Raf kinases have been linked to endothelial cell survival. Here, we show that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) differentially activate Raf, resulting in protection from distinct pathways of apoptosis in human endothelial cells and chick embryo vasculature. bFGF activated Raf-1 via p21-activated protein kinase-1 (PAK-1) phosphorylation of serines 338 and 339, resulting in Raf-1 mitochondrial translocation and endothelial cell protection from the intrinsic pathway of apoptosis, independent of the mitogen-activated protein kinase kinase-1 (MEK1). In contrast, VEGF activated Raf-1 via Src kinase, leading to phosphorylation of tyrosines 340 and 341 and MEK1-dependent protection from extrinsic-mediated apoptosis. These findings implicate Raf-1 as a pivotal regulator of endothelial cell survival during angiogenesis.

Genotoxic stress induced by anticancer drugs can lead to apoptosis of both angiogenic endothelial cells (ECs) and proliferating tumor cells.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma (MM) and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drugconjugate approved for the treatment of relapsed/refractory MM. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo. GSK2857916 treatment enhances intra-tumor immune cell infiltration and activation, delays tumor growth and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to re-challenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T-cells.Combinations with OX40/OX86, an immune agonist antibody, significantly enhance anti-tumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

In a screen for mouse mutations with dominant behavioral anomalies, we identified Wheels, a mutation associated with circling and hyperactivity in heterozygotes and embryonic lethality in homozygotes. Mutant Wheels embryos die at E10.5-E11.5 and exhibit a host of morphological anomalies which include growth retardation and anomalies in vascular and hindbrain development. The latter includes perturbation of rhombomeric boundaries as detected by Krox20 and Hoxb1. PECAM-1 staining of embryos revealed normal formation of the primary vascular plexus. However, subsequent stages of branching and remodeling do not proceed normally in the yolk sac and in the embryo proper. To obtain insights into the circling behavior, we examined development of the inner ear by paint-filling of membranous labyrinths of Whl/+ embryos. This analysis revealed smaller posterior and lateral semicircular canal primordia and a delay in the canal fusion process at E12.5. By E13.5, the lateral canal was truncated and the posterior canal was small or absent altogether. Marker analysis revealed an early molecular phenotype in heterozygous embryos characterized by perturbed expression of Bmp4 and Msx1 in prospective lateral and posterior cristae at E11.5. We have constructed a genetic and radiation hybrid map of the centromeric portion of mouse Chromosome 4 across the Wheels region and refined the position of the Wheels locus to the approximately 1.1-cM region between D4Mit104 and D4Mit181. We have placed the locus encoding Epha7, in the Wheels candidate region; however, further analysis showed no mutations in the Epha7-coding region and no detectable changes in mRNA expression pattern. In summary, our findings indicate that Wheels, a gene which is essential for the survival of the embryo, may link diverse processes involved in vascular, hindbrain, and inner ear development.

Chromosome deletions have several applications in the genetic analysis of complex organisms. They can be used as reagents in region-directed mutagenesis, for mapping of simple or complex traits, or to identify biological consequences of segmental haploidy, the latter being relevant to human contiguous gene syndromes and imprinting. We have generated three deletion complexes in ES (Embryonic Stem) cells that collectively span ∼ 40 cM of proximal mouse chromosome 5. The deletion complexes were produced by irradiation of F 1 hybrid ES cells containing herpes simplex virus thymidine kinase genes (tk) integrated at the Dpp6, Hdh (Huntington disease locus), or Gabrb1 loci, followed by selection for tk-deficient clones. Deletions centered at the adjacent Hdh and Dpp6 loci ranged up to ∼ 20 cM or more in length and overlapped in an interdigitated fashion. However, the interval between Hdh and Gabrb1 appeared to contain a locus haploinsufficient for ES cell viability, thereby preventing deletions of either complex from overlapping. In some cases, the deletions resolved the order of markers that were previously genetically inseparable. A subset of the ES cell-bearing deletions was injected into blastocysts to generate germline chimeras and establish lines of mice segregating the deletion chromosomes. At least 11 of the 26 lines injected were capable of producing germline chimeras. In general, those that failed to undergo germline transmission bore deletions larger than the germline-competent clones, suggesting that certain regions of chromosome 5 contain haploinsufficient developmental genes, and/or that overall embryonic viability is cumulatively decreased as more genes are rendered hemizygous. Mice bearing deletions presumably spanning the semidominant hammertoe locus (Hm) had no phenotype, suggesting that the classic allele is a dominant, gain-of-function mutation. Overlapping deletion complexes generated in the fashion described in this report will be useful as multipurpose genetic tools and in systematic functional mapping of the mouse genome.