Enforced expression of PPP1R13L increases tumorigenesis and invasion through p53-dependent and p53-independent mechanisms.

PPP1R13L was initially identified as a protein that binds to the NF-[kappa]B subunit p65/RelA and inhibits its transcriptional activity. It also binds p53 and inhibits its action. One set of experimental findings based on over-expression of PPP1R13L indicates that PPP1R13L blocks apoptosis. Another set of experiments, based on endogenous production of PPP1R13L, suggests that the protein may sometimes be pro-apoptotic. We have used primary mouse embryonic fibroblasts (MEFs), dually transformed by H-ras and Adenovirus E1A and differing in their p53 status, to explore the effects of PPP1R13L over-expression, thus examining the ability of PPP1R13L to act as an oncoprotein. We found that over-expression of PPP1R13L strongly accelerated tumor formation by ras/E1A and also resulted in an increased metastatic potential of the tumors. PPP1R13L over-expressing cells were depleted for both p53 and active p65/RelA and we found that both p53 dependent and independent apoptosis pathways were regulated by PPP1R13L. Finally, studies with the proteasome inhibitor MG132 revealed that over-expression of PPP1R13L causes faster p53 degradation, a likely explanation for the depletion of p53. Taken together, our results show that increased levels of PPP1R13L can increase tumorigenesis and furthermore pinpoint PPP1R13L as a gene that influences metastasis

Functional Identification of Tumor Suppressor Genes Through an in vivo RNA Interference Screen in a Mouse Lymphoma Model

2010 April 6Short hairpin RNAs (shRNAs) capable of stably suppressing gene function by RNA interference (RNAi) can mimic tumor-suppressor-gene loss in mice. By selecting for shRNAs capable of accelerating lymphomagenesis in a well-characterized mouse lymphoma model, we identified over ten candidate tumor suppressors, including Sfrp1, Numb, Mek1, and Angiopoietin 2. Several components of the DNA damage response machinery were also identified, including Rad17, which acts as a haploinsufficient tumor suppressor that responds to oncogenic stress and whose loss is associated with poor prognosis in human patients. Our results emphasize the utility of in vivo RNAi screens, identify and validate a diverse set of tumor suppressors, and have therapeutic implications

DR5 Knockout Mice Are Compromised in Radiation-Induced Apoptosis

DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM)

The discovery of SWI/SNF chromatin remodeling activity as a novel and targetable dependency in uveal melanoma

Uveal melanoma is a rare and aggressive cancer that originates in the uveal tissue of the eye. Currently, there are no approved targeted therapies for this cancer, and very few effective treatments are available. While activating mutations in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, other targetable molecular players are only partially understood. Through new analysis of unbiased, functional genomics screens and comprehensive validation studies in a panel of uveal melanoma cell lines, we find evidence that the SWI/SNF complex is essential in uveal melanoma. The mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) are often mutated in cancers and described as tumor suppressors, yet context specific roles for these complexes in the maintenance of certain cancers are beginning to emerge. We determined that the catalytic activity of SWI/SNF is critical, and further translated these findings with our recently described small molecule inhibitors of BRM and BRG1, the closely related catalytic subunits of the SWI/SNF complexes. Finally, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage specific transcription factor MITF, suggesting that SWI/SNF cooperates with MITF to drive a lineage specific transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path to the treatment of this cancer

Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants

The c-Myc oncoprotein promotes proliferation and apoptosis, such that mutations that disable apoptotic programmes often cooperate with MYC during tumorigenesis. Here we report that two common mutant MYC alleles derived from human Burkitt's lymphoma uncouple proliferation from apoptosis and, as a result, are more effective than wild-type MYC at promoting B cell lymphomagenesis in mice. Mutant MYC proteins retain their ability to stimulate proliferation and activate p53, but are defective at promoting apoptosis due to a failure to induce the BH3-only protein Bim (a member of the B cell lymphoma 2 (Bcl2) family) and effectively inhibit Bcl2. Disruption of apoptosis through enforced expression of Bcl2, or loss of either Bim or p53 function, enables wild-type MYC to produce lymphomas as efficiently as mutant MYC. These data show how parallel apoptotic pathways act together to suppress MYC-induced transformation, and how mutant MYC proteins, by selectively disabling a p53-independent pathway, enable tumour cells to evade p53 action during lymphomagenesis

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM)

Discovery of Darovasertib (NVP-LXS196), a Pan-PKC Inhibitor for the Treatment of Metastatic Uveal Melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy in the adult eye. Despite the aggressive local management of primary UM, the development of metastases is common with no effective treatment options for metastatic disease. Genetic analysis of UM samples reveals the presence of mutually exclusive activating mutations in the Gq alpha subunits GNAQ and GNA11. One of the key downstream targets of the constitutively active Gq alpha subunits is the protein kinase C (PKC) signaling pathway. Herein, we describe the discovery of darovasertib (NVP-LXS196), a potent pan-PKC inhibitor with high whole kinome selectivity. The lead series was optimized for kinase and off target selectivity to afford a compound that is rapidly absorbed and well tolerated in preclinical species. LXS196 is being investigated in the clinic as a monotherapy and in combination with other agents for the treatment of uveal melanoma (UM), including primary UM and metastatic uveal melanoma (MUM)