We describe the design of a set of inhibitors to investigate the relationship between cyclin G associated kinase (GAK) and epidermal growth factor receptor (EGFR) in chordoma bone cancers. These compounds were characterized both in vitro and using in cell target engagement assays. The most potent chordoma inhibitors were further characterized in a kinome-wide screen demonstrating narrow spectrum profiles..

Summary Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in C. elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion that enables protrusion growth. Together these studies define a netrin dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Stabilization of oscillatory UNC-40 clustering by its ligand UNC-6 directs localized F-actin generation and polarization of cells.

Localized F-actin disassembly by ADF/cofilin drives invadopodial membrane recycling through endolysosomes, which promotes efficient cell transmigration through the basement membrane.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

Cardiovascular defects, injuries, and degenerative diseases often require surgical intervention and the use of implantable replacement material and conduits. Traditional vascular grafts made of synthetic polymers, animal and cadaveric tissues, or autologous vasculature have been utilized for almost a century with well-characterized outcomes, leaving areas of unmet need for the patients in terms of durability and long-term patency, susceptibility to infection, immunogenicity associated with the risk of rejection, and inflammation and mechanical failure. Research to address these limitations is exploring avenues as diverse as gene therapy, cell therapy, cell reprogramming, and bioengineering of human tissue and replacement organs. Tissue-engineered vascular conduits, either with viable autologous cells or decellularized, are the forefront of technology in cardiovascular reconstruction and offer many benefits over traditional graft materials, particularly in the potential for the implanted material to be adopted and remodeled into host tissue and thus offer safer, more durable performance. This review discusses the key advances and future directions in the field of surgical vascular repair, replacement, and reconstruction, with a focus on the challenges and expected benefits of bioengineering human tissues and blood vessels.

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