The R-Spondin (RSpo) family of secreted proteins is implicated in the activation of the Wnt signaling pathway. Despite the high structural homology between the four members, expression patterns and phenotypes in knockout mice have demonstrated striking differences. Here we dissected and compared the molecular and cellular function of all RSpo family members. Although all four RSpo proteins activate the canonical Wnt pathway, RSpo2 and 3 are more potent than RSpo1, whereas RSpo4 is relatively inactive. All RSpo members require Wnt ligands and LRP6 for activity and amplify signaling of Wnt3A, Wnt1, and Wnt7A, suggesting that RSpo proteins are general regulators of canonical Wnt signaling. Like RSpo1, RSpo2-4 antagonize DKK1 activity by interfering with DKK1 mediated LRP6 and Kremen association. Analysis of RSpo deletion mutants indicates that the cysteine-rich furin domains are sufficient and essential for the amplification of Wnt signaling and inhibition of DKK1, suggesting that Wnt amplification by RSpo proteins may be a direct consequence of DKK1 inhibition. Together, these findings indicate that RSpo proteins modulate the Wnt pathway by a common mechanism and suggest that coexpression with specific Wnt ligands and DKK1 may determine their biological specificity in vivo.

We describe the cloning and characterization of Trident , a novel member of the fork head/winged-helix family, from murine thymus. In the mouse embryo, the gene was expressed in all tissues, whereas in adult mice expression was only detected in the thymus. Further analysis revealed that Trident expression strictly correlated with cell cycling, independent of cell type. Timing of [3H]thymidine incorporation showed that mRNA and protein expression were strongly upregulated upon entry into the S phase of the cell cycle. Moreover, the protein was phosphorylated in M phase. PCR-mediated selection of optimal binding sites yielded a consensus motif resembling that of other family members. These results identify Trident as a transcription factor, which is likely involved in cell cycle-specific gene regulation.

Aurora2 is a cell cycle regulated serine/threonine protein kinase which is overexpressed in many tumor cell lines. We demonstrate that Aurora2 is regulated by phosphorylation in a cell cycle dependent manner. This phosphorylation occurs on a conserved residue, Threonine 288, within the activation loop of the catalytic domain of the kinase and results in a signi®cant increase in the enzymatic activity. Threonine 288 resides within a consensus motif for the cAMP dependent kinase and can be phosphorylated by PKA in vitro. The protein phosphatase 1 is shown to dephosphorylate this site in vitro, and in vivo the phosphorylation of T288 is induced by okadaic acid treatment. Furthermore, we show that the Aurora2 kinase is regulated by proteasome dependent degradation and that Aurora2 phosphorylated on T288 may be targeted for degradation during mitosis. Our experiments suggest that phosphorylation of T288 is important for regulation of the Aurora2 kinase both for its activity and its stability. Oncogene (2000) 19, 4906 ± 4916.

In order to maintain a stable karyotype, the eukaryotic cell cycle is coordinated such that only one round of S phase precedes each mitosis, and mitosis is not initiated until DNA replication is completed. Several checkpoints and regulatory proteins have been defined in lower eukaryotes that govern this coordination, but little is known about the proteins that are involved in mammalian cells. Previously, we have shown that the winged-helix transcription factor Trident - also known as HFH-11, FKL16 and WIN [1] [2] [3] - is exclusively expressed in cycling cells and is phosphorylated during mitosis [1] [4]. The cellular function of Trident has yet to be described, however. Here, we have shown that disruption of the Trident gene in mice resulted in postnatal death, most probably because of circulatory failure. Histological analysis of Trident -/- embryos from embryonic day 10 (E10) onwards revealed a specific, characteristic defect in the developing myocardium. The orientation of the myocytes was highly irregular and the nuclei of these disorganized cardiomyocytes were clearly polyploid with up to a 50-fold increase in DNA content. Polyploidy was also observed in embryonic hepatocytes. Our results indicate that expression of Trident is required to prevent multiple rounds of S phase in the heart and the liver. Trident therefore appears to have a role in preventing DNA re-replication during the G2 and M phases.

The online version of this article has a supplementary appendix.Background C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models. Design and MethodsC-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated. -stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemiaderived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to Ctype lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells. ConclusionsOur results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.Key words: C-type, lectin-like molecule-1, immunotherapy, acute myeloid leukemia.Citation: Zhao X, Singh S, Pardoux C, Zhao J, Hsi ED, Abo A, and Korver W. Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia. Haematologica. 2010;95:71-78. doi:10.3324/haematol.2009 This is an open-access paper.Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia