RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here, we describe a functionally critical region of RAS located outside the effector lobe that can be targeted for inhibition. We developed a synthetic binding protein (monobody), termed NS1, that bound with high affinity to both GTP- and GDP-bound states of H- and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS disrupting RAS dimerization/nanoclustering, which in turn blocked CRAF:BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Intersectins (ITSNs) are a family of multi-domain proteins involved in regulation of diverse cellular pathways. These scaffold proteins are well known for regulating endocytosis but also play important roles in cell signaling pathways including kinase regulation and Ras activation. ITSNs participate in several human cancers, such as neuroblastomas and glioblastomas, while its downregulation is associated with lung injury. Alterations in ITSN expression have been found in neurodegenerative diseases such as Down Syndrome and Alzheimer’s disease. Binding proteins for ITSNs include endocytic regulatory factors, cytoskeleton related proteins (i.e. actin or dynamin), signaling proteins as well as herpes virus proteins. This review will summarize recent studies on ITSNs, highlighting the importance of these scaffold proteins in the aforementioned processes.

Neuroblastoma accounts for 15% of all pediatric cancer deaths. Intersectin 1(ITSN1), a scaffold protein involved in phosphoinositide 3-kinase(PI3K) signaling, regulates neuroblastoma cells independent of MYCN status. We hypothesize that by silencing ITSN1 in neuroblastoma cells, tumor growth will be decreased in an orthotopic mouse tumor model. SK-N-AS neuroblastoma cells transfected with empty vector(pSR), vectors expressing scrambled shRNA(pSCR), or shRNAs targeting ITSN1(sh#1 and sh#2) were used to create orthotopic neuroblastoma tumors in mice. Volume was monitored weekly with ultrasound. End-point was tumor volume >1000mm3. Tumor cell lysates were analyzed with anti-ITSN1 antibody by Western blot. Orthotopic tumors were created in all cell lines. Twenty-five days post injection, pSR tumor size was 917.6±247.7mm3, pSCR was 1180±159.9mm3, sh#1 was 526.3±212.8mm3, and sh#2 was 589.2±74.91mm3. sh#1-tumors and sh#2-tumors were smaller than pSCR(p=0.02), no difference between sh#1 and sh#2. Survival was superior in sh#2-tumors(p=0.02), trended towards improved survival in sh#1-tumors(p=0.09), compared to pSCR-tumors, no difference in pSR tumors. Western blot showed decreased ITSN1 expression in sh#1 and sh#2 compared to pSR and pSCR. Silencing ITSN1 in neuroblastoma cells led to decreased tumor growth in an orthotopic mouse model. Orthotopic animal models can provide insight into the role of ITSN1 pathways in neuroblastoma tumorigenesis.