ABSTRACT KRAS is the most frequently mutated oncogene in human cancer. In multiple types of cancer, a missense mutation at codon 12 substitutes a glycine for a cysteine, causing hyperactivation of the GTPase and enhanced MAPK signalling. Recent drug discovery efforts culminating from work during the past decade have resulted in two FDA-approved inhibitors, sotorasib and adagrasib, which target the KRASG12C mutant allele. Ongoing medicinal chemistry efforts across academia and industry have continued developing more potent and efficacious KRASG12C inhibitors. One agent in late-stage clinical trials, divarasib, has demonstrated robust overall response rates, in some cases greater than currently approved agents. Divarasib also exhibits enhanced covalent target engagement in vitro and significant specificity for KRASG12C, yet the structural details of its binding have not been published. Here we report a high-resolution crystal structure of cysteine-light KRAS-4BG12C in complex with divarasib. Though it binds in the same allosteric pocket as sotorasib and adagrasib, the switch-II loop in each crystal structure takes on a distinct conformation differing as much as 5.6 Å between the Cα atom of residue 65 with sotorasib. Additionally, we highlight structural features of the drug complex that may guide future medicinal chemistry efforts targeting various KRAS alleles.

ABSTRACT Morphogenesis plays a pivotal role in the infection process of Trichophyton rubrum, a primary aetiological agent of dermatophytosis that inhabits superficial human tissues. T. rubrum proliferates by extending filamentous structures, or hyphae, which are composed of highly polarized cells. In response to environmental stimuli, T. rubrum also produces asexual spores called microconidia, consisting of individual cells. Although these dynamic morphological changes are critical for T. rubrum proliferation and environmental adaptation, the molecular mechanisms underlying these processes remain poorly understood. In previous research, we demonstrated that repressing Cdc24, a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42, disrupts fungal cell polarity and impairs hyphal formation in T. rubrum. In this study, we show that Rac deficiency in the Δrac strain minimally affects hyphal formation, as indicated by the cell polarity index (the ratio of a cell’s long to short diameter in hyphae). However, simultaneous Rac deficiency and Cdc42 repression in the Δrac/P ctr4 cdc42 strain significantly disrupted cell polarity, suggesting that Rac and Cdc42 perform overlapping functions in hyphal morphogenesis. Interestingly, Rac deficiency inhibited microconidia formation, whereas cdc42 repression had no detectable impact. Furthermore, adding cysteine, a radical scavenger abundant in keratins, to the growth medium reduced microconidia production in the wild-type strain but not in the Δrac strain. These findings suggest that cysteine in host tissues inhibits Rac-mediated microconidia formation. Overall, this study identifies Rac as a key regulator of T. rubrum morphogenesis, with specific roles in both hyphal development and microconidia formation.

RAC1 is a small 21 kDa RHO GTPase that plays a pivotal role in regulating actin cytoskeletal dynamics and cell growth. Alterations in the activity of RAC1 are implicated in a range of diseases, including cancer. Increased RAC1 activity, due to overexpression and/or activating mutations, drives transcriptional upregulation, reactive oxygen species production, mesenchymal-to-epithelial transition, membrane ruffling, and uncontrolled cell proliferation, which are hallmarks of an oncogenic phenotype. While RAC1-activating mutations alone do not appear sufficient to transform cells, their combination with other common mutations, such as BRAF, NRAS, or NF1, have been linked to drug resistance and significantly worsen patient prognosis and hinder treatment responses. The precise mechanisms underlying drug resistance, and the regulation of RAC1 splicing remain poorly understood. RAC1 is a challenging therapeutic target due to its ubiquitous presence and essential cellular functions. To date, there are no established standard treatments for cancers that harbour an additional RAC1 mutation or for RAC1-mediated drug resistance. Current experimental strategies aim to target RAC1 localization, its activators (e.g. guanine nucleotide exchange factors) and downstream effectors. Regulating RAC1 expression by targeting epigenetic regulators, and direct targeting of RAC1 itself, may also be possible in the near future.

ABSTRACT RhoA, Rac1 and CDC42 are small G proteins that play a crucial role in regulating various cellular processes, such as the formation of actin cytoskeleton, cell shape and cell migration. Our recent results suggest that MLL is responsible for maintaining the balance of these small Rho GTPases. MLL depletion affects the stability of Rho GTPases, leading to a decrease in their protein levels and loss of activity. These changes manifest in the form of abnormal cell shape and disrupted actin cytoskeleton, resulting in reduced cell spreading and migration. Interestingly, their chaperone protein RhoGDI1 but not the Rho GTPases, is under the direct transcriptional regulation of MLL. Here, we comment on the possible implications of these observations on the signalling by Rho GTPases protein network.

ABSTRACT The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.

ABSTRACT Next year marks one-quarter of a century since the discovery of the so-called COPI-independent pathway, which operates between the Golgi apparatus and the endoplasmic reticulum (ER) in eukaryotic cells. Unlike almost all other intracellular trafficking pathways, this pathway is not regulated by the physical accumulation of multisubunit proteinaceous coat molecules, but instead by the small GTPase Rab6. What also sets it apart from other pathways is that the transport carriers themselves often take the form of tubules, rather than conventional vesicles. In this review, we assess the relevant literature that has accumulated to date, in an attempt to provide a concerted description of how this pathway is regulated. We discuss the possible cargo molecules that are carried in this pathway, and the likely mechanism of Rab6 tubule biogenesis, including how the cargo itself may play a critical role. We also provide perspective surrounding the various molecular motors of the kinesin, myosin and dynein families that have been implicated in driving Rab6-coated tubular membranes long distances through the cell prior to delivering their cargo to the ER. Finally, we also raise several important questions that require resolution, if we are to ultimately provide a comprehensive molecular description of how the COPI-independent pathway is controlled.

ABSTRACT Activated Cdc42-associated kinase (ACK), a non-receptor tyrosine kinase, is an effector for the small GTPase Cdc42. ACK is emerging as an important component of the cancer landscape and thus, a promising target for the treatment of many malignancies. ACK is also being increasingly recognized as a potentially influential player in the regulation of protein homoeostasis. The delicate equilibrium between protein synthesis and protein degradation is crucial for healthy cell function and dysregulation of protein homoeostasis is a common occurrence in human disease. Here, we review the molecular mechanisms by which ACK regulates the stability of diverse cellular proteins (e.g. EGFR, p27, p53, p85 isoforms and RhoGDI-3), some of which rely on the kinase activity of ACK while others, interestingly, do not. Ultimately, further research will be required to bridge our knowledge gaps and determine if ACK regulates the stability of further cellular proteins but collectively, such mechanistic interrogation would contribute to determining whether ACK is a promising target for anti-cancer therapy. In therapeutics, proteasome inhibitors are an efficacious but problematic class of drugs. Targeting other modulators of proteostasis, like ACK, could open novel avenues for intervention.

ABSTRACT Rho proteins are part of the Ras superfamily, which function to modulate cytoskeletal dynamics including cell adhesion and motility. Recently, an activating mutation in Cdc42, a Rho family GTPase, was found in a patient sample of melanoma. Previously, our work had shown the PI3K was important downstream of mutationally active Cdc42. Our present study sought to determine whether PI3K was a crucial downstream partner for Cdc42 in a melanoma cells line with a BRAF mutation, which is the most common mutation in cutaneous melanoma. In this work we were able to show that Cdc42 contributes to proliferation, anchorage-independent growth, cell motility and invasion. Treatment with a pan-PI3K inhibitor was able to effectively ameliorate all these cancer phenotypes. These data suggest that PI3K may be an important target downstream of Cdc42 in melanoma.

ABSTRACT P-Rex1 and P-Rex2 are guanine-nucleotide exchange factors (GEFs) that activate Rac small GTPases in response to the stimulation of G protein-coupled receptors and phosphoinositide 3-kinase. P-Rex Rac-GEFs regulate the morphology, adhesion and migration of various cell types, as well as reactive oxygen species production and cell cycle progression. P-Rex Rac-GEFs also have pathogenic roles in the initiation, progression or metastasis of several types of cancer. With one exception, all P-Rex functions are known or assumed to be mediated through their catalytic Rac-GEF activity. Thus, inhibitors of P-Rex Rac-GEF activity would be valuable research tools. We have generated a panel of small-molecule P-Rex inhibitors that target the interface between the catalytic DH domain of P-Rex Rac-GEFs and Rac. Our best-characterized compound, P-Rex inhibitor 1 (PREX-in1), blocks the Rac-GEF activity of full-length P-Rex1 and P-Rex2, and of their isolated catalytic domains, in vitro at low-micromolar concentration, without affecting the activities of several other Rho-GEFs. PREX-in1 blocks the P-Rex1 dependent spreading of PDGF-stimulated endothelial cells and the production of reactive oxygen species in fMLP-stimulated mouse neutrophils. Structure-function analysis revealed critical structural elements of PREX-in1, allowing us to develop derivatives with increased efficacy, the best with an IC50 of 2 µM. In summary, we have developed PREX-in1 and derivative small-molecule compounds that will be useful laboratory research tools for the study of P-Rex function. These compounds may also be a good starting point for the future development of more sophisticated drug-like inhibitors aimed at targeting P-Rex Rac-GEFs in cancer.

ABSTRACT The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.