines or growth factor-independent cell strains were obtained. The results indicate that introduction of mutant p53 into normal hematopoietic cells in vitro contributes to transformation, including enhanced proliferative potential, modified differentiation and the suppression of apoptosis in these cells.

An internal deletion enhances the transcriptional activity of a recombinant retrovirus in hematopoietic cells in vivo.

Lv-myc is a recombinant retrovirus that spontaneously arose during experiments designed to express the provirus LNAv-myc in the hematopoietic system of bone marrow-reconstituted mice (L. Bonham, K. MacKenzie, S. Wood, P. B. Rowe, and G. Symonds, Oncogene 7:2219-2229, 1992). The recombinant provirus is of interest because it is able to promote long terminal repeat-initiated transcription in hematopoietic cells in vivo, whereas the parental provirus, LNAv-myc, is transcriptionally repressed in the same cells. Here we report that Lv-myc was generated by precise deletion of the neomycin resistance gene (neo) and the human gamma-actin promoter from LNAv-myc. In comparison with LNAv-myc, no sequence alterations in the viral regulatory regions of Lv-myc were detected. Thus, it appears that neo and/or the gamma-actin promoter exerted a cis-acting repressor effect on the long terminal repeat of LNAv-myc in vivo. The origin of Lv-myc was also investigated, and it was shown that Lv-myc was harbored as a productive provirus in a G418-resistant subpopulation of the LNAv-myc producer cell line, psi 2AV. It appears that Lv-myc arose during propagation of the psi 2AV cell line. Repeated sequence detected at the sites of the deletion suggest that Lv-myc was generated by a template misalignment during reverse transcription of LNAv-myc.