Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: it is rare in lung and colorectal cancers (~1%), yet relatively common (~20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRAS G12R is functionally distinct from the more common KRAS G12D or KRAS G12V mutant proteins (KRAS G12D/V ). We found that KRAS G12D/V but not KRAS G12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRAS G12D/V but not KRAS G12R -mutant PDAC. Surprisingly, we found that KRAS G12R is defective for interaction with a key effector, p110α phosphoinositide 3-kinase (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRAS G12R -mutant PDAC. Finally, we determined that KRAS G12R -mutant PDAC displayed a distinct drug sensitivity profile compared with KRAS G12D -mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy.Hobbs et al.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOA Y42C , the most common RHOA mutation in DGC, is a gain-offunction oncogenic mutant, and that expression of RHOA Y42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOA Y42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and β-catenin. RHOA Y42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE:The functional signifi cance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOA Y42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and defi ne how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.

Concomitant chemoradiation therapy, should be the treatment of choice for carcinoma of the cervical esophagus.

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Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry–based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62. Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.

Rho GTPases play central roles in a wide variety of cellular processes, including cytoskeletal dynamics, cell adhesion and cell polarity. RhoU and RhoV are Rho GTPases that have some atypical properties compared with classical Rho family members, such as the presence of N-and C-terminal extension regions, unusual GDP/GTP cycling and post-translational modification by palmitoylation but not prenylation. Their activity and localization is regulated by the N-terminal and C-terminal regions, and so far no GEFs or GAPs have been identified for them. Similar to Rac and Cdc42, they interact with PAK serine/threonine kinases, and in the case of PAK4, this interaction leads to RhoU protein stabilization. In cells, RhoU and RhoV alter cell shape and cell adhesion, which probably underlies some of the phenotypes reported for these proteins in vivo, for example in heart development and epithelial morphogenesis. However, the molecular basis for these functions of RhoU and RhoV remains to be characterized.

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