5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine zipper kinase inhibitors.

Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved, increasing evidence suggest that inhibition of MELK would constitute a promising approach for cancer therapy. A weak high-throughput screening hit (17, IC50 ≈ 5 µM) with lead-like properties was optimized for MELK inhibition. The early identification of a plausible binding mode by molecular modeling offered guidance in the choice of modifications towards compound 52 which displayed a 98 nM IC50. A good selectivity profile was achieved for a representative member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the potential to deliver compounds for further development with chemotherapeutic aims.

SOX7 expression is critically required in FLK1-expressing cells for vasculogenesis and angiogenesis during mouse embryonic development.

The transcriptional program that regulates the differentiation of endothelial precursor cells into a highly organized vascular network is still poorly understood. Here we explore the role of SOX7 during this process, performing a detailed analysis of the vascular defects resulting from either a complete deficiency in Sox7 expression or from the conditional deletion of Sox7 in FLK1-expressing cells. We analysed the consequence of Sox7 deficiency from E7.5 onward to determine from which stage of development the effect of Sox7 deficiency can be observed. We show that while Sox7 is expressed at the onset of endothelial specification from mesoderm, Sox7 deficiency does not impact the emergence of the first endothelial progenitors. However, by E8.5, clear signs of defective vascular development are already observed with the presence of highly unorganised endothelial cords rather than distinct paired dorsal aorta. By E10.5, both Sox7 complete knockout and FLK1-specific deletion of Sox7 lead to widespread vascular defects. In contrast, while SOX7 is expressed in the earliest specified blood progenitors, the VAV-specific deletion of Sox7 does not affect the hematopoietic system. Together, our data reveal the unique role of SOX7 in vasculogenesis and angiogenesis during embryonic development.

Endothelial precursor cell-based therapy to target the pathologic angiogenesis and compensate tumor hypoxia.

Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected: MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.

The Flk1-Cre-mediated deletion of ETV2 defines its narrow temporal requirement during embryonic hematopoietic development.

During embryonic development, the emergence of hematopoiesis and vasculogenesis is tightly associated, with many transcription factors implicated in both developmental processes. Among those factors, ETV2 acts at the top of the hierarchy and controls the formation of both lineages. However, it is not known at which stage of mesoderm development ETV2 is acting and whether ETV2 activity is further required once mesodermal precursors have been specified to the hematopoietic and endothelial fates. In this study, we characterize the developmental window during which ETV2 expression is required for hematopoietic and endothelial development. Using cre-mediated deletion of ETV2, we demonstrate that ETV2 is acting prior to or at the time of FLK1 expression in mesodermal precursors to initiate the hematopoietic and endothelial program. Using the in vitro differentiation of embryonic stem cells as a model system, we further show that ETV2 re-expression in Etv2(-/-) Flk1-negative precursors drives hematopoiesis specification and switches on the expression of most genes known to be implicated in hematopoietic and endothelial development. Among the downstream targets of ETV2, we identify the transcription factors SCL, GATA2, and FLI1 known to operate a recursive loop controlling hematopoietic development. Surprisingly, SCL re-expression in Etv2(-/-) cells fully rescues hematopoiesis, while the re-expression of FLI1 or GATA2 promotes only a very limited rescue. Altogether, our data establish that ETV2 is required very transiently to specify mesodermal precursors to hematopoiesis and vasculogenesis and that SCL is one of the key downstream targets of ETV2 in controlling hematopoietic specification.

SOX7 regulates the expression of VE-cadherin in the haemogenic endothelium at the onset of haematopoietic development.

At early stages of vertebrate ontogeny, blood and endothelial cells develop from a common mesodermal progenitor, the haemangioblast. Upon haematopoietic commitment, the haemangioblast generates blood precursors through populations of endothelial cells with haemogenic properties. Although several transcription factors have been implicated in haemangioblast differentiation, the precise mechanisms governing cell fate decisions towards the generation of haemogenic endothelium precursors remain largely unknown. Under defined conditions, embryonic stem (ES) cells can be differentiated into haemangioblast-like progenitors that faithfully recapitulate early embryonic haematopoiesis. Here, we made use of mouse ES cells as a model system to understand the role of SOX7, a member of a large family of transcription factors involved in a wide range of developmental processes. During haemangioblast differentiation, SOX7 is expressed in haemogenic endothelium cells and is downregulated in nascent blood precursors. Gain-of-function assays revealed that the enforced expression of Sox7 in haemangioblast-derived blast colonies blocks further differentiation and sustains the expression of endothelial markers. Thus, to explore the transcriptional activity of SOX7, we focused on the endothelial-specific adhesion molecule VE-cadherin. Similar to SOX7, VE-cadherin is expressed in haemogenic endothelium and is downregulated during blood cell formation. We show that SOX7 binds and activates the promoter of VE-cadherin, demonstrating that this gene is a novel downstream transcriptional target of SOX7. Altogether, our findings suggest that SOX7 is involved in the transcriptional regulation of genes expressed in the haemogenic endothelium and provide new clues to decipher the molecular pathways that drive early embryonic haematopoiesis.

Effects of heme oxygenase-1 on induction and development of chemically induced squamous cell carcinoma in mice.

Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1(+/+), HO-1(+/-), and HO-1(-/-) mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1(-/-) and HO-1(+/-) than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1(-/-) lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.