Development of a high-throughput TR-FRET screening assay for a fast-cycling KRAS mutant.

Mutations in the small GTPase protein KRAS are one of the leading drivers of cancers including lung, pancreatic, and colorectal, as well as a group of developmental disorders termed "Rasopathies". Recent breakthroughs in the development of mutant-specific KRAS inhibitors include the FDA approved drug Lumakras (Sotorasib, AMG510) for KRAS G12C-mutated non-small cell lung cancer (NSCLC), and MRTX1133, a promising clinical candidate for the treatment of KRAS G12D-mutated cancers. However, there are currently no FDA approved inhibitors that target KRAS mutations occurring at non-codon 12 positions.

A randomized controlled trial of glycopyrrolate administered by metered dose inhaler in patients with uncontrolled asthma despite ICS/LABA treatment.

Objective: To evaluate the efficacy and safety of three doses of glycopyrrolate metered dose inhaler (GP MDI) in patients with uncontrolled asthma despite treatment with inhaled corticosteroid/long-acting β-agonists (ICS/LABA) with or without tiotropium, to characterize the benefit of triple therapy.

Method: This phase II/III, double-blind study randomized patients to 24 weeks' treatment with twice-daily GP MDI 36 µg, 18 µg, 9 µg, or placebo MDI (all delivered Aerosphere inhalers), or once-daily open-label tiotropium 2.5 µg.

Dose-Ranging and Cumulative Dose Studies of Albuterol Sulfate MDI in Co-Suspension Delivery™ Technology (AS MDI; PT007) in Patients with Asthma: the ASPEN and ANTORA Trials.

Background And Objectives: Co-suspension Delivery™ Technology has been developed for the administration of albuterol sulfate pressurised inhalation suspension via metered-dose inhaler (AS MDI, PT007). We assessed the efficacy and safety of AS MDI versus Proventil in order to determine the optimal dose of AS MDI to take to Phase III clinical trials.

Methods: ASPEN (NCT03371459) and ANTORA (NCT03364608) were Phase II, randomised, crossover, multicentre studies of AS MDI versus Proventil in patients with persistent asthma.

Reduced All-Cause Mortality in the ETHOS Trial of Budesonide/Glycopyrrolate/Formoterol for Chronic Obstructive Pulmonary Disease. A Randomized, Double-Blind, Multicenter, Parallel-Group Study.

In the phase III, 52-week ETHOS (Efficacy and Safety of Triple Therapy in Obstructive Lung Disease) trial in chronic obstructive pulmonary disease (COPD) (NCT02465567), triple therapy with budesonide/glycopyrrolate/formoterol fumarate (BGF) significantly reduced all-cause mortality compared with glycopyrrolate/formoterol fumarate (GFF). However, 384 of 8,509 patients were missing vital status at Week 52 in the original analyses. To assess the robustness of the ETHOS mortality findings after additional data retrieval for patients missing Week 52 vital status in the original analyses.

Discernment between candidate mechanisms for KRAS G13D colorectal cancer sensitivity to EGFR inhibitors.

Phase three clinical trial evidence suggests that colorectal cancers with the KRAS G13D mutation may benefit from EGFR inhibitors, like cetuximab, in contrast to the other most common KRAS mutations. A mechanism to explain why this mutation behaves differently from other KRAS mutations had long been lacking. Two recent studies have reproduced KRAS G13D specific sensitivity to cetuximab in cellular models, and both have implicated the tumor suppressor NF1 as a critical variable in determining sensitivity and resistance.

Efficacy and safety of two doses of budesonide/formoterol fumarate metered dose inhaler in COPD.

Inhaled corticosteroid/long-acting β-agonist combination therapy is a recommended treatment option for patients with chronic obstructive pulmonary disease (COPD) and increased exacerbation risk, particularly those with elevated blood eosinophil levels. SOPHOS (NCT02727660) evaluated the efficacy and safety of two doses of budesonide/formoterol fumarate dihydrate metered dose inhaler (BFF MDI) formoterol fumarate dihydrate (FF) MDI, each delivered using co-suspension delivery technology, in patients with moderate-to-very severe COPD and a history of exacerbations. In this phase 3, randomised, double-blind, parallel-group, 12-52-week, variable length study, patients received twice-daily BFF MDI 320/10 µg or 160/10 µg, or FF MDI 10 µg.

Barriers to Gender-Affirming Care for Transgender and Gender Nonconforming Individuals.

Gender-affirming care, including hormone therapy, "top" (e.g., chest reconstruction surgery) and "bottom" (e.

A Qualitative Study Examining Young Adults' Experiences of Disclosure and Nondisclosure of LGBTQ Identity to Health Care Providers.

Shifting cultural attitudes and legislation have increased focus on the health care needs of lesbian, gay, bisexual, transgender, and queer (LGBTQ) patients. However, patient nondisclosure of LGBTQ identity creates a barrier to accessing care. This qualitative study examined a diverse sample of LGBTQ young adults and their experiences of disclosure and nondisclosure to medical providers.

Rho GTPase Transcriptome Analysis Reveals Oncogenic Roles for Rho GTPase-Activating Proteins in Basal-like Breast Cancers.

The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis.

RAS isoforms and mutations in cancer at a glance.

RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application.

Substrate trapping proteomics reveals targets of the βTrCP2/FBXW11 ubiquitin ligase.

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex.

P-Rex1 directly activates RhoG to regulate GPCR-driven Rac signalling and actin polarity in neutrophils.

G-protein-coupled receptors (GPCRs) regulate the organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine-nucleotide-exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and phosphoinositide 3-kinase (PI3K) in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils.

Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease.

The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs).

The RhoGEF TEM4 Regulates Endothelial Cell Migration by Suppressing Actomyosin Contractility.

Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells.

Identification of a novel actin-binding domain within the Rho guanine nucleotide exchange factor TEM4.

Spatio-temporal activation of Rho GTPases is essential for their function in a variety of biological processes and is achieved in part by regulating the localization of their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). In this study, we provide the first characterization of the full-length protein encoded by RhoGEF TEM4 and delineate its domain structure, catalytic activity, and subcellular localization. First, we determined that TEM4 can stimulate guanine nucleotide exchange on RhoA and the related RhoB and RhoC isoforms.

The RalB small GTPase mediates formation of invadopodia through a GTPase-activating protein-independent function of the RalBP1/RLIP76 effector.

Our recent studies implicated key and distinct roles for the highly related RalA and RalB small GTPases (82% sequence identity) in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis and invasive and metastatic growth, respectively. How RalB may promote PDAC invasion and metastasis has not been determined. In light of known Ral effector functions in regulation of actin organization and secretion, we addressed a possible role for RalB in formation of invadopodia, actin-rich membrane protrusions that contribute to tissue invasion and matrix remodeling.

Oncogenic activity of Ect2 is regulated through protein kinase C iota-mediated phosphorylation.

The Rho GTPase guanine nucleotide exchange factor Ect2 is genetically and biochemically linked to the PKCι oncogene in non-small cell lung cancer (NSCLC). Ect2 is overexpressed and mislocalized to the cytoplasm of NSCLC cells where it binds the oncogenic PKCι-Par6 complex, leading to activation of the Rac1 small GTPase. Here, we identify a previously uncharacterized phosphorylation site on Ect2, threonine 328, that serves to regulate the oncogenic activity of Ect2 in NSCLC cells.

Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?

There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase-activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state.

Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins.

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. Although the exchange potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases.

Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression.

Autoinhibition of the Rho guanine nucleotide exchange factor ASEF is relieved by interaction with the APC tumor suppressor. Here we show that binding of the armadillo repeats of APC to a 'core APC-binding' (CAB) motif within ASEF, or truncation of the SH3 domain of ASEF, relieves autoinhibition, allowing the specific activation of CDC42. Structural determination of autoinhibited ASEF reveals that the SH3 domain forms an extensive interface with the catalytic DH and PH domains to obstruct binding and activation of CDC42, and the CAB motif is positioned adjacent to the SH3 domain to facilitate activation by APC.

Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif.

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood.

The Dbs PH domain contributes independently to membrane targeting and regulation of guanine nucleotide-exchange activity.

Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent membrane targeting modules.

Signaling interplay in Ras superfamily function.

Ras proteins function as signaling hubs that are activated by convergent signaling pathways initiated by extracellular stimuli. Activated Ras in turn regulates a diversity of downstream cytoplasmic signaling cascades. Ras proteins are founding members of a large superfamily of small GTPases that have significant sequence and biochemical similarities.

Larger than Dbl: new structural insights into RhoA activation.

Dbl homology (DH) domains are almost always followed immediately by pleckstrin homology (PH) domains in Dbl family proteins, and these DH-PH fragments directly activate GDP-bound Rho GTPases by catalyzing the exchange of GDP for GTP. New crystal structures of the DH-PH domains from leukemia-associated Rho guanine nucleotide exchange factor (RhoGEF) and PDZ-RhoGEF bound to RhoA reveal how DH-PH domains cooperate to specifically activate Rho GTPases.