Large-scale phosphoproteomics reveals activation of the MAPK/GADD45ß/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells.

BACKGROUND: BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. METHODS: To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry. RESULTS: Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45ß. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1. CONCLUSIONS: Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes.

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.

Temporal Gene Expression Profiles Reflect the Dynamics of Lymphoid Differentiation.

Understanding the emergence of lymphoid committed cells from multipotent progenitors (MPP) is a great challenge in hematopoiesis. To gain deeper insight into the dynamic expression changes associated with these transitions, we report the quantitative transcriptome of two MPP subsets and the common lymphoid progenitor (CLP). While the transcriptome is rather stable between MPP2 and MPP3, expression changes increase with differentiation. Among those, we found that pioneer lymphoid genes such as Rag1, Mpeg1, and Dntt are expressed continuously from MPP2. Others, such as CD93, are CLP specific, suggesting their potential use as new markers to improve purification of lymphoid populations. Notably, a six-transcription factor network orchestrates the lymphoid differentiation program. Additionally, we pinpointed 24 long intergenic-non-coding RNA (lincRNA) differentially expressed through commitment and further identified seven novel forms. Collectively, our approach provides a comprehensive landscape of coding and non-coding transcriptomes expressed during lymphoid commitment.

Battle of the Sex Chromosomes: Competition between X and Y Chromosome-Encoded Proteins for Partner Interaction and Chromatin Occupancy Drives Multicopy Gene Expression and Evolution in Muroid Rodents.

Transmission distorters (TDs) are genetic elements that favor their own transmission to the detriments of others. Slx/Slxl1 (Sycp3-like-X-linked and Slx-like1) and Sly (Sycp3-like-Y-linked) are TDs, which have been coamplified on the X and Y chromosomes of Mus species. They are involved in an intragenomic conflict in which each favors its own transmission, resulting in sex ratio distortion of the progeny when Slx/Slxl1 versus Sly copy number is unbalanced. They are specifically expressed in male postmeiotic gametes (spermatids) and have opposite effects on gene expression: Sly knockdown leads to the upregulation of hundreds of spermatid-expressed genes, whereas Slx/Slxl1-deficiency downregulates them. When both Slx/Slxl1 and Sly are knocked down, sex ratio distortion and gene deregulation are corrected. Slx/Slxl1 and Sly are, therefore, in competition but the molecular mechanism remains unknown. By comparing their chromatin-binding profiles and protein partners, we show that SLX/SLXL1 and SLY proteins compete for interaction with H3K4me3-reader SSTY1 (Spermiogenesis-specific-transcript-on-the-Y1) at the promoter of thousands of genes to drive their expression, and that the opposite effect they have on gene expression is mediated by different abilities to recruit SMRT/N-Cor transcriptional complex. Their target genes are predominantly spermatid-specific multicopy genes encoded by the sex chromosomes and the autosomal Speer/Takusan. Many of them have coamplified with not only Slx/Slxl1/Sly but also Ssty during muroid rodent evolution. Overall, we identify Ssty as a key element of the X versus Y intragenomic conflict, which may have influenced gene content and hybrid sterility beyond Mus lineage since Ssty amplification on the Y predated that of Slx/Slxl1/Sly.

Bdf1 Bromodomains Are Essential for Meiosis and the Expression of Meiotic-Specific Genes.

Bromodomain and Extra-terminal motif (BET) proteins play a central role in transcription regulation and chromatin signalling pathways. They are present in unicellular eukaryotes and in this study, the role of the BET protein Bdf1 has been explored in Saccharomyces cerevisiae. Mutation of Bdf1 bromodomains revealed defects on both the formation of spores and the meiotic progression, blocking cells at the exit from prophase, before the first meiotic division. This phenotype is associated with a massive deregulation of the transcription of meiotic genes and Bdf1 bromodomains are required for appropriate expression of the key meiotic transcription factor NDT80 and almost all the Ndt80-inducible genes, including APC complex components. Bdf1 notably accumulates on the promoter of Ndt80 and its recruitment is dependent on Bdf1 bromodomains. In addition, the ectopic expression of NDT80 during meiosis partially bypasses this dependency. Finally, purification of Bdf1 partners identified two independent complexes with Bdf2 or the SWR complex, neither of which was required to complete sporulation. Taken together, our results unveil a new role for Bdf1 -working independently from its predominant protein partners Bdf2 and the SWR1 complex-as a regulator of meiosis-specific genes.

The Arabidopsis hnRNP-Q Protein LIF2 and the PRC1 Subunit LHP1 Function in Concert to Regulate the Transcription of Stress-Responsive Genes.

LHP1-INTERACTING FACTOR2 (LIF2), a heterogeneous nuclear ribonucleoprotein involved in Arabidopsis thaliana cell fate and stress responses, interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a Polycomb Repressive Complex1 subunit. To investigate LIF2-LHP1 functional interplay, we mapped their genome-wide distributions in wild-type, lif2, and lhp1 backgrounds, under standard and stress conditions. Interestingly, LHP1-targeted regions form local clusters, suggesting an underlying functional organization of the plant genome. Regions targeted by both LIF2 and LHP1 were enriched in stress-responsive genes, the H2A.Z histone variant, and antagonistic histone marks. We identified specific motifs within the targeted regions, including a G-box-like motif, a GAGA motif, and a telo-box. LIF2 and LHP1 can operate both antagonistically and synergistically. In response to methyl jasmonate treatment, LIF2 was rapidly recruited to chromatin, where it mediated transcriptional gene activation. Thus, LIF2 and LHP1 participate in transcriptional switches in stress-response pathways.

The spatiotemporal program of DNA replication is associated with specific combinations of chromatin marks in human cells.

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about [Formula: see text] predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.

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.

A microfluidic platform integrating functional vascularized organoids-on-chip.

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics.

A new scaffold-free tumoroid model provides a robust preclinical tool to investigate invasion and drug response in Renal Cell Carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent kidney cancers, which is often asymptomatic and thus discovered at a metastatic state (mRCC). mRCC are highly heterogeneous tumors composed of subclonal populations that lead to poor treatment response rate. Several recent works explored the potential of ccRCC tumoroids culture derived from patients. However, these models were produced following a scaffold-based method using collagen I or Matrigel that exhibit lot variability and whose complexity could induce treatment response modifications and phenotypic alterations. Following the observation that ccRCC tumoroids can create their own niche by secreting extracellular matrix components, we developed the first scaffold-free tumoroid model of ccRCC tumors. Tumoroids from mice as well as from human tumors were generated with high success rate (≥90%) using a magnetic suspension method and standard culture media. Immunofluorescence analysis revealed their self-organization capacities to maintain multiple tumor-resident cell types, including endothelial progenitor cells. Transcriptomic analysis showed the reproducibility of the method highlighting that the majority of gene expression patterns was conserved in tumoroids compared to their matching tumor tissue. Moreover, this model enables to evaluate drug effects and invasiveness of renal cancer cells in a 3D context, providing a robust preclinical tool for drug screening and biomarker assessment in line with alternative ex vivo methods like tumor tissue slice culture or in vivo xenograft models.

The Immunopeptidome from a Genomic Perspective: Establishing the Noncanonical Landscape of MHC Class I-Associated Peptides.

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I-associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development.

CK2ß Is a Gatekeeper of Focal Adhesions Regulating Cell Spreading.

CK2 is a hetero-tetrameric serine/threonine protein kinase made up of two CK2α/α' catalytic subunits and two CK2ß regulatory subunits. The free CK2α subunit and the tetrameric holoenzyme have distinct substrate specificity profiles, suggesting that the spatiotemporal organization of the individual CK2 subunits observed in living cells is crucial in the control of the many cellular processes that are governed by this pleiotropic kinase. Indeed, previous studies reported that the unbalanced expression of CK2 subunits is sufficient to drive epithelial to mesenchymal transition (EMT), a process involved in cancer invasion and metastasis. Moreover, sub-stoichiometric expression of CK2ß compared to CK2α in a subset of breast cancer tumors was correlated with the induction of EMT markers and increased epithelial cell plasticity in breast carcinoma progression. Phenotypic changes of epithelial cells are often associated with the activation of phosphotyrosine signaling. Herein, using phosphotyrosine enrichment coupled with affinity capture and proteomic analysis, we show that decreased expression of CK2ß in MCF10A mammary epithelial cells triggers the phosphorylation of a number of proteins on tyrosine residues and promotes the striking activation of the FAK1-Src-PAX1 signaling pathway. Moreover, morphometric analyses also reveal that CK2ß loss increases the number and the spatial distribution of focal adhesion signaling complexes that coordinate the adhesive and migratory processes. Together, our findings allow positioning CK2ß as a gatekeeper for cell spreading by restraining focal adhesion formation and invasion of mammary epithelial cells.

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.

RNA sequencing of chronic GVHD skin lesions defines shared and unique inflammatory pathways characterizing lichen planus and morphea.

Cutaneous involvement of chronic graft-versus-host disease (cGVHD) has a wide range of manifestations including a lichenoid form with a currently assumed mixed Th1/Th17 signature and a sclerotic form with Th1 signature. Despite substantial heterogeneity of innate and adaptive immune cells recruited to the skin and of the different clinical manifestations, treatment depends mainly on the severity of the skin involvement and relies on systemic, high-dose glucocorticoids alone or in combination with a calcineurin inhibitor. We performed the first study using RNA sequencing to profile and compare the transcriptome of lichen planus cGVHD (n = 8), morphea cGVHD (n = 5), and healthy controls (n = 6). Our findings revealed shared and unique inflammatory pathways to each cGVHD subtype that are both pathogenic and targetable. In particular, the deregulation of IFN signaling pathway was strongly associated with cutaneous cGVHD, whereas the triggering receptor expressed on myeloid cells 1 pathway was found to be specific of lichen planus and likely contributes to its pathogenesis. The results were confirmed at a protein level by performing immunohistochemistry staining and at a transcriptomic level using real-time quantitative polymerase chain reaction.

Region-specific expression of young small-scale duplications in the human central nervous system.

BACKGROUND: The duplication of genes is one of the main genetic mechanisms that led to the gain in complexity of biological tissue. Although the implication of duplicated gene expression in brain evolution was extensively studied through comparisons between organs, their role in the regional specialization of the adult human central nervous system has not yet been well described. RESULTS: Our work explored intra-organ expression properties of paralogs through multiple territories of the human central nervous system (CNS) using transcriptome data generated by the Genotype-Tissue Expression (GTEx) consortium. Interestingly, we found that paralogs were associated with region-specific expression in CNS, suggesting their involvement in the differentiation of these territories. Beside the influence of gene expression level on region-specificity, we observed the contribution of both duplication age and duplication type to the CNS region-specificity of paralogs. Indeed, we found that small scale duplicated genes (SSDs) and in particular ySSDs (SSDs younger than the 2 rounds of whole genome duplications) were more CNS region-specific than other paralogs. Next, by studying the two paralogs of ySSD pairs, we observed that when they were region-specific, they tend to be specific to the same region more often than for other paralogs, showing the high co-expression of ySSD pairs. The extension of this analysis to families of paralogs showed that the families with co-expressed gene members (i.e. homogeneous families) were enriched in ySSDs. Furthermore, these homogeneous families tended to be region-specific families, where the majority of their gene members were specifically expressed in the same region. CONCLUSIONS: Overall, our study suggests the involvement of ySSDs in the differentiation of human central nervous system territories. Therefore, we show the relevance of exploring region-specific expression of paralogs at the intra-organ level.

Transcriptomic Characterization of Postmolar Gestational Choriocarcinoma.

The human placenta shares properties with solid tumors, such as rapid growth, tissue invasion, cell migration, angiogenesis, and immune evasion. However, the mechanisms that drive the evolution from premalignant proliferative placental diseases-called hydatidiform moles-to their malignant counterparts, gestational choriocarcinoma, as well as the factors underlying the increased aggressiveness of choriocarcinoma arising after term delivery compared to those developing from hydatidiform moles, are unknown. Using a 730-gene panel covering 13 cancer-associated canonical pathways, we compared the transcriptomic profiles of complete moles to those of postmolar choriocarcinoma samples and those of postmolar to post-term delivery choriocarcinoma. We identified 33 genes differentially expressed between complete moles and postmolar choriocarcinoma, which revealed TGF-ß pathway dysregulation. We found the strong expression of SALL4, an upstream regulator of TGF-ß, in postmolar choriocarcinoma, compared to moles, in which its expression was almost null. Finally, there were no differentially expressed genes between postmolar and post-term delivery choriocarcinoma samples. To conclude, the TGF-ß pathway appears to be a crucial step in the progression of placental malignancies. Further studies should investigate the value of TGF- ß family members as biomarkers and new therapeutic targets.

NLRP7 Promotes Choriocarcinoma Growth and Progression through the Establishment of an Immunosuppressive Microenvironment.

The inflammatory gene NLRP7 is the major gene responsible for recurrent complete hydatidiform moles (CHM), an abnormal pregnancy that can develop into gestational choriocarcinoma (CC). However, the role of NLRP7 in the development and immune tolerance of CC has not been investigated. Three approaches were employed to define the role of NLRP7 in CC development: (i) a clinical study that analyzed human placenta and sera collected from women with normal pregnancies, CHM or CC; (ii) an in vitro study that investigated the impact of NLRP7 knockdown on tumor growth and organization; and (iii) an in vivo study that used two CC mouse models, including an orthotopic model. NLRP7 and circulating inflammatory cytokines were upregulated in tumor cells and in CHM and CC. In tumor cells, NLRP7 functions in an inflammasome-independent manner and promoted their proliferation and 3D organization. Gravid mice placentas injected with CC cells invalidated for NLRP7, exhibited higher maternal immune response, developed smaller tumors, and displayed less metastases. Our data characterized the critical role of NLRP7 in CC and provided evidence of its contribution to the development of an immunosuppressive maternal microenvironment that not only downregulates the maternal immune response but also fosters the growth and progression of CC.

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.