Frank McCormick

Oncogenic K-Ras mutation occurs frequently in several types of cancers, including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage-independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage-independent culture conditions, we discovered the involvement of Met in K-Ras–dependent, anchorage-independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage-independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global downregulation of mRNA translation during anchorage-independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras–mediated Met expression drives “K-Ras addiction” in anchorage-independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage-independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. Cancer Res; 75(14); 2851–62. ©2015 AACR.

Significance: Lung cancer is the leading cause of cancer death worldwide. About 10% harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with EGFR inhibitors, only 5% of patients achieve tumor reduction >90%, even though all of those treated have EGFR mutations. Our study addressed this discrepant response by investigating the mechanism of innate drug resistance, i.e. resistance inherent in the tumor cells even before treatment begins and not acquired over its course. Because overcoming innate resistance will increase the primary response, our findings may provide an opportunity to develop new therapies to reduce the probability of emergent resistance to EGFR inhibitors.

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas^G12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas^G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. Significance Rat sarcoma (Ras) proteins play central roles in both normal and oncogenic signaling. Mechanisms of how Ras interacts with its effectors on the cell membrane, however, are still poorly understood, significantly hampering efforts to target this molecule in human cancer. Here we have used quantitative superresolution fluorescence microscopy in combination with carefully engineered biological systems to show that Ras dimers drive oncogenic signaling through the Raf-MAPK pathway. Our study suggests a new, dimer model of Ras-Raf signaling and the potential value of Ras dimers as a therapeutic target.

Summary: Data-driven analyses of scientific abstracts with web apps such as “abstract interactomes” provide a new visualization tool for the biomedical research community to interactively navigate a rich assembly of investigators and identify common research topics. Alternative conference formats such as “social interactomes,” with structured, albeit informal, discussions among attendees, are able to engage fellows and top investigators, facilitate the exchange of ideas, and encourage data sharing and future collaborations. Cancer Discov; 4(11); 1265–8. ©2014 AACR.

RNAi is a powerful tool for target identification and can lead to novel therapies for pharmacologically intractable targets such as KRAS. RNAi therapy must combine potent siRNA payloads with reliable in vivo delivery for efficient target inhibition. We used a functional “Sensor” assay to establish a library of potent siRNAs against RAS pathway genes and to show that they efficiently suppress their targets at low dose. This reduces off-target effects and enables combination gene knockdown. We administered Sensor siRNAs in vitro and in vivo and validated the delivery of KRAS siRNA alone and siRNA targeting the complete RAF effector node (A/B/CRAF) as promising strategies to treat KRAS-mutant colorectal cancer. We further demonstrate that improved therapeutic efficacy is achieved by formulating siRNA payloads that combine both single-gene siRNA and node-targeted siRNAs (KRAS + PIK3CA/B). The customizable nature of Sensor siRNA payloads offers a universal platform for the combination target identification and development of RNAi therapeutics. Significance: To advance RNAi therapy for KRAS-mutant cancer, we developed a validated siRNA library against RAS pathway genes that enables combination gene silencing. Using an in vivo model for real-time siRNA delivery tracking, we show that siRNA-mediated inhibition of KRAS as well as RAF or PI3K combinations can impair KRAS-mutant colorectal cancer in xenograft models. Cancer Discov; 4(10); 1182–97. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1103

Mutationally activated BRAF is expressed in melanoma, glioblastoma, thyroid, lung and colon cancers and in a subset of haematological malignancies. The most common BRAF mutation leads to the substitution of a glutamic acid for valine at amino acid 600 (V600E) in the kinase domain of the protein. This substitution mimics phosphorylation of the activation loop, thereby inducing constitutive BRAF protein kinase activity. Point mutations in the related ARAF and CRAF protein kinases, although very rare, have been reported as oncogenic drivers in some human cancers. In addition to point mutation, gene fusion events are reported to activate BRAF and CRAF. Numerous non-V600E alterations in BRAF have been reported in cancer and in a rare developmental disorder. Many of these promote kinase activity by relieving autoinhibitory mechanisms or promote activation of other RAF isoforms in a RAS-dependent manner. ATP-competitive BRAF kinase inhibitors are currently under investigation for the treatment of BRAF-mutated cancers. However, to date, efficacy is limited to a subset of melanomas owing to primary or adaptive resistance mechanisms in colorectal and thyroid cancers that reactivate signalling downstream of receptor tyrosine kinases. In clinical trials of BRAF-mutated melanoma, BRAF inhibitors tend to induce high rates of response that show transient durability due to the onset of drug-resistant disease. Acquired resistance to BRAF-V600E inhibitors is strikingly complex but frequently involves reactivation of MEK–ERK MAP kinase signalling. Drug resistance due to overexpression of oncogenic BRAF-V600E leads to 'oncogene overdose' following cessation of drug administration — a phenomenon that could be clinically exploitable to forestall the onset of drug resistance. The efficacy of RAF inhibitors in tumours with other RAF mutations is mostly unknown, although preclinical studies indicate varied responses that are inhibitor-specific and depend on the biochemical mechanism of oncogene activation.

Ras genes are frequently activated in cancer. Attempts to develop drugs that target mutant Ras proteins have, so far, been unsuccessful. Tumors bearing these mutations, therefore, remain among the most difficult to treat. Most efforts to block activated Ras have focused on pathways downstream. Drugs that inhibit Raf kinase have shown clinical benefit in the treatment of malignant melanoma. However, these drugs have failed to show clinical benefit in Ras mutant tumors. It remains unclear to what extent Ras depends on Raf kinase for transforming activity, even though Raf proteins bind directly to Ras and are certainly major effectors of Ras action in normal cells and in development. Furthermore, Raf kinase inhibitors can lead to paradoxical activation of the MAPK pathway. MEK inhibitors block the Ras-MAPK pathway, but often activate the PI3’-kinase, and have shown little clinical benefit as single agents. This activation is mediated by EGF-R and other receptor tyrosine kinases through relief of a negative feedback loop from ERK. Drug combinations that target multiple points within the Ras signaling network are likely to be necessary to achieve substantial clinical benefit. Other effectors may also contribute to Ras signaling and provide a source of targets. In addition, unbiased screens for genes necessary for Ras transformation have revealed new potential targets and have added to our understanding of Ras cancer biology.

Viruses bind to specific cellular receptors in order to infect their hosts. The specific receptors a virus uses are important factors in determining host range, cellular tropism, and pathogenesis. For adenovirus, the existing model of entry requires two receptor interactions. First, the viral fiber protein binds Coxsackie and Adenovirus Receptor (CAR), its primary cellular receptor, which docks the virus to the cell surface. Next, viral penton base engages cellular integrins, coreceptors thought to be required exclusively for internalization and not contributing to binding. However, a number of studies reporting data which conflicts with this simple model have been published. These observations have led us to question the proposed two-step model for adenovirus infection.