Phosphorylation of BECLIN-1 by BCR-ABL suppresses autophagy in chronic myeloid leukemia

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL+ cells

APC/CCdh1-Mediated Degradation of the F-Box Protein NIPA Is Regulated by Its Association with Skp1

NIPA (Nuclear Interaction Partner of Alk kinase) is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCFNIPA E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/CCdh1-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination

Cks1 Is Required for Tumor Cell Proliferation but Not Sufficient to Induce Hematopoietic Malignancies

The Cks1 component of the SCFSkp2 complex is necessary for p27Kip1 ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27Kip1. To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo

The epigenetics of breast cancer – Opportunities for diagnostics, risk stratification and therapy

The stage and molecular pathology-dependent prognosis of breast cancer, the limited treatment options for triple-negative carcinomas, as well as the development of resistance to therapies illustrate the need for improved early diagnosis and the development of new therapeutic approaches. Increasing data suggests that some answers to these challenges could be found in the area of epigenetics. In this study, we focus on the current research of the epigenetics of breast cancer, especially on the potential of epigenetics for clinical application in diagnostics, risk stratification and therapy. The differential DNA methylation status of specific gene regions has been used in the past to differentiate breast cancer cells from normal tissue. New technologies as detection of circulating nucleic acids including microRNAs to early detect breast cancer are emerging. Pattern of DNA methylation and expression of histone-modifying enzymes have been successfully used for risk stratification. However, all these epigenetic biomarkers should be validated in larger clinical studies. Recent preclinical and clinical studies show a therapeutic benefit of epigenetically active drugs for breast cancer entities that are still difficult to treat (triple negative, UICC stage IV). Remarkably, epigenetic therapies combined with chemotherapies or hormone-based therapies represent the most promising strategy. At the current stage, the integration of epigenetic substances into established breast cancer therapy protocols seems to hold the greatest potential for a clinical application of epigenetic research

NIPA (Nuclear Interaction Partner of ALK) Is Crucial for Effective NPM-ALK Mediated Lymphomagenesis

The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK(+) cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK(+) Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK(+) bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-Cre(TG/wt)Nipa(flox/flox) NPM-ALK(+) BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer stem-cell-like features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models

The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts

Hartmann D, de Strooper B, Serneels L, et al. The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts. Human Molecular Genetics. 2002;11(21):2615-2624.The metalloprotease ADAM 10 is an important APP α-secretase candidate, but in vivo proof of this is lacking. Furthermore, invertebrate models point towards a key role of the ADAM 10 orthologues Kuzbanian and sup-17 in Notch signalling. In the mouse, this function is, however, currently attributed to ADAM 17/TACE, while the role of ADAM 10 remains unknown. We have created ADAM 10-deficient mice. They die at day 9.5 of embryogenesis with multiple defects of the developing central nervous system, somites, and cardiovascular system. In situ hybridization revealed a reduced expression of the Notch target gene hes-5 in the neural tube and an increased expression of the Notch ligand dll-1, supporting an important role for ADAM 10 in Notch signalling in the vertebrates as well. Since the early lethality precluded the establishment of primary neuronal cultures, APPsα generation was analyzed in embryonic fibroblasts and found to be preserved in 15 out of 17 independently generated ADAM 10-deficient fibroblast cell lines, albeit at a quantitatively more variable level than in controls, whereas a severe reduction was found in only two cases. The variability was not due to differences in genetic background or to variable expression of the alternative α-secretase candidates ADAM 9 and ADAM 17. These results indicate, therefore, either a regulation between ADAMs on the post-translational level or that other, not yet known, proteases are able to compensate for ADAM 10 deficiency. Thus, the observed variability, together with recent reports on tissue-specific expression patterns of ADAMs 9, 10 and 17, points to the existence of tissue-specific ‘teams’ of different proteases exerting α-secretase activity