EphB1 Suppression in Acute Myelogenous Leukemia: Regulating the DNA Damage Control System.

UNLABELLED: Loss of ephrin receptor (EphB1) expression may associate with aggressive cancer phenotypes; however, the mechanism of action remains unclear. To gain detailed insight into EphB1 function in acute myelogenous leukemia (AML), comprehensive analysis of EphB1 transcriptional regulation was conducted. In AML cells, EphB1 transcript was inversely correlated with EphB1 promoter methylation. The presence of EphB1 allowed EfnB1 ligand-mediated p53 DNA binding, leading to restoration of the DNA damage response (DDR) cascade by the activation of ATR, Chk1, p53, p21, p38, CDK1(tyr15), and Bax, and downregulation of HSP27 and Bcl2. Comparatively, reintroduction of EphB1 expression in EphB1-methylated AML cells enhanced the same cascade of ATR, Chk1, p21, and CDK1(tyr15), which consequently enforced programmed cell death. Interestingly, in pediatric AML samples, EphB1 peptide phosphorylation and mRNA expression were actively suppressed as compared with normal bone marrow, and a significant percentage of the primary AML specimens had EphB1 promoter hypermethylation. Finally, EphB1 repression associated with a poor overall survival in pediatric AML. Combined, the contribution of EphB1 to the DDR system reveals a tumor-suppressor function for EphB1 in pediatric AML. IMPLICATIONS: The tumor-suppressor function of EphB1 is clinically relevant across many malignancies, suggesting that EphB1 is an important regulator of common cancer cell transforming pathways.

Insights in dynamic kinome reprogramming as a consequence of MEK inhibition in MLL-rearranged AML.

Single kinase-targeted cancer therapies often failed prolonged responses because cancer cells bypass through alternative routes. In this study, high-throughput kinomic and proteomic approaches enabled to identify aberrant activity profiles in mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML) that defined druggable targets. This approach revealed impaired activity of proteins belonging to the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway. Pharmacological druggable MAPK pathway targets tested in primary MLL-rearranged AML included MAPKK1/2 (MEK), cyclic AMP-responsive element-binding protein (CREB) and MAPK8/9 (JNK). MEK inhibition showed to severely decrease MLL-rearranged AML cell survival without showing cytotoxicity in normal controls, whereas inhibition of CREB and JNK failed to exhibit MLL selectivity. Exploring the working mechanism of MEK inhibition, we assessed proteome activity in response to MEK inhibition in THP-1. MAPK1/3 (Erk) phosphorylation was instantly decreased in concurrence with a sustained Akt/mammalian target of rapamycin (mTOR) phosphorylation that enabled a subpopulation of cells to survive MEK inhibition. After exhaustion of MEK inhibition the AML cells recovered via increased activity of vascular endothelial growth factor receptor-2 (VEGFR-2) and Erk proteins to resume their proliferative state. Combined MEK and VEGFR-2 inhibition strengthened the reduction in MLL-rearranged AML cell survival by blocking the Akt/mTOR and MAPK pathways simultaneously. The generation of insights in cancerous altered activity profiles and alternative escape mechanisms upon targeted therapy allows the rational design of novel combination strategies.

Stromal interaction essential for vascular endothelial growth factor A-induced tumour growth via transforming growth factor-ß signalling.

BACKGROUND: High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment. METHODS: HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n=7) or VEGFA165 cells (n=7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays. RESULTS: In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P=0.001), combined with increased angiogenesis (P=0.002) and enhanced tumour cell proliferation (P=0.001). Gene expression profiling revealed human genes involved in TGF-ß signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression. CONCLUSION: These results suggest a role for VEGFA-driven tumour growth by TGF-ß signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype.

Tumour vasculature and angiogenic profile of paediatric pilocytic astrocytoma; is it much different from glioblastoma?

AIMS: Pilocytic astrocytomas are the most frequent brain tumours in children. Because of their high vascularity, this study aimed to obtain insights into potential angiogenic related therapeutic targets in these tumours by characterization of the vasculature and the angiogenic profile. In this study 59 paediatric pilocytic astrocytomas were compared with 62 adult glioblastomas, as a prototype of tumour angiogenesis. METHODS: Microvessel density, vessel maturity in terms of basement membrane and pericyte coverage, and turnover of both endothelial and tumour cells, and vascular endothelial growth factor (VEGF) expression were evaluated in tumour tissue, immunohistochemically stained with, respectively, CD34, collagen IV, smooth muscle actin, Ki67/CD34, caspase-3/CD34 and VEGF(-A-D). As an indicator for vessel stability the angiopoietin (ANGPT)-1/ANGPT-2 balance was calculated using Real Time RT-PCR. RESULTS: Pilocytic astrocytoma and glioblastoma showed similar fractions of vessels covered with basement membrane and pericytes. Overlapping ANGPT-1/ANGPT-2 balance and VEGF-A expression were found. Pilocytic astrocytoma had fewer but wider vessels compared with glioblastoma. Turnover of endothelial and tumour cells were relatively lower in pilocytic astrocytoma. Within pilocytic astrocytoma, higher ANGPT-1/ANGPT-2 balance was correlated with fewer apoptotic endothelial cells. Lower numbers of vessels were correlated with higher VEGF-A expression. CONCLUSIONS: Despite the fact that pilocytic astrocytoma showed a different vessel architecture compared with glioblastoma, a critical overlap in vessel immaturity/instability and the angiogenic profile was seen between both tumours. These findings suggest encouraging possibilities for targeting angiogenesis (for instance with anti-VEGF) as a therapeutic strategy in pilocytic astrocytoma.