BMP9 is a key player in endothelial identity and its loss is sufficient to induce arteriovenous malformations.

AIMS: BMP9 is a high affinity ligand of ALK1 and endoglin receptors that are mutated in the rare genetic vascular disorder hereditary hemorrhagic telangiectasia (HHT). We have previously shown that loss of Bmp9 in the 129/Ola genetic background leads to spontaneous liver fibrosis via capillarization of liver sinusoidal endothelial cells (LSEC) and kidney lesions. We aimed to decipher the molecular mechanisms downstream of BMP9 to better characterize its role in vascular homeostasis in different organs. METHODS AND RESULTS: For this, we performed an RNA-seq analysis on LSEC from adult WT and Bmp9-KO mice and identified over 2000 differentially expressed genes. Gene ontology analysis showed that Bmp9 deletion led to a decrease in BMP and Notch signalling, but also LSEC capillary identity while increasing their cell cycle. The gene ontology term 'glomerulus development' was also negatively enriched in Bmp9-KO mice vs. WT supporting a role for BMP9 in kidney vascularization. Through different imaging approaches (electron microscopy, immunostainings), we found that loss of Bmp9 led to vascular enlargement of the glomeruli capillaries associated with alteration of podocytes. Importantly, we also showed for the first time that the loss of Bmp9 led to spontaneous arteriovenous malformations (AVMs) in the liver, gastrointestinal tract, and uterus. CONCLUSION: Altogether, these results demonstrate that BMP9 plays an important role in vascular quiescence both locally in the liver by regulating endothelial capillary differentiation markers and cell cycle but also at distance in many organs via its presence in the circulation. It also reveals that loss of Bmp9 is sufficient to induce spontaneous AVMs, supporting a key role for BMP9 in the pathogenesis of HHT.

Cancer selective cell death induction by a bivalent CK2 inhibitor targeting the ATP site and the allosteric αD pocket.

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity.

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

Different cardiovascular and pulmonary phenotypes for single- and double-knock-out mice deficient in BMP9 and BMP10.

AIMS: BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension, but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10. METHODS AND RESULTS: Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-knock-out (KO) mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After 3 weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single- and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay. CONCLUSIONS: Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodelling, whereas BMP10 plays a role in hypoxia-induced cardiac remodelling in mice.

Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction.

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL- cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug-gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

Ex-Vivo Treatment of Tumor Tissue Slices as a Predictive Preclinical Method to Evaluate Targeted Therapies for Patients with Renal Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the third type of urologic cancer. At time of diagnosis, 30% of cases are metastatic with no effect of chemotherapy or radiotherapy. Current targeted therapies lead to a high rate of relapse and resistance after a short-term response. Thus, a major hurdle in the development and use of new treatments for ccRCC is the lack of good pre-clinical models that can accurately predict the efficacy of new drugs and allow the stratification of patients into the correct treatment regime. Here, we describe different 3D cultures models of ccRCC, emphasizing the feasibility and the advantage of ex-vivo treatment of fresh, surgically resected human tumor slice cultures of ccRCC as a robust preclinical model for identifying patient response to specific therapeutics. Moreover, this model based on precision-cut tissue slices enables histopathology measurements as tumor architecture is retained, including the spatial relationship between the tumor and tumor-infiltrating lymphocytes and the stromal components. Our data suggest that acute treatment of tumor tissue slices could represent a benchmark of further exploration as a companion diagnostic tool in ccRCC treatment and a model to develop new therapeutic drugs.

Protein kinase CK2 contributes to placental development: physiological and pathological implications.

Preeclampsia (PE) is the most threatening pathology of human pregnancy. Its development is thought to be due to a failure in the invasion of trophoblast cells that establish the feto-maternal circulation. Protein kinase CK2 is a ubiquitous enzyme reported to be involved in the control of cell invasion. CK2 consists of two subunits, a catalytic subunit, CK2α, and a regulatory subunit, CK2ß. To date, no data exist regarding the expression and role of this enzyme in normal and PE pregnancies. We performed studies, at the clinical level using distinctive cohorts from early pregnancy (n = 24) and from PE (n = 23) and age-matched controls (n = 28); in vitro, using trophoblast cell lines; ex vivo, using placental explants; and in vivo, using PE mouse models. We demonstrated that (i) CK2 is more expressed during the late first trimester of pregnancy and is mainly localized in differentiated trophoblast cells, (ii) the inhibition of its enzymatic activity decreased the proliferation, migration, invasion, and syncytialization of trophoblast cells, both in 2D and 3D culture systems, and (iii) CK2 activity and the CK2α/CK2ß protein ratio were increased in PE human placentas. The pattern and profile of CK2 expression were confirmed in gravid mice along with an increase in the PE mouse models. Altogether, our results demonstrate that CK2 plays an essential role in the establishment of the feto-maternal circulation and that its deregulation is associated with PE development. The increase in CK2 activity in PE might constitute a compensatory mechanism to ensure proper pregnancy progress.

Design on a Rational Basis of High-Affinity Peptides Inhibiting the Histone Chaperone ASF1.

Anti-silencing function 1 (ASF1) is a conserved H3-H4 histone chaperone involved in histone dynamics during replication, transcription, and DNA repair. Overexpressed in proliferating tissues including many tumors, ASF1 has emerged as a promising therapeutic target. Here, we combine structural, computational, and biochemical approaches to design peptides that inhibit the ASF1-histone interaction. Starting from the structure of the human ASF1-histone complex, we developed a rational design strategy combining epitope tethering and optimization of interface contacts to identify a potent peptide inhibitor with a dissociation constant of 3 nM. When introduced into cultured cells, the inhibitors impair cell proliferation, perturb cell-cycle progression, and reduce cell migration and invasion in a manner commensurate with their affinity for ASF1. Finally, we find that direct injection of the most potent ASF1 peptide inhibitor in mouse allografts reduces tumor growth. Our results open new avenues to use ASF1 inhibitors as promising leads for cancer therapy.

Targeting AU-rich element-mediated mRNA decay with a truncated active form of the zinc-finger protein TIS11b/BRF1 impairs major hallmarks of mammary tumorigenesis.

Altered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnCS334D, by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnCS334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnCS334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnCS334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnCS334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnCS334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer.

Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma.

Potent inhibitors of PI3K (GDC-0941) and Src (Saracatinib) exhibit as individual agents, excellent oral anticancer activity in preclinical models and have entered phase II clinical trials in various cancers. We found that PI3K and Src kinases are dysregulated in clear cell renal carcinomas (ccRCCs), an aggressive disease without effective targeted therapies. In this study we addressed this challenge by testing GDC-0941 and Saracatinib as either single agents or in combination in ccRCC cell lines, as well as in mouse and PDX models. Our findings demonstrate that combined inhibition of PI3K and Src impedes cell growth and invasion and induces cell death of renal carcinoma cells providing preclinical evidence for a pairwise combination of these anticancer drugs as a rational strategy to improve renal cancer treatment.

The tyrosine-kinase inhibitor sunitinib targets vascular endothelial (VE)-cadherin: a marker of response to antitumoural treatment in metastatic renal cell carcinoma.

BACKGROUND: Vascular endothelial (VE)-cadherin is an endothelial cell-specific protein responsible for endothelium integrity. Its adhesive properties are regulated by post-translational processing, such as tyrosine phosphorylation at site Y685 in its cytoplasmic domain, and cleavage of its extracellular domain (sVE). In hormone-refractory metastatic breast cancer, we recently demonstrated that sVE levels correlate to poor survival. In the present study, we determine whether kidney cancer therapies had an effect on VE-cadherin structural modifications and their clinical interest to monitor patient outcome. METHODS: The effects of kidney cancer biotherapies were tested on an endothelial monolayer model mimicking the endothelium lining blood vessels and on a homotypic and heterotypic 3D cell model mimicking tumour growth. sVE was quantified by ELISA in renal cell carcinoma patients initiating sunitinib (48 patients) or bevacizumab (83 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials). RESULTS: Human VE-cadherin is a direct target for sunitinib which inhibits its VEGF-induced phosphorylation and cleavage on endothelial monolayer and endothelial cell migration in the 3D model. The tumour cell environment modulates VE-cadherin functions through MMPs and VEGF. We demonstrate the presence of soluble VE-cadherin in the sera of mRCC patients (n = 131) which level at baseline, is higher than in a healthy donor group (n = 96). Analysis of sVE level after 4 weeks of treatment showed that a decrease in sVE level discriminates the responders vs. non-responders to sunitinib, but not bevacizumab. CONCLUSIONS: These data highlight the interest for the sVE bioassay in future follow-up of cancer patients treated with targeted therapies such as tyrosine-kinase inhibitors.

The size-speed-force relationship governs migratory cell response to tumorigenic factors.

Tumor development progresses through a complex path of biomechanical changes leading first to cell growth and contraction and then cell deadhesion, scattering, and invasion. Tumorigenic factors may act specifically on one of these steps or have a wider spectrum of actions, leading to a variety of effects and thus sometimes to apparent contradictory outcomes. Here we used micropatterned lines of collagen type I/fibronectin on deformable surfaces to standardize cell behavior and measure simultaneously cell size, speed of motion and magnitude of the associated traction forces at the level of a single cell. We analyzed and compared the normal human breast cell line MCF10A in control conditions and in response to various tumorigenic factors. In all conditions, a wide range of biomechanical properties was identified. Despite this heterogeneity, normal and transformed motile cells followed a common trend whereby size and contractile forces were negatively correlated with cell speed. Some tumorigenic factors, such as activation of ErbB2 or loss of the ßsubunit of casein kinase 2, shifted the whole population toward a faster speed and lower contractility state. Treatment with transforming growth factor ß induced some cells to adopt opposing behaviors such as extremely high versus extremely low contractility. Thus tumor transformation amplified preexisting population heterogeneity and led some cells to exhibit biomechanical properties that were more extreme than those observed with normal cells.