Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.cerebral cavernous malformation | signaling | MAP kinase | expression | endothelium C erebral cavernous malformations (CCMs) are characterized by endothelial cell channels in low-blood flow venous capillaries with poor coverage of pericytes and smooth cells and poorly developed tight and adherens junctions, resulting in increased permeability, hemorrhage, and subsequent neurologic deficits. Germline loss-of-function mutations in any one of three CCM genes-CCM1 (Krit1), CCM2 (OSM), or CCM3 (PDCD10)-cause the familial and sporadic forms of CCMs. The proteins encoded by these genes interact in a cytosolic complex. Critical insights into their in vivo functions have been obtained from genetic manipulation in fish and mice.In zebrafish, null mutations in either ccm1 (santa, san) or ccm2 (valentine, vtn) result in an enlarged heart ("big heart") and dilation of the subintestinal vessels and posterior cardinal vein, and morpholino (MO) studies indicate that ccm1 and ccm2 are in the same pathway during zebrafish cardiovascular development (1, 2). The contribution of ccm3 or its downstream effectors in the ccm1/2 pathway in zebrafish is less clear (3, 4). CCM proteins have been shown to regulate the signal strength of several pathways acting both as down-regulators and as activators. Reduced expression of CCM1, CCM2, or CCM3 increases RhoA activation (5). CCM2 and CCM3 siRNAs increase phosphorylation of AKT and the MAP kinases p38 and ERK1/2 (6). CCM1 promotes NOTCH activation (7) and deficiency in CCM proteins leads to endothelial-mesenchymal transition (EMT) by up-regulating TGF-β/BMP signaling (8). ccm1 and ccm2 deletion in zebrafish leads to the up-regulation of β1-integrin signaling and klf2 expression that results in a proangiogenic program (9). Ccm2-like (Ccm2l), a recently described paralog of Ccm2, was sho...

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25–100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis.