Evaluating downstream targets of cullin4-dependent E3 ligases and of D-type cyclins: implications for the dysregulation of ubiquitination and cell growth in cancer

Cyclin D1 and cullin4 (CUL4) are two proteins known to be upregulated in cancer. Cyclin D1 functions to regulate the cell division cycle and cell growth, while CUL4 assembles E3 ubiquitin ligase complexes that function to ubiquitinate target proteins, often marking them for degradation. However, the downstream effectors of their oncogenic activities are not fully characterized. Therefore, the aim of this study was to discover novel targets of the D-type cyclins and of CUL4, and to better describe the E3 ubiquitin ligase complexes assembled by CUL4. We identified the TSC1- TSC2 tumor suppressor complex, a key negative regulator of cell growth, as a cyclin D-interacting complex, and demonstrated that D-type cyclins could down-regulate the activity of TSC1-TSC2 by both CDK (Cyclin Dependent Kinase) -dependent and -independent mechanisms. In a separate line of studies, I conducted a genetic analysis of mutants of Cul4 and one of its putative substrate receptor molecules, Ddb1 (Damaged DNA Binding protein 1) in Drosophila, and established that CUL4DDB1 plays an essential role in cell growth, proliferation, and development. These studies suggested a number of novel substrates of the CUL4DDB1 ligase, and also served to clarify the role of CUL4DDB1 in controlling the degradation of the replication licensing factor CDT1/DUP during the cell cycle. Collectively, these analyses of the D-type cyclins and CUL4 broaden our understanding of the consequence of their disruption in cancer development

PLA2 promotes fusion between PMN‐specific granules and complex liposomes

Neutrophil stimulation results in the activation of a variety of phospholipases, including phospholipase A2 (PLA2), which releases arachidonic acid from the 2 position of membrane phospholipids, leaving a lysophospholipid. Because arachidonic acid is known to be a potent fusogen in vitro, we examined the effect of metabolism by PLA2 on the fusion of complex liposomes (liposomes prepared with a phospholipid composition similar to that found in neutrophil plasma membrane). We observed that PLA2 augmented the fusion of complex liposomes with each other as well as with specific granules isolated from human neutrophils, lowering the Ca2+ requirement for fusion by three orders of magnitude. Furthermore, although lysophospholipids inhibited fusion, the incorporation of arachidonic acid into liposome membranes overcame the inhibitory effects of the lysophospholipids. Thus with PLA2 and annexins we were able to obtain fusion of complex liposomes at concentations of Ca2+ that are close to physiological. Our data suggest that the activation of PLA2 and the generation of arachidonic acid may be the major fusion‐promoting event mediating neutrophil degranulation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141645/1/jlb0663.pd

Direct Recruitment of Polycomb Repressive Complex 1 to Chromatin by Core Binding Transcription Factors

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.Stem Cell and Regenerative Biolog

Cell Type–dependent Requirement for PIP Box–regulated Cdt1 Destruction During S Phase

Previous studies have shown that Cdt1 overexpression in cultured cells can trigger re-replication, but not whether CRL4Cdt2-triggered destruction of Cdt1 is required for normal mitotic cell cycle progression in vivo. We demonstrate that PIP box–mediated destruction of Cdt1Dup during S phase is necessary for the cell division cycle in Drosophila

Spatial Mapping and Immunomodulatory Role of the OX40/OX40L Pathway in Human Non-Small Cell Lung Cancer

Purpose: To evaluate the tissue distribution and clinical significance of OX40 and OX40L in human non-small cell lung cancer (NSCLC). Experimental design: Using multiplexed quantitative immunofluorescence, we conducted simultaneous and localized measurements of OX40 and OX40L proteins, major T-cell subsets, and conventional type 1 dendritic cells (cDC1) in 614 primary NSCLCs from three independent cohorts represented in tissue microarrays. We also measured OX40L protein in samples from a phase I clinical trial of intratumor administration of a lipid nanoparticle encapsulated mRNA encoding OX40L (mRNA-2416) in human solid tumors. Finally, we studied the OX40 pathway in 212 uterine/ovarian serous carcinomas. Results: OX40 protein was expressed in approximately 90% of NSCLCs, and OX40L was detected in approximately 10% of cases. Increased expression of OX40 was associated with higher CD4(+) and CD8(+) T lymphocytes, as well as cDC1s. Elevated expression of OX40L was consistently associated with increased CD4(+) tumor-infiltrating lymphocytes and longer overall survival. No association was found between OX40 or OX40L levels and oncogenic driver mutations in EGFR and KRAS in lung adenocarcinomas. Delivering OX40L mRNA using intratumor mRNA-2416 injection mediated increased local OX40L protein levels that was most prominent in a patient with ovarian serous carcinoma. Detectable OX40L protein levels were observed in 15% of primary uterine/ovarian serous malignancies and associated with longer survival. Conclusions: The OX40 pathway is expressed in a fraction of NSCLCs and is associated with a favorable immune contexture. Although OX40L is uncommonly expressed in NSCLC and serous malignancies, it is associated with better prognosis and can be introduced using exogenous mRNA