RASopathy mutations provide functional insight into the BRAF cysteine-rich domain and reveal the importance of autoinhibition in BRAF regulation.

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.

Regulation of GTPase function by autophosphorylation.

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.

Distinct Binding Preferences between Ras and Raf Family Members and the Impact on Oncogenic Ras Signaling.

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.

Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Inhibition of Ras/Raf/MEK/ERK Pathway Signaling by a Stress-Induced Phospho-Regulatory Circuit.

Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.

Effect of Neonatal Abstinence Syndrome Treatment Status and Maternal Depressive Symptomatology on Maternal Reports of Infant Behaviors.

OBJECTIVE: The objective of this study is to investigate the effects of maternal perinatal depression symptoms and infant treatment status for neonatal abstinence syndrome (NAS) on maternal perceptions of infant regulatory behavior at 6 weeks of age. METHODS: Mothers and their infants (N = 106; 53 dyads) were recruited from a rural, White cohort in Northeast Maine. Mothers in medication-assisted treatment (methadone) and their infants (n = 35 dyads) were divided based on the infant's NAS pharmacological treatment (n = 20, NAS+ group; n = 15, NAS- group) and compared with a demographically similar, nonexposed comparison group (n = 18 dyads; COMP group). At 6 weeks postpartum, mothers reported their depression symptoms Beck Depression Inventory-2nd Edition) and infant regulatory behaviors [Mother and Baby Scales (MABS)]. Infant neurobehavior was assessed during the same visit using the Neonatal Network Neurobehavioral Scale (NNNS). RESULTS: Mothers in the NAS+ group showed significantly higher depression scores than the COMP group (p < .05) while the NAS- group did not. Across the sample, mothers with higher depression scores reported higher infant "unsettled-irregularity" MABS scores, regardless of group status. Agreement between maternal reports of infant regulatory behaviors and observer-assessed NNNS summary scares was poor in both the NAS+ and COMP groups. CONCLUSIONS: Postpartum women in opioid recovery with infants requiring pharmacological intervention for NAS are more at risk for depression which may adversely influence their perceptions of their infants' regulatory profiles. Unique, targeted attachment interventions may be needed for this population.

The association between adverse childhood experiences and treatment response for adults with alcohol and other drug use disorders.

BACKGROUND AND OBJECTIVES: Adverse events during childhood increase the risk for the development of substance use disorders (SUDs). This study examined the association between adverse childhood experiences (ACEs) and SUD treatment response. METHODS: This cohort analysis included data from longitudinal clinical assessments extracted from the records of 438 consenting individuals undergoing SUD treatment (63% male; 88.8% White). Mixed effects models evaluated the relationship between scores on the ACE questionnaire and indicators of treatment response (i.e., alcohol and drug abstinence self-efficacy; symptoms of depression, anxiety, and posttraumatic stress disorder) for individuals with alcohol-related (n = 332) and other drug-related (n = 275) diagnoses, with some participants included in both groups. RESULTS: Treatment response varied as a function of ACEs, with the magnitude of differences varying across time in treatment. Relative to those with no ACE history, those who experienced ≥2 ACEs reported worse depression, anxiety, PTSD symptoms, and alcohol/drug abstinence self-efficacy at baseline, with many differences remaining at the 30-day assessment. All differences abated by discharge, with the exception of PTSD symptoms among those in the drug use group with a history of ≥4 ACEs. Male gender and older age were generally associated with lower symptomology and higher abstinence self-efficacy. DISCUSSION AND CONCLUSIONS: Assessing ACE history early in SUD treatment may improve treatment planning and prognosis. Future studies should evaluate the role of trauma-informed programming and individual interventions to improve treatment response. SCIENTIFIC SIGNIFICANCE: This study demonstrates the association between adverse childhood experiences and symptom severity among patients across participation in SUD treatment.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

M-Ras/Shoc2 signaling modulates E-cadherin turnover and cell-cell adhesion during collective cell migration.

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.

COVID-19 Reflections: We Are Not OK: Safety Net Primary Care Access in a Non-Expansion State Amid COVID-19.

In North Carolina, rural health care-especially the primary care safety net-is a remarkable but under-resourced vital support system. COVID-19 stressed that already precarious system. While the acute COVID-19 crisis may be receding, we are concerned about the long-term effects of the pandemic on both individuals and the rural primary care safety net.

Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling.

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

Advancing RAS/RASopathy therapies: An NCI-sponsored intramural and extramural collaboration for the study of RASopathies.

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.

Regulatory and health technology assessment advice on postlicensing and postlaunch evidence generation is a foundation for lifecycle data collection for medicines.

The understanding of the benefit risk profile, and relative effectiveness of a new medicinal product, are initially established in a circumscribed patient population through clinical trials. There may be uncertainties associated with the new medicinal product that cannot be, or do not need to be resolved before launch. Postlicensing or postlaunch evidence generation (PLEG) is a term for evidence generated after the licensure or launch of a medicinal product to address these remaining uncertainties. PLEG is thus part of the continuum of evidence development for a medicinal product, complementing earlier evidence, facilitating further elucidation of a product's benefit/risk profile, value proposition, and/or exploring broader aspects of disease management and provision of healthcare. PLEG plays a role in regulatory decision making, not only in the European Union but also in other jurisdictions including the USA and Japan. PLEG is also relevant for downstream decision-making by health technology assessment bodies and payers. PLEG comprises studies of different designs, based on data collected in observational or experimental settings. Experience to date in the European Union has indicated a need for improvements in PLEG. Improvements in design and research efficiency of PLEG could be addressed through more systematic pursuance of Scientific Advice on PLEG with single or multiple decision makers. To date, limited information has been available on the rationale, process or timing for seeking PLEG advice from regulators or health technology assessment bodies. This article sets out to address these issues and to encourage further uptake of PLEG advice.

Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge Theonella swinhoei That Inhibit Ras/Raf Interaction.

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.

LAT-independent Erk activation via Bam32-PLC-γ1-Pak1 complexes: GTPase-independent Pak1 activation.

In T cells, the adaptor Bam32 is coupled to Erk activation downstream of the TCR by an unknown mechanism. We characterized in Jurkat cells and primary T lymphocytes a pathway dependent on Bam32-PLC-γ1-Pak1 complexes, in which Pak1 kinase activates Raf-1 and Mek-1, both upstream of Erk. In the Bam32-PLC-γ1-Pak1 complex, catalytically inactive PLC-γ1 is used as a scaffold linking Bam32 to Pak1. PLC-γ1(C-SH2) directly binds S141 of Bam32, preventing LAT-mediated activation of Ras by PLC-γ1. The Bam32-PLC-γ1 interaction enhances the binding of the SH3 domain of the phospholipase with Pak1. The PLC-γ1(SH3)-Pak1 interaction activates Pak1 independently of the small GTPases Rac1/Cdc42, previously described as being the only activators of Pak1 in T cells. Direct binding of the SH3 domain of PLC-γ1 to Pak1 dissociates inactive Pak1 homodimers, a mechanism required for Pak1 activation. We have thus uncovered a LAT/Ras-independent, Bam32-nucleated pathway that activates Erk signaling in T cells.

Complexity in the signaling network: insights from the use of targeted inhibitors in cancer therapy.

Cancer often arises when normal cellular growth goes awry due to defects in critical signal transduction pathways. A growing number of inhibitors that target specific components of these pathways are in clinical use, but the success of these agents has been limited by the resistance to inhibitor therapy that ultimately develops. Studies have now shown that cancer cells respond to chronic drug treatment by adapting their signaling circuitry, taking advantage of pathway redundancy and routes of feedback and cross-talk to maintain their function. This review focuses on the compensatory signaling mechanisms highlighted by the use of targeted inhibitors in cancer therapy.

Targeting the Raf kinases in human cancer: the Raf dimer dilemma.

The Raf protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the Ras GTPases and as the initiating kinases in the ERK cascade. In human cancer, Raf activity is frequently dysregulated due to mutations in the Raf family member B-Raf or to alterations in upstream Raf regulators, including Ras and receptor tyrosine kinases. First-generation Raf inhibitors, such as vemurafenib and dabrafenib, have yielded dramatic responses in malignant melanomas containing B-Raf mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In particular, issues related to the dimerisation of the Raf kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. Here, we will review the importance of Raf dimerisation in cell signalling as well as its effects on Raf inhibitor therapy, and we will present the new strategies that are being pursued to overcome the 'Raf Dimer Dilemma'.

Macrophilones from the Marine Hydroid Macrorhynchia philippina Can Inhibit ERK Cascade Signaling.

Six new macrophilone-type pyrroloiminoquines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton-deficient and heteroatom-rich structures of macrophilones B-G (2-7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1-7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macrophilones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal transduction pathway.

The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling.

Protein scaffolds play an important role in signal transduction, regulating the localization of signaling components and mediating key protein interactions. Here, we report that the major binding partners of the Connector Enhancer of KSR 1 (CNK1) scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors, and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream from insulin and insulin-like growth factor 1 (IGF-1) receptors. We identified a domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins, and found that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and rescue experiments, we found that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) to generate a PIP(2)-rich microenvironment that is critical for the membrane recruitment of insulin receptor substrate 1 (IRS1) and signal transmission to the PI3K/AKT cascade. These findings identify CNK1 as a new positive regulator of insulin signaling.

Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma.

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.