Ral Activation Research Articles

The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer.

Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

Obesity causes mitochondrial fragmentation and dysfunction in white adipocytes due to RalA activation

Mitochondrial dysfunction is a characteristic trait of human and rodent obesity, insulin resistance and fatty liver disease. Here we show that high-fat diet (HFD) feeding causes mitochondrial fragmentation in inguinal white adipocytes from male mice, leading to reduced oxidative capacity by a process dependent on the small GTPase RalA. RalA expression and activity are increased in white adipocytes after HFD. Targeted deletion of RalA in white adipocytes prevents fragmentation of mitochondria and diminishes HFD-induced weight gain by increasing fatty acid oxidation. Mechanistically, RalA increases fission in adipocytes by reversing the inhibitory Ser637 phosphorylation of the fission protein Drp1, leading to more mitochondrial fragmentation. Adipose tissue expression of the human homolog of Drp1, DNM1L, is positively correlated with obesity and insulin resistance. Thus, chronic activation of RalA plays a key role in repressing energy expenditure in obese adipose tissue by shifting the balance of mitochondrial dynamics toward excessive fission, contributing to weight gain and metabolic dysfunction.

Impaired Insulin Signaling Mediated by the Small GTPase Rac1 in Skeletal Muscle of the Leptin-Deficient Obese Mouse.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4. The small GTPase Rac1 acts as a switch of signal transduction that regulates GLUT4 translocation to the plasma membrane following insulin stimulation. However, it remains obscure whether signaling cascades upstream and downstream of Rac1 in skeletal muscle are impaired by obesity that causes insulin resistance and type 2 diabetes. In an attempt to clarify this point, we investigated Rac1 signaling in the leptin-deficient (Lepob/ob) mouse model. Here, we show that insulin-stimulated GLUT4 translocation and Rac1 activation are almost completely abolished in Lepob/ob mouse skeletal muscle. Phosphorylation of the protein kinase Akt2 and plasma membrane translocation of the guanine nucleotide exchange factor FLJ00068 following insulin stimulation were also diminished in Lepob/ob mice. On the other hand, the activation of another small GTPase RalA, which acts downstream of Rac1, by the constitutively activated form of Akt2, FLJ00068, or Rac1, was partially abrogated in Lepob/ob mice. Taken together, we conclude that insulin-stimulated glucose uptake is impaired by two mechanisms in Lepob/ob mouse skeletal muscle: one is the complete inhibition of Akt2-mediated activation of Rac1, and the other is the partial inhibition of RalA activation downstream of Rac1.

Impact of novel pan-RAS inhibitors on efficacy and resistance to AMG-510 and MRTX-1133 in pancreatic cancer cell lines.

4155 Background: Mutant-activated RAS genes are the most frequently mutated gene family associated with cancer (almost 30% of all cancers contain a mutant RAS gene). KRAS is the predominant isoform mutated in cancer and is the isoform exclusively mutated in pancreatic ductal carcinoma (PDAC). Since almost all PDAC cases harbor a mutant RAS, it is arguably the most RAS-addicted tumor type. There is now considerable evidence implicating mutant KRAS as a driver of PDAC. Recently, several mutant KRAS-targeted therapies (sotorasib and adagrasib) have been developed and show promise in PDAC patients. We have developed a direct inhibitor of RAS with a predicted unique interaction region capable of directly binding to wild-type H- and K-RAS, but which shows preferential binding for KRAS G12D and G12C mutants. This novel inhibitor disrupts the RAS effector domain and blocks the ability of RAS to signal through its effectors. Methods: We used in silico virtual library screening to identify an initial candidate inhibitor which was effective at inhibiting the 3D growth of PDAC cells without affecting their growth in 2D. Subsequent iterative rounds of medicinal chemistry was then performed to identify a series of derivatives with enhanced activity, as determined by 3D growth inhibition assays and effects on Ras signaling, as determined by Western blot analysis of phosphor-ERK, phosphor-Akt and activation of Ral A. Results: Our series of RAS inhibitors effectively block PDAC cell growth in 3D without impacting their 2D proliferation and suppress the interaction of KRAS with its effector cRAF. The inhibitors also effectively inhibit RAS signaling in mutant RAS containing PDAC cells. Since our novel compounds are predicted to bind to RAS at a different site to either AMG-510 (G12C specific inhibitor) and MRTX-1133 (G12D specific inhibitor), we tested the co-operativity of our compounds with these existing agents. Our compounds enhanced the anti-proliferative effects of both MRTX-113 and AMG-510 in mutant KRAS G12D and G12C PDAC cells, respectively. Conclusions: We have developed a series of novel RAS inhibitors that directly bind preferentially to mutant KRAS that may serve as new mutant KRAS-targeted therapeutics, and that may also have the potential to enhance the efficacy or suppress the resistance of AMG-510 and MRTX-1133.

Regulation of Ras p21 and RalA GTPases activity by quinine in mammary epithelial cells.

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.

RILP inhibits proliferation, migration, and invasion of PC3 prostate cancer cells

RILP (Rab-interacting lysosomal protein) is a key regulator of lysosomal transport and a potential tumor suppressor. However, the role of RILP in prostate cancer and the underlying mechanism of RILP in regulating the proliferation, migration, and invasion of prostate cancer cells remain to be studied. In this study, we confirmed RalGDS (Ral guanine nucleotide dissociation stimulator) as the interaction partner of RILP in PC3 prostate cancer cells. Immunofluorescence microscopy showed that RILP recruits RalGDS to the lysosomal compartment. We found that RILP inhibits the activation of RalA and downstream effector RalBP1, and negatively regulates the downstream molecular phosphorylation of Ras. We showed that RILP inhibits the proliferation, migration, and invasion of PC3 prostate cancer cells, which may give rise to novel ideas for cancer treatment.

Antitumor Effects of Ral-GTPases Downregulation in Glioblastoma.

Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.

The Full Spectrum of Behavioral Activation in Older Adults: A Scoping Review

<h3>Introduction</h3> Mental health concerns are common in older adults, and include many symptoms that may not meet criteria for specific DSM-5 diagnoses but which nevertheless affect patients' overall functioning and quality of life. However, timely access to quality mental health care remains inadequate in many communities. Behavioral activation (BA) is a brief psychotherapeutic treatment modality that is gaining momentum as a readily deployable intervention for addressing mental health needs in older adults. Early in its development, BA was primarily conceptualized as a treatment for patients with depression. Since its inception, the intervention has found utility in a broad range of patient populations and conditions beyond what most clinicians and researchers originally imagined. BA has the advantage of being able to be implemented by a range of provider types—e.g., health educators, physicians, mental health providers, family caregivers and community volunteers. Although several reviews have summarized the literature on BA for depression in older adults, there has not been a comprehensive scoping review that has looked broadly at the full spectrum of BA in older adults (i.e., beyond its use for depression). Therefore, the primary objective of this scoping review is to provide a comprehensive overview of the literature on the use of behavioral activation with older adults. This overview includes a categorization of the target conditions (e.g., depression, anxiety), specific populations studied (e.g., caregivers, ethnic minorities, veterans), context or setting (e.g., outpatient vs. inpatient), modality of BA use (i.e., stand-alone vs. incorporated into a larger treatment protocol), and type of individuals delivering the intervention. The aim of this full spectrum review is to provide a comprehensive snapshot of the work done to-date while also highlighting areas of growth for targeted dissemination and knowledge gaps for future study. <h3>Methods</h3> In order to conduct a comprehensive, scoping review of the literature on BA, we utilized both a wide date range as well as broad search criteria. We searched for manuscripts published from 1980 to 2020 in PubMed, PsycInfo, Cochrane, and EMBASE. All types of literature were eligible for inclusion (e.g., reviews, randomized clinical trials, case studies, book chapters). The search strategy consisted of selected keywords and combinations of keywords, which were selected to be inclusive of most scholarly work in the field (e.g., behavio(u)ral activation, older adult, geriatric, depression, anxiety). We did not exclude any specific diagnoses or conditions, as we sought to paint as comprehensive a picture as possible of the landscape related to BA. <h3>Results</h3> The database search strategy yielded 3953 articles; after removing duplicates, 1335 unique publications remained (see attached Figure, PRISMA diagram). Selection of publications for the final scoping review consists of two stages of screening. Initial inclusion criteria were: 1) title or abstract included specific and clear reference to behavioral activation; AND 2) clear focus of article or mean age of participants ≥ 50, OR unclear age of participants (leading to a need for additional screening). These initial inclusion criteria resulted in 640 records which were identified as either: 1) meeting inclusion criteria; OR 2) in need of additional screening in the second stage. Our team is currently conducting the second stage of screening for final inclusion, by retrieving identified manuscripts and reviewing the full text to determine age of participants. The final number, scope, and nature of records/publications included in the scoping review will be presented in the poster. <h3>Conclusions</h3> By casting a very wide net with our search strategy, we have uncovered a collection of articles whose breadth and depth were eye-opening—even to the seasoned BA provider. This broad spectrum of work to date with older adults, as well as their caregivers, underscores the transdiagnostic applicability of BA; targets of intervention or study included mental health conditions (e.g., depression, anxiety, substance use, PTSD, OCD, complicated bereavement, eating and sleep disorders), physical conditions (e.g. high blood pressure, diabetes, obesity, frailty) and psychosocial issues (e.g. loneliness, isolation, grief and loss, spiritual connectedness). This broad search revealed a diverse and surprisingly extensive range of life challenges addressed, in an array of contexts (e.g., in-home, community based, outpatient and inpatient settings), and with adaptations to render BA appropriate for a multitude of languages and cultures. It is hoped that this scoping review will not only identify areas in need of further study, but will thoroughly document the state of the field, giving a boost to dissemination efforts aimed at addressing the growing mental health needs of older adults. <h3>This research was funded by</h3> N/A. Chart: https://apps.aagponline.org/abstracts/uploads/2022/08n7lpw0gg4oebo.pdf

Covalent Fragment Screening Identifies Rgl2 RalGEF Cysteine for Targeted Covalent Inhibition of Ral GTPase Activation.

Ral GTPases belong to the RAS superfamily, and they are directly activated by K-RAS. The RalGEF pathway is one of the three major K-RAS signaling pathways. Ral GTPases do not possess a cysteine nucleophile to develop a covalent inhibitor following the strategy that led to a K-RAS G12C therapeutic agent. However, several cysteine amino acids exist on the surface of guanine exchange factors that activate Ral GTPases, such as Rgl2. Here, we screen a library of cysteine electrophile fragments to determine if covalent bond formation at one of the Rgl2 surface cysteines could inhibit Ral GTPase activation. We found several chloroacetamide and acrylamide fragments that inhibited Ral GTPase exchange by Rgl2. Site-directed mutagenesis showed that covalent bond formation at Cys-284, but not other cysteines, leads to inhibition of Ral activation by Rgl2. Follow-up time- and concentration-dependent studies of derivatives identified by substructure search of commercial libraries further confirmed Cys-284 as the reaction site and identified the indoline fragments as the most promising series for further development. Cys-284 is located outside of the Ral ⋅ Rgl2 interface on a loop that has several residues that come in direct contact with Ral GTPases. Our allosteric covalent fragment inhibitors provide a starting point for the development of small-molecule covalent inhibitors to probe Ral GTPases in animal models.

Dysregulation of RalA signaling through dual regulatory mechanisms exerts its oncogenic functions in hepatocellular carcinoma.

Background and AimsRas‐like (Ral) small guanosine triphosphatases (GTPases), RalA and RalB, are proto‐oncogenes directly downstream of Ras and cycle between the active guanosine triphosphate‐bound and inactive guanosine diphosphate‐bound forms. RalGTPase‐activating protein (RalGAP) complex exerts a negative regulation. Currently, the role of Ral up‐regulation in cancers remains unclear. We aimed to examine the clinical significance, functional implications, and underlying mechanisms of RalA signaling in HCC.Approach and ResultsOur in‐house and The Cancer Genome Atlas RNA sequencing data and quantitative PCR data revealed significant up‐regulation of RalA in patients’ HCCs. Up‐regulation of RalA was associated with more aggressive tumor behavior and poorer prognosis. Consistently, knockdown of RalA in HCC cells attenuated cell proliferation and migration in vitro and tumorigenicity and metastasis in vivo. We found that RalA up‐regulation was driven by copy number gain and uncovered that SP1 and ETS proto‐oncogene 2 transcription factor cotranscriptionally drove RalA expression. On the other hand, RalGAPA2 knockdown increased the RalA activity and promoted intrahepatic and extrahepatic metastasis in vivo. Consistently, we observed significant RalGAPA2 down‐regulation in patients’ HCCs. Intriguingly, HCC tumors showing simultaneous down‐regulation of RalGAPA2 and up‐regulation of RalA displayed a significant association with more aggressive tumor behavior in terms of more frequent venous invasion, more advanced tumor stage, and poorer overall survival. Of note, Ral inhibition by a Ral‐specific inhibitor RBC8 suppressed the oncogenic functions in a dose‐dependent manner and sensitized HCC cells to sorafenib treatment, with an underlying enhanced inhibition of mammalian target of rapamycin signaling.ConclusionsOur results provide biological insight that dysregulation of RalA signaling through dual regulatory mechanisms supports its oncogenic functions in HCC. Targeting RalA may serve as a potential alternative therapeutic approach alone or in combination with currently available therapy.

Low expression of RalGAPs associates with the poorer overall survival of head and neck squamous cell carcinoma.

BackgroundThe role of Ral and RalGAPs on the progression of head and neck squamous cell carcinoma (HNSC) remains unclear.MethodsThe predesigned siRNAs against RalGAPs were transfected into cells to evaluate the effect on RalA activation. The Data from TCGA and GTEx were combined to analyze the pan-cancer gene expression of RalA and RalGAPs in cancer and adjacent normal tissues. Kaplan-Meier analysis was used to assess the predictive value of RalA and RalGAPs expression on the overall survival of patients with HNSC. Methylation-specific PCR in vitro and next-generation bisulfite sequencing in vivo were used to evaluate the association between DNA methylation and the down-regulation of RalGAPs.ResultsRalGAPs negatively regulated RalA activation. HNSC patients with low level of RalGAPα2 had worse overall survival. The promoter of RalGAPα2 was widely methylated in comparison to RalGAPα1 and the DNA methylation level of RalGAPα2 promoter was increased in HNSC tissues and associated with the presence of neck lymph node metastasis.ConclusionsRalA and RalGAPs could act as a specific predictor to assess the prognosis of HNSC. DNA methylation might be a potential mechanism that downregulated the RalGAPα2 expression.

RAL Mediates Multiple Myeloma Cell Survival Independently of Oncogenic RAS

IntroductionMutation of RAS is one of the most frequent oncogenic lesions in human cancers and is present in up to half of primary multiple myeloma (MM) cases. Despite its therapeutic relevance, pharmacological inhibition of oncogenic RAS has so far been unsuccessful. Therefore, downstream effectors and RAS-associated pathways may serve as alternative therapeutic targets. The small GTPase RAL is a putative downstream effector of RAS and promotes malignant transformation and tumor cell survival in solid neoplasias. We set out to analyze the functional role of RAL in MM using shRNA-mediated knockdown of the two RAL isoforms, RALA and RALB. Our data demonstrate that RAL promotes MM cell survival and is activated independently of oncogenic RAS.MethodsExpression of RALA and RALB was analyzed by immunohistochemistry in bone marrow trephines from MM patients (n=24) and by Western blotting in purified primary MM cells (n=10). To abrogate RAL function in MM cell lines, two sequence-specific shRNA expression vectors targeting either RALA or RALB were generated. Cell viability after RAL knockdown was measured by flow cytometry using annexin V/propidium iodide staining. Effects on MEK/MAPK and PI3K/Akt signaling were determined by Western analysis. Additionally, RAL pulldown assays were used to analyze the functional link between RAL activation and oncogenic RAS. Moreover, we performed RNA sequencing to compare RAS- and RAL-dependent gene expression profiles. Finally, potential RAL interaction partners were identified by mass spectrometry.ResultsIn comparison to MGUS or normal plasma cells RALA showed strong expression in all MM cells tested, whereas RALB was expressed more heterogeneously. RNAi-mediated knockdown of RALA or RALB significantly reduced cell survival in L-363 (25% and 40%, respectively) and MM.1S cells (32% and 52%, respectively). Phospho-Akt and phospho-ERK1/2 levels remained unaffected by RAL depletion. Interestingly, abrogation of oncogenic RAS did not alter RAL activation in pulldown assays. Moreover, RNA sequencing after RAS or RAL knockdown revealed differential expression of 1473 KRAS-regulated genes versus 771 RALA-regulated genes, with an overlap of 235 genes, underscoring the notion that both targets serve different signaling pathways. Mass spectrometry analysis revealed interaction partners of RAL which are currently evaluated.ConclusionOur data indicate that RAL GTPases serve as promising therapeutic targets in MM, which mediate tumor cell survival independently of oncogenic RAS. Development of pharmacological inhibitors targeting RAL and its downstream interaction partners is, therefore, warranted for potential clinical translation. DisclosuresNo relevant conflicts of interest to declare.

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

Scoping Review of Geriatric Depression and Behavioral Activation

<h3>Introduction</h3> A substantial proportion of older adults will experience depression (including major depressive disorder and subsyndromal depressive symptoms), yet depression remains underdiagnosed and undertreated among older adults. Behavioral Activation is an effective, empirically-validated, first-line treatment for mild to moderate depression. Behavioral activation is a patient-centered intervention designed to decrease depressive behavior (e.g. social isolation, physical inactivity) and increase engagement in pleasurable activities (e.g. hobbies, family time). The objective of this scoping review is to evaluate the gaps in the literature and knowledge base regarding the implementation contexts, target populations, delivery modalities, assessment methods, and effectiveness of behavioral activation as a treatment for depression and/or anxiety among older adults, including both inpatient and outpatient settings. <h3>Methods</h3> We performed a scoping review and searched for peer-reviewed articles published from 1990 to 2020. The keywords and their combinations (e.g., depression, anxiety, elder(s), elderly, geriatric, older adult(s), age, aged, aging, behavio(u)ral activation) were used in search engines including the following but not limited to PubMed, PsycINFO, and Cochrane. Our search strategy identified 423 results; after removing duplicates, 232 results remained. Currently, we are evaluating the titles and abstracts using broad inclusion criteria to ensure that all relevant behavioral activation relevant to geriatric depression and/or anxiety are included. All types of studies and publications (e.g. randomized clinical trials, cohort studies, case controls, reviews, etc.) that focus on behavioral activation as an intervention or primary focus are being included in this comprehensive search. Studies have to include participants > 50 years old with either clinically diagnosed depression and/or anxiety at or above a pre-defined cut-point on a validated symptom scale (e.g., PHQ-9: >5 and GAD-7: >5). <h3>Results</h3> This scoping review will chart the landscape of literature relevant to behavioral activation for older adults with depression and/or anxiety. The literature will be reviewed in terms of study setting (e.g., community or outpatient settings, inpatient psychiatric units, assisted living facilities); study type (including randomized clinical trials, as well as observational studies, cohort studies, case controls, cross sectional, etc.); mode, frequency, and duration of intervention delivery; type and training of providers; assessment methods; and outcomes. Reviews will be examined for themes and consistency in synthesis of findings. Particular attention will be paid to identifying gaps in the literature where further work is needed to broaden the reach and impact of this promising intervention. <h3>Conclusions</h3> Scoping reviews represent a compelling method for evaluating existing literature and identifying gaps for future study. The present scoping review will synthesize the identified literature, in the hope of helping not only clinicians who work closely with older adults, but also researchers wishing to pursue studies in this area. Given a burgeoning older adult population and a paucity of geriatric mental health providers, clinical guidelines offering alternative nonpharmacologic therapy can be effective and cost-effective for clinicians and healthcare systems. Given the COVID-19 pandemic and the consequent escalation in social isolation among older adults, it is also hoped that this scoping review will identify promising avenues and methods for adapting behavioral activation in myriad ways—i.e., through implementation via telehealth platforms—to the older adult population. <h3>Funding</h3> Not applicable

Abstract 2589: Dysregulation of RalGAPA2 in hepatocellular carcinoma

Abstract RalGTPases are Ras-like G-proteins which cycle between the active GTP-bound and the inactive GDP-bound forms. Elevation of expression and activity of RalGTPase has been demonstrated to play an oncogenic role in human cancers. RalA activity could be positively regulated by RalGEF and negatively regulated by RalGAP. The functional RalGAP complex consists of either one of the catalytic subunits, RalGAPA1 or RalGAPA2, together with the regulatory subunit, RalGAPB. In this study, we aimed to investigate the potential role of RalGAPA2 dysregulation and its functional significance in hepatocellular carcinoma (HCC). By RNA-sequencing analysis in human HCC tissues and cell lines, we found that RalGAPA2 was the predominantly expressed RalGAP catalytic subunit in the liver. To examine the RalGAPA2 expression in HCC, we performed qPCR analysis in paired HCC tissues (n=90), and 37.8% of the HCC samples showed a 2-fold downregulation of RalGAPA2. Clinically, downregulation of RalGAPA2 was significantly correlated with a lower cellular differentiation (P=0.034) and a shorter overall survival rate (P=0.045). Interestingly, HCC tumors showing combined upregulation of RalA and downregulation of RalGAPA2 tended to be associated with more aggressive tumor behaviour, including higher occurrence of venous invasion (P=0.001) and more advanced TNM stage (P=0.007). On the other hand, by targeted-DNA sequencing, RalGAPA2 was identified to be recurrently mutated in 7.4% of the patients in a HBV-associated HCC cohort (n=95). Functionally, CRISPR/Cas9-mediated RalGAPA2 knockout HCC cells showed an enhanced migratory ability when compared to the control cells, underlying an upregulation of RalA activity. In HCC, expression of RalA isoform was found to be specifically upregulated and functionally supporting HCC metastasis. Also, inhibition of RalGAP has been demonstrated to promote RalA activity and enhance cell invasion in several cancer types, including oral squamous cell carcinoma, prostate and bladder cancer. Consistently, the inverse correlation between the RalA activity and RalGAPA2 expression was also observed in a panel of HCC cell lines. Furthermore, HCC cells possessing a higher RalA activity were demonstrated to be more prone to RBC8 treatment, a RalGTPase-specific inhibitor. Taken together, our results suggested that RalGAPA2 might play a potential tumor suppressive role in HCC by negatively regulating RalA activity. Citation Format: Lu Tian, Lo Kong Chan, Daniel Wai Hung HO, LuQing Zhao, Irene Oi Lin NG. Dysregulation of RalGAPA2 in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2589.

Bi-allelic Variants in RALGAPA1 Cause Profound Neurodevelopmental Disability, Muscular Hypotonia, Infantile Spasms, and Feeding Abnormalities

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.

A Crucial Role for the Small GTPase Rac1 Downstream of the Protein Kinase Akt2 in Insulin Signaling that Regulates Glucose Uptake in Mouse Adipocytes.

Insulin-stimulated glucose uptake is mediated by translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes and skeletal muscle cells. In both types of cells, phosphoinositide 3-kinase and the protein kinase Akt2 have been implicated as critical regulators. In skeletal muscle, the small GTPase Rac1 plays an important role downstream of Akt2 in the regulation of insulin-stimulated glucose uptake. However, the role for Rac1 in adipocytes remains controversial. Here, we show that Rac1 is required for insulin-dependent GLUT4 translocation also in adipocytes. A Rac1-specific inhibitor almost completely suppressed GLUT4 translocation induced by insulin or a constitutively activated mutant of phosphoinositide 3-kinase or Akt2. Constitutively activated Rac1 also enhanced GLUT4 translocation. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was abrogated by inhibition of phosphoinositide 3-kinase or Akt2. On the other hand, constitutively activated Akt2 caused Rac1 activation, and insulin-induced Rac1 activation was suppressed by an Akt2-specific inhibitor. Moreover, GLUT4 translocation induced by a constitutively activated mutant of Akt2 or Rac1 was diminished by knockdown of another small GTPase RalA. RalA was activated by a constitutively activated mutant of Akt2 or Rac1, and insulin-induced RalA activation was suppressed by an Akt2- or Rac1-specific inhibitor. Collectively, these results suggest that Rac1 plays an important role in the regulation of insulin-dependent GLUT4 translocation downstream of Akt2, leading to RalA activation in adipocytes.

Down-regulation of RalGTPase-Activating Protein Promotes Colitis-Associated Cancer via NLRP3 Inflammasome Activation.

Background & AimsRal guanosine triphosphatase–activating protein α2 (RalGAPα2) is the major catalytic subunit of the negative regulators of the small guanosine triphosphatase Ral, a member of the Ras subfamily. Ral regulates tumorigenesis and invasion/metastasis of some cancers; however, the role of Ral in colitis-associated cancer (CAC) has not been investigated. We aimed to elucidate the role of Ral in the mechanism of CAC.MethodsWe used wild-type (WT) mice and RalGAPα2 knockout (KO) mice that showed Ral activation, and bone marrow chimeric mice were generated as follows: WT to WT, WT to RalGAPα2 KO, RalGAPα2 KO to WT, and RalGAPα2 KO to RalGAPα2 KO mice. CAC was induced in these mice by intraperitoneal injection of azoxymethane followed by dextran sulfate sodium intake. Intestinal epithelial cells were isolated from colon tissues, and we performed complementary DNA microarray analysis. Cytokine expression in normal colon tissues and CAC was analyzed by quantitative polymerase chain reaction.ResultsBone marrow chimeric mice showed that immune cell function between WT mice and RalGAPα2 KO mice was not significantly different in the CAC mechanism. RalGAPα2 KO mice had a significantly larger tumor number and size and a significantly higher proportion of tumors invading the submucosa than WT mice. Higher expression levels of matrix metalloproteinase-9 and matrix metalloproteinase-13 were observed in RalGAPα2 KO mice than in WT mice. The expression levels of interleukin 1β, NLRP3, apoptosis associated speck-like protein containing a CARD, and caspase-1 were apparently increased in the tumors of RalGAPα2 KO mice compared with WT mice. NLRP3 inhibitor reduced the number of invasive tumors.ConclusionsRal activation participates in the mechanism of CAC development via NLRP3 inflammasome activation.

RAL GTPases mediate multiple myeloma cell survival and are activated independently of oncogenic RAS.

Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma (MM) cases, but has so far remained a clinically undruggable target. RAS-like protein (RAL) is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary MM, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes MM cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.

Ral GTPase Activation by Downregulation of RalGAP Enhances Oral Squamous Cell Carcinoma Progression

Ral small GTPases, consisting of RalA and RalB, are members of the Ras family. Their activity is upregulated by RalGEFs. Since several RalGEFs are downstream effectors of Ras, Ral is activated by the oncogenic mutant Ras. Ral is negatively regulated by RalGAP complexes that consist of a catalytic α1 or α2 subunit and its common partner β subunit and similarly regulate the activity of RalA as well as RalB in vitro. Ral plays an important role in the formation and progression of pancreatic and lung cancers. However, the involvement of Ral in oral squamous cell carcinoma (OSCC) is unclear. In this study, we investigated OSCC by focusing on Ral. OSCC cell lines with high Ral activation exhibited higher motility. We showed that knockdown of RalGAPβ increased the activation level of RalA and promoted the migration and invasion of HSC-2 OSCC cells in vitro. In contrast, overexpression of wild-type RalGAPα2 in TSU OSCC cells attenuated the activation level of RalA and inhibited cell migration and invasion. Real-time quantitative polymerase chain reaction analysis of samples from patients with OSCC showed that RalGAPα2 was downregulated in oral cancer tissues as compared with normal epithelia. Among patients with OSCC, those with a lower expression of RalGAPα2 showed a worse overall survival rate. A comparison of DNA methylation and histone modifications of the RalGAPα2 gene in OSCC cell lines suggested that crosstalk among DNA methylation, histone H4Ac, and H3K27me2 was involved in the downregulation of RalGAPα2. Thus, activation of Ral GTPase by downregulation of RalGAP expression via a potential epigenetic mechanism may enhance OSCC progression.