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Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms. Editor's summary: Mutations in the GTPase RHOA are common in a type of aggressive, hereditary gastric cancer caused by loss of CDH1, which encodes a cell adhesion receptor. Schaefer et al. found that the two most common RHOA mutations drive the development and progression of this cancer in distinct ways (see also the Focus by Benton and Chernoff). Both the Y42C and L57V mutations conferred a similar gain-of-function phenotype that promoted the interaction of RHOA with the actin cytoskeleton and activation of the transcriptional coactivator YAP1 to promote malignant transformation, growth, and cell motility in Cdh1-deficient mouse-derived gastric organoids. However, distinct from Y42C, L57V engaged the kinases PAK1 and IGF1R, and inhibition of either kinase suppressed the growth of organoids. These results reveal potential drug targets in the treatment of gastric cancer. —Leslie K. Ferrarelli [ABSTRACT FROM AUTHOR]

Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192). Editor's summary: Glioblastoma is the most aggressive and common primary brain tumor in adults, and chloroethyl-cyclohexyl-nitrosourea (CCNU), which is given at disease progression, often fails. To overcome this, Look et al. previously found that combination of CCNU with the L19 antibody fused to tumor necrosis factor or L19TNF was able to prolong survival in glioma mouse models. Here, the authors demonstrated that this combination in mice induced a T cell–dependent antitumor immune response, and a clinical trial was initiated where three out of five patients have shown objective responses. This supports further evaluation of the combination of L19TNF and CCNU for patients with glioblastoma. —Dorothy Hallberg [ABSTRACT FROM AUTHOR]

Escherichia coli are part of the normal intestinal microbiome, but some enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) strains can cause potentially life-threatening gastroenteritis. Virulence factors underlying the ability of EHEC and EPEC to cause disease include those encoded in the locus of the enterocyte effacement (LEE) pathogenicity island. Here, we demonstrated that EsrL, a small RNA present in many E. coli strains, promoted pathogenicity, adhesion, and biofilm formation in EHEC and EPEC. PhoB, the response regulator of the two-component system that controls cellular responses to phosphate, directly repressed esrL expression under low-phosphate conditions. A phosphate-rich environment, similar to that of the human intestine, relieved PhoB-mediated repression of esrL. EsrL interacted with and stabilized the LEE-encoded regulator (ler) transcript, which encodes a transcription factor for LEE genes, leading to increased bacterial adhesion to cultured cells and colonization of the rabbit colon. EsrL also bound to and stabilized the fimC transcript, which encodes a chaperone that is required for the assembly of type 1 pili, resulting in enhanced cell adhesion in pathogenic E. coli and enhanced biofilm formation in pathogenic and nonpathogenic E. coli. Our findings demonstrate that EsrL stimulates the expression of virulence genes in both EHEC and EPEC under phosphate-rich conditions, thus promoting the pathogenicity of EHEC and EPEC in the nutrient-rich gut environment. Phosphate-stimulated virulence: To cause disease, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) must first adhere to intestinal cells (enterocytes). Adhesion requires the expression of genes in the LEE pathogenicity island driven by the master regulator Ler. Jia et al. identified a small RNA, EsrL, that promoted EHEC and EPEC adhesion to cultured human cells and EHEC colonization of the rabbit colon. Under high-phosphate conditions similar to those in the gut, EsrL expression was derepressed and EsrL bound to and stabilized the ler transcript. Stabilization of another transcript by EsrL ultimately enhanced the capacity of EPEC and nonpathogenic E. coli to form biofilms, which protects the bacteria from antibiotics and host defenses. Stabilization of both transcripts likely contributes to E. coli virulence and biofilm formation in the high-phosphate environment of the gut.–AMV [ABSTRACT FROM AUTHOR]

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR-null (BVR−/−) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N-methyl-d-aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling. Scaffolding adhesion signaling for learning: Biliverdin reductase (BVR) and its product bilirubin have critical functions in the liver and antioxidant, neuroprotective functions in the brain. Here, Vasavda et al. found an additional synaptic role for BVR that was independent of either bilirubin or biliverdin. The authors found that BVR physically linked focal adhesion kinases to an effector kinase at synapses in the mouse hippocampus. This molecular bridge enabled signaling between these kinases and subsequently to NMDA receptors that mediate changes in synaptic strength in response to changing inputs—a cellular process called synaptic plasticity that underlies learning. Mice lacking BVR had impaired focal adhesion signaling and showed performance deficits on various cognitive tests. This mechanism did not require the catalytic function of BVR, thus revealing a scaffolding function for the enzyme in the brain. [ABSTRACT FROM AUTHOR]