Karen L. MacKenzie

SIRT1 Promotes N-Myc Oncogenesis through a Positive Feedback Loop Involving the Effects of MKP3 and ERK on N-Myc Protein Stability

The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3), leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc–induced neuroblastoma.

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.

Multiple stages of malignant transformation of human endothelial cells modelled by co-expression of telomerase reverse transcriptase, SV40 T antigen and oncogenic N-ras.

We have modelled multiple stages of malignant transformation of human endothelial cells (ECs) by overexpressing the catalytic subunit of human telomerase (hTERT), together with SV40 T antigen (SV40T) and oncogenic N-ras. Transfection with hTERT alone, led to the immortalization of two out of three cultures of bone marrow-derived ECs (BMECs). One hTERT transduced BMEC culture underwent a long proliferative lag before resuming proliferation. BMECs transfected with hTERT alone were functionally and phenotypically normal. BMECs transfected with SV40T (BMSVTs) had an extended lifespan, but eventually succumbed to crisis. BMSVTs exhibited a partially transformed phenotype, demonstrating growth factor independence, altered antigen expression and forming tiny, infrequent colonies in vitro. Transduction of BMSVTs with hTERT resulted in immortalization of 4 out of 4 cultures. BMSVTs immortalized with hTERT formed large colonies in vitro and small transient tumours in vivo. BMECs co-expressing SV40T, hTERT and N-ras exhibited an overtly transformed phenotype; forming very large colonies with an altered morphology and generating rapidly growing tumours in vivo. These investigations demonstrate transformation of human ECs to an overtly malignant phenotype. This model will be useful for understanding mechanisms underlying vascular and angiogenic neoplasias, as well as for testing drugs designed to curtail aberrant EC growth.

ERG promotes T-acute lymphoblastic leukemia and is transcriptionally regulated in leukemic cells by a stem cell enhancer

The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.

Concentration- and schedule-dependent effects of chemotherapy on the angiogenic potential and drug sensitivity of vascular endothelial cells.

The anti-angiogenic activity of chemotherapy is both dose- and schedule-dependent. While conventional maximum tolerated dose (MTD) chemotherapy exerts only mild and reversible anti-angiogenic effects, low-dose metronomic (LDM) chemotherapy was developed to specifically target tumour angiogenesis. However, the long-term effects of either MTD or LDM chemotherapy on vascular endothelial cells have never been investigated. Here, we demonstrated that repeated exposure to MTD and LDM chemotherapy differentially impact on the angiogenic potential and chemosensitivity of immortalized endothelial cells. Repeated MTD vinblastine treatment of vascular endothelial cells led to an increased proliferation rate and resistance to paclitaxel. In contrast, repeated LDM treatment with vinblastine or etoposide impaired the angiogenic potential of endothelial cells and increased their chemosensitivity. This effect was associated with a significant decrease in βII- and βIII-tubulin expression. Functional analysis using siRNA showed that silencing the expression of βIII-tubulin in endothelial cells significantly decreased their capacity to form vascular structures and increased their sensitivity to the anti-angiogenic and vascular-disrupting effects of chemotherapy, whereas silencing βII-tubulin expression had no effect. Collectively our results show that LDM chemotherapy impairs the angiogenic potential of endothelial cells while increasing their chemosensitivity—an effect at least in part mediated by the down-regulation of βIII-tubulin expression. Furthermore, our study suggests that βIII-tubulin represents an attractive therapeutic target to increase the anti-angiogenic effects of chemotherapy and overall anti-tumour efficacy.

Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization

Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

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.

Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand.

To optimize conditions for ex vivo expansion of adult hematopoietic stem cells, we evaluated the co-culture of G-CSF mobilized human peripheral blood (PB) CD34(+) cells with endothelial cells engineered to overexpress various hematopoietic growth factors. Immortalized human bone marrow endothelial cells (BMEC) transfected with an expression vector carrying cDNA encoding the human telomerase reverse transcriptase (hTERT) and human umbilical vein endothelial cells (HUVEC) were transfected with combinations of adenovectors expressing murine c-kit ligand (KL), human thrombopoietin (TPO), human Flt3 ligand (FL), and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Ex vivo expansion of PB CD34(+) cells from normal donors and non-Hodgkin lymphoma (NHL) patients in endothelial co-culture was evaluated weekly for total cell production, progenitor (CFU-GM, BFU-E) cell production, and stem cell production as measured by Week-5 Cobblestone Area Forming Cell assay (Wk-5 CAFC). HUVEC transfected with adenovectors expressing TPO, KL, and FL provided the best co-culture system for expanding CD34(+) cells. Maximal total nuclear cell, CFU-GM, and Wk-5 CAFC production occurred between weeks 2 and 3 with 113-fold, 25-fold, and 2.2-5.5-fold expansions, respectively. We did not detect significant differences when GM-CSF was added to the co-culture system. Expansion was also obtained using recombinant human cytokines, but was not maintained beyond 3 weeks. We demonstrated that continuous generation of high levels of TPO, FL, and KL as well as other factors secreted by endothelium provided a clinically relevant co-culture method for ex vivo expansion of stem and progenitor cells from cryopreserved CD34(+) populations.

New prospects for targeting telomerase beyond the telomere

Telomerase activity is responsible for the maintenance of chromosome end structures (telomeres) and cancer cell immortality in most human malignancies, making telomerase an attractive therapeutic target. The rationale for targeting components of the telomerase holoenzyme has been strengthened by accumulating evidence indicating that these molecules have extra-telomeric functions in tumour cell survival and proliferation. This Review discusses current knowledge of the biogenesis, structure and multiple functions of telomerase-associated molecules intertwined with recent advances in drug discovery approaches. We also describe the fertile ground available for the pursuit of next-generation small-molecule inhibitors of telomerase.

Effective Management of Advanced Angiosarcoma by the Synergistic Combination of Propranolol and Vinblastine-based Metronomic Chemotherapy: A Bench to Bedside Study

Background Angiosarcomas are rare malignant tumors of vascular origin that represent a genuine therapeutic challenge. Recently, the combination of metronomic chemotherapy and drug repositioning has been proposed as an attractive alternative for cancer patients living in developing countries. Methods In vitro experiments with transformed endothelial cells were used to identify synergistic interactions between anti-hypertensive drug propranolol and chemotherapeutics. This led to the design of a pilot treatment protocol combining oral propranolol and metronomic chemotherapy. Seven consecutive patients with advanced/metastatic/recurrent angiosarcoma were treated with this combination for up to 12 months, followed by propranolol-containing maintenance therapy. Findings Gene expression analysis showed expression of ADRB1 and ADRB2 adrenergic receptor genes in transformed endothelial cells and in angiosarcoma tumors. Propranolol strongly synergized with the microtubule-targeting agent vinblastine in vitro, but only displayed additivity or slight antagonism with paclitaxel and doxorubicin. A combination treatment using bi-daily propranolol (40 mg) and weekly metronomic vinblastine (6 mg/m2) and methotrexate (35 mg/m2) was designed and used in 7 patients with advanced angiosarcoma. Treatment was well tolerated and resulted in 100% response rate, including 1 complete response and 3 very good partial responses, based on RECIST criteria. Median progression-free and overall survival was 11 months (range 5–24) and 16 months (range 10–30), respectively. Interpretation Our results provide a strong rationale for the combination of β-blockers and vinblastine-based metronomic chemotherapy for the treatment of advanced angiosarcoma. Furthermore, our study highlights the potential of drug repositioning in combination with metronomic chemotherapy in low- and middle-income country setting. Funding This study was funded by institutional and philanthropic grants.