Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition.

Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.

Role of the E3 ubiquitin ligase RNF157 as a novel downstream effector linking PI3K and MAPK signaling pathways to the cell cycle.

The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome-CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G2/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition.

Targeted therapeutics that block signal transduction through the RAS-RAF-MEK and PI3K-AKT-mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941-mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.

FDG-PET is a good biomarker of both early response and acquired resistance in BRAFV600 mutant melanomas treated with vemurafenib and the MEK inhibitor GDC-0973.

BACKGROUND: The BRAF inhibitor, vemurafenib, has recently been approved for the treatment of metastatic melanoma in patients harboring BRAFV600 mutations. Currently, dual BRAF and MEK inhibition are ongoing in clinical trials with the goal of overcoming the acquired resistance that has unfortunately developed in some vemurafenib patients. FDG-PET measures of metabolic activity are increasingly employed as a pharmacodynamic biomarker for guiding single-agent or combination therapies by gauging initial drug response and monitoring disease progression. However, since tumors are inherently heterogeneous, investigating the effects of BRAF and MEK inhibition on FDG uptake in a panel of different melanomas could help interpret imaging outcomes. METHODS: 18 F-FDG uptake was measured in vitro in cells with wild-type and mutant (V600) BRAF, and in melanoma cells with an acquired resistance to vemurafenib. We treated the cells with vemurafenib alone or in combination with MEK inhibitor GDC-0973. PET imaging was used in mice to measure FDG uptake in A375 melanoma xenografts and in A375 R1, a vemurafenib-resistant derivative. Histological and biochemical studies of glucose transporters, the MAPK and glycolytic pathways were also undertaken. RESULTS: We demonstrate that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous BRAFV600 but ineffective in cells with acquired resistance or having WT BRAF status. However, combination with GDC-0973 results in a highly significant increase of efficacy and inhibition of FDG uptake across all twenty lines. Drug-induced changes in FDG uptake were associated with altered levels of membrane GLUT-1, and cell lines harboring RAS mutations displayed enhanced FDG uptake upon exposure to vemurafenib. Interestingly, we found that vemurafenib treatment in mice bearing drug-resistant A375 xenografts also induced increased FDG tumor uptake, accompanied by increases in Hif-1α, Sp1 and Ksr protein levels. Vemurafenib and GDC-0973 combination efficacy was associated with decreased levels of hexokinase II, c-RAF, Ksr and p-MEK protein. CONCLUSIONS: We have demonstrated that 18 F-FDG-PET imaging reflects vemurafenib and GDC-0973 action across a wide range of metastatic melanomas. A delayed post-treatment increase in tumor FDG uptake should be considered carefully as it may well be an indication of acquired drug resistance. TRIAL REGISTRATION: ClinicalTrials.gov NCT01271803.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility.

Inhibitor of apoptosis proteins (IAP) are evolutionarily conserved anti-apoptotic regulators. C-RAF protein kinase is a direct RAS effector protein, which initiates the classical mitogen-activated protein kinase (MAPK) cascade. This signalling cascade mediates diverse biological functions, such as cell growth, proliferation, migration, differentiation and survival. Here we demonstrate that XIAP and c-IAPs bind directly to C-RAF kinase and that siRNA-mediated silencing of XIAP and c-IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitylation of C-RAF in vivo through the Hsp90-mediated quality control system, independently of its E3 ligase activity. In addition, XIAP or c-IAP-1/2 knockdown cells showed enhanced cell migration in a C-RAF-dependent manner. XIAP promotes binding of CHIP (carboxy terminal Hsc70-interacting protein), a chaperone-associated ubiquitin ligase, to the C-RAF-Hsp90 complex in vivo. Interfering with CHIP expression resulted in stabilization of C-RAF and enhanced cell migration, as observed in XIAP knockdown cells. Our data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signalling pathway and cell migration.