Ras Superfamily Of Small GTPases Research Articles

Presynaptic Rac1 in the hippocampus selectively regulates working memory.

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

ARL15 and its Multiple Disease Association: Emerging Functions and Potential Therapeutic Application.

ARL15 is a member of the RAS superfamily of small GTPases and is associated with several metabolic traits, including increased risk of diabetes, rheumatoid arthritis and lipid metabolism disorders. The ARL15 gene encodes for an uncharacterized small GTP binding protein. Its precise role in human physiology remains unknown, but several genetic association studies have recognized different variants in this gene to be statistically associated with numerous traits and complex diseases. Here, we provided the unique features of ARL15 small G protein, its association with varied metabolic and lifestyle diseases, its function in vesicular and lipid trafficking, and its binding partners. We outlined this protein as a promising and emerging therapeutic target to combat metabolic disorders like cardiovascular diseases, diabetes and rheumatoid arthritis. The review provides a comprehensive description of the current advancements in ARL15 research with a perspective that focused research will position this small GTPase as a viable target for the treatment of rheumatoid arthritis.

Addiction to Small Gtpase ARF6-Mediated Sphingolipid Homeostasis By AML but Not the Host

Background Cancer cells' unique metabolic demand has emerged as a promising therapeutic target in Acute Myeloid Leukemia (AML). The vesicular trafficking and signaling gatekeeper, ADP-Ribosylation Factor 6 (ARF6), belongs to the RAS superfamily of small GTPases and has been shown to regulate the homeostasis of lipids such as PIP2, phosphatidic acids, and cholesterol. Here we describe AML cells' unusual addiction to ARF6-mediated sphingolipid homeostasis and its clinical significance. Experiments and Results ARF6 knockdown (KD) or CRISPR-mediated ARF6 knockout (KO) inhibited the proliferation of AML cell lines, THP-1 and MV4-11, as well as the colony formation of primary AML CD34 + cells. Surprisingly, Tamoxifen-induced whole body KO of Arf6 in adult Arf6 f/f;Rosa-CreERT 2 mice led to healthy and grossly normal mice with a normal lifespan. Adult mice lacking ARF6 show no abnormality in complete blood count, urine test, or histology. Hence, ARF6 is genetically indispensable for AML, but not for the host. To confirm AML's addition to ARF6 in vivo, we introduced Arf6 KO in an MLL-AF9-induced AML model in C57BL6/J mice. We are currently following up with the disease progression between Arf6-KO AML group and control AML group, and expect to obtain results before the 2023 ASH meeting. After discovering that ARF6 is genetically necessary for AML but not needed for the host, we analyzed its clinical significance. Since ARF6 mutations are not detected in AML samples, we focused on analyzing its expression. With univariate analysis, we show that high ARF6 expression (top 50%) is significantly associated with worse overall survival (OS) and worse disease-free survival (DFS) of AML patients in the TCGA-NEJM-2013 (TCGA) cohort (N=200) and the Münchner Leukämielabor (MLL) cohort (N=579). In multi-variate analysis, high ARF6 expression is significantly associated with worse OS in the BeatAML cohort (N=562) and shows a trend towards worse OS and DFS in the TCGA cohort. Following a hypothesis-driven approach, we investigated the role of ARF6 in regulating sphingolipid homeostasis by HPLC-MS lipidomic analyses of AML cells (THP-1) with or without ARF6 depletion. Lipidomic profiles revealed that ARF6 KD led to an increase of the overall ceramide/sphingomyelin ratio, suggesting a defect in sphingolipid homeostasis. We then analyzed the expression of sphingolipid metabolic enzymes and observed that ARF6 KD downregulated sphingomyelin synthase 1 and 2 (SGMS1 and SGMS2) expression, hindering ceramide-to-sphingomyelin conversion. Supporting this, we found a positive linear correlation between ARF6 and SGMS1/2 expression among the patients in TCGA cohort, confirming their correlation. High expression of the ARF6 effectors SGMS1/2 is also associated with worse OS and DFS in the TCGA cohort and the MLL cohort in univariate analysis, resembling that of ARF6. Downstream of ceramide accumulation, ARF6 KD inhibited the pro-survival AKT pathway, confirming the pro-apoptotic roles of ceramide in cancers. The anti-proliferative effect of ARF6 depletion can be rescued by sphingomyelinase inhibitor GW4869 (blocking ceramide production) or by expressing a constitutively active form of AKT (AKT1 E17K). To pharmacologically target ARF6, we developed two generations of ARF6 small molecular inhibitor pro-drugs, named NAV-2729 and A6-4471. NAV-2729 and A6-4471 showed high specificity to ARF6 without inhibiting other small GTPases, with in vitro IC 50 in the lower μM range. Both NAV-2729 and A6-4471 inhibited AML cell line proliferation and reduced primary AML cell colony formation in lower μM range. Cord blood normal CD34 + cells are not sensitive to ARF6 inhibitors, but non-specific toxicity was observed at higher concentrations. When tested in vivo with THP-1 xenografts in NSG mice, both inhibitors reduced circulating AML cells without obvious toxicity to the host. Conclusion ARF6 is necessary for AML proliferation and its higher expression is associated with worse patient survival. However, ARF6 is dispensable for normal host physiology. Mechanistically, ARF6 maintains critical ceramide/sphingomyelin balance in AML by regulating SGMS1/2 expression. Pharmacologically inhibiting ARF6 suppressed AML proliferation in vitro and in vivo in a genotype-agnostic fashion. Our study implicates ARF6 as a potentially actionable and safe target in AML.

Phase 1 Pharmacodynamic and Pharmacokinetic Study of the Geranylgeranyltransferase I Inhibitor PTX-100 (GGTI-2418) in Patients with Advanced Malignancies

Introduction: The RAS superfamily of small GTPases mediate hallmarks of cancer cell proliferation, viability, migration, invasion and metastasis. Several GTPases such as RAL, RHO and RAC are required for RAS to induce malignant transformation. RAS post-translational modification (PTM) by farnesyltransferase (FT) as well as RAL, RHO and RAC PTM by geranylgeranyltransferase 1 (GGT-1) are critical for the membrane localization and oncogenicity of these GTPases. Mutations in HRAS, NRAS, KRAS and RHOA are prevalent in solid tumors as well as T-cell lymphomas (TCL) such as peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), PTCL-not otherwise specified and cutaneous T-cell lymphoma (CTCL). PTX-100 is a small molecule inhibitor of GGT-1 that disrupts the oncogenic activity by inhibiting RAL, RHO and RAC PTM. Methods: This ongoing phase 1 study (NCT03900442) in 2 Australian sites, consists of a 3+3 dose escalation with an expansion phase. The aim is to evaluate the pharmacodynamic (PD), pharmacokinetics (PK), preliminary efficacy and safety of 500, 1000 and 2000 mg/m 2 PTX-100 in patients (pts) with advanced malignancies. PTX-100 is administered by IV infusion over 60 minutes days 1 to 5 of a 14 day cycle for 4 cycles. Disease response is assessed via CT, MRI or PET-CT or per SOC after 4 cycles. Pts with a complete response (CR), partial response (PR) or stable disease (SD) may be eligible for continued treatment with PTX 100, dosed in 21 day cycles with response assessment every 3 months. Results: The first pt was enrolled in October 2019 and enrollment is nearly completed in the expansion cohort. The data cutoff was 10 th July 2023. Ten pts were enrolled into Part 1 with 3 dosed at 500 mg/m 2, 3 at 1000 mg/m 2 and 4 at 2000 mg/m 2. The expansion cohort at 2000 mg/m 2 began in May 2022 and there have been 10/15 enrolled to date. Positive long-term responses (32 and 13.8 months) were observed for 2 TCL pts in Part 1 so the decision was made to enroll TCL pts into the expansion cohort. The median age was 67 years (44 to 81), the median number of prior lines was 3 (1-6). The TCL subtypes were PCTL NOS (n=6), AITL (n=3), and CTCL (n=5). Of the 14 enrolled TCL pts, 10 pts had response assessments after 4 cycles, with an overall response rate (ORR) of 40% (4pts), including 10% (1 pt) CR, 30% (3pts) PR. Two CTCL pts (20%) had durable SD >6 months, contributing to a 60% Disease Control Rate. The median Progression Free Survival (PFS) for all TCL pts was 5.3 mos, CTCL pts 13.6 mos and PTCL pts 2.5 mos. No responses were observed in the 5 solid tumor pts. There were 23 Grade ≥3 adverse events in 8 pts including laboratory neutropenia in 2 pts with 1 deemed possibly related. Neutrophil count decreased in 2 pts with 1 deemed possibly related and the platelet count decreased in 2 pts with 1 deemed possibly related. Grade 1-2 adverse events deemed possibly related included, nausea reported in 4 pts and diarrhea in 3 pts. For the PD studies, we determined the effects of PTX-100 on the geranylgeranylation (GG) of the small GTPase RAP1 in peripheral blood monocytes (PBMCs) isolated from pts on cycle 1, day 1 (prior to treatment) and subsequently on days 2, 3, 4, 5, and 8. PTX-100 inhibited GG of RAP1A as early as 1 day after treatment, and with the majority of the pts, the inhibition of RAP1A GG persisted three days after the last dose of PTX-100. Inhibition of RAP1 GG was observed with all doses including the lowest dose of 500 mg/m 2. PK analysis is underway at the time of abstract preparation and the PK parameters will be presented at the meeting. Conclusions: Preliminary data from this phase 1 dose escalation study of PTX-100 indicates promising safety profile in this difficult to treat pt population. PTX-100 demonstrated clinical activity in TCL with an ORR of 40%, and a median PFS for all TCL pts of 5.3 months. PD studies demonstrated that the GGT-1 target was engaged with sustained inhibition of RAP1 GG.

Abstract 3492: The small GTPase ARF6 augments BRAFV600E-MAPK signaling in melanomagenesis

Abstract ARF6 is a member of the Ras superfamily of small GTPases and has critical functions in endo-membrane trafficking pathways. We previously reported that ARF6 is aberrantly activated in metastatic melanoma and that activation of ARF6 stimulates WNT5A/beta-catenin and PI3K/AKT signaling to promote invasion and accelerate metastasis. Herein, we discovered an ARF6 activation signature in primary melanomas that is associated with inferior overall survival, supporting a novel role for ARF6 in early-stage progression. Conversely, melanocyte-specific deletion of Arf6 in Dct;TVA::BrafV600E;Cdkn2aNull murine melanoma significantly reduced tumorigenesis and progression, and improved survival. Importantly, bulk tumor transcriptome analysis showed reduced expression of ERK-induced genes in these BRAFV600E tumors. Likewise, immunofluorescent staining of tumor sections showed a significant reduction of cytoplasmic and nuclear p-ERK levels. Together these data demonstrate that loss of ARF6 reduces BRAFV600E-MAPK signaling output in vivo. This is a provocative finding because it implies that endomembrane trafficking has a major role in promoting BRAFV600E-driven tumorigenesis. More specifically, ARF6 may be critical for augmenting BRAFV600E signaling. Consistent with this hypothesis, expression of activated ARF6(ARF6Q67L) in our murine BRAFV600E melanoma cells was associated with upregulation of BRAFV600E, p-MEK, and p-ERK levels, detected by Reverse Phase Protein Array analysis. In addition, ARF6Q67L tumor cells showed an increase in anti-apoptotic proteins with a reciprocal decrease in pro-apoptotic proteins. Knockdown of Arf6 in BRAFV600E human melanoma cells significantly reduced proliferation and sensitized cells to vemurafenib-induced apoptosis. Overall, our data reveal that ARF6 augments MAPK signaling in BRAFV600E-driven tumorigenesis. Further work is needed to understand if ARF6 mediates endomembrane-dependent signaling of the BRAFV600E-MAPK pathway. Citation Format: Junhua Wang, Aaron Rogers, Coulson Rich, Lise Sorensen, Sheri Holmen, Roger Wolff, Allie Grossmann. The small GTPase ARF6 augments BRAFV600E-MAPK signaling in melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3492.

RANBP1 (RAN Binding Protein 1): The Missing Genetic Piece in Cancer Pathophysiology and Other Complex Diseases

RANBP1 encoded by RANBP1 or HTF9A (Hpall Tiny Fragments Locus 9A), plays regulatory functions of the RAN-network, belonging to the RAS superfamily of small GTPases. Through this function, RANBP1 regulates the RANGAP1 activity and, thus, the fluctuations between GTP-RAN and GDP-RAN. In the light of this, RANBP1 take actions in maintaining the nucleus-cytoplasmic gradient, thus making nuclear import-export functional. RANBP1 has been implicated in the inter-nuclear transport of proteins, nucleic acids and microRNAs, fully contributing to cellular epigenomic signature. Recently, a RANBP1 diriment role in spindle checkpoint formation and nucleation has emerged, thus constituting an essential element in the control of mitotic stability. Over time, RANBP1 has been demonstrated to be variously involved in human cancers both for the role in controlling nuclear transport and RAN activity and for its ability to determine the efficiency of the mitotic process. RANBP1 also appears to be implicated in chemo-hormone and radio-resistance. A key role of this small-GTPases related protein has also been demonstrated in alterations of axonal flow and neuronal plasticity, as well as in viral and bacterial metabolism and in embryological maturation. In conclusion, RANBP1 appears not only to be an interesting factor in several pathological conditions but also a putative target of clinical interest.

Geranylgeranyl diphosphate synthase: Role in human health, disease and potential therapeutic target.

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthesis pathway, is responsible for the production of geranylgeranyl pyrophosphate (GGPP). GGPP serves as a substrate for the post-translational modification (geranylgeranylation) of proteins, including those belonging to the Ras superfamily of small GTPases. These proteins play key roles in signalling pathways, cytoskeletal regulation and intracellular transport, and in the absence of the prenylation modification, cannot properly localise and function. Aberrant expression of GGDPS has been implicated in various human pathologies, including liver disease, type 2 diabetes, pulmonary disease and malignancy. Thus, this enzyme is of particular interest from a therapeutic perspective. Here, we review the physiological function of GGDPS as well as its role in pathophysiological processes. We discuss the current GGDPS inhibitors under development and the therapeutic implications of targeting this enzyme.

Systematic pan-cancer analysis identifies RALA as a tumor targeting immune therapeutic and prognostic marker.

RALA is a member of the small GTPase Ras superfamily and has been shown to play a role in promoting cell proliferation and migration in most tumors, and increase the resistance of anticancer drugs such as imatinib and cisplatin. Although many literatures have studied the cancer-promoting mechanism of RALA, there is a lack of relevant pan-cancer analysis. This study systematically analyzed the differential expression and mutation of RALA in pan-cancer, including different tissues and cancer cell lines, and studied the prognosis and immune infiltration associated with RALA in various cancers. Next, based on the genes co-expressed with RALA in pan-cancer, we selected 241 genes with high correlation for enrichment analysis. In terms of pan-cancer, we also analyzed the protein-protein interaction pathway of RALA and the application of small molecule drug Guanosine-5'-Diphosphate. We screened hepatocellular cancer (HCC) to further study RALA. The results indicated that RALA was highly expressed in most cancers. RALA was significantly correlated with the infiltration of B cells and macrophages, as well as the expression of immune checkpoint molecules such as CD274, CTLA4, HAVCR2 and LAG3, suggesting that RALA can be used as a kind of new pan-cancer immune marker. The main functions of 241 genes are mitosis and protein localization to nucleosome, which are related to cell cycle. For HCC, the results displayed that RALA was positively correlated with common intracellular signaling pathways such as angiogenesis and apoptosis. In summary, RALA was closely related to the clinical prognosis and immune infiltration of various tumors, and RALA was expected to become a broad-spectrum molecular immune therapeutic target and prognostic marker for pan-cancer.

Crystal Structure of Schizosaccharomyces pombe Rho1 Reveals Its Evolutionary Relationship with Other Rho GTPases.

The Rho protein, a homolog of Ras, is a member of the Ras superfamily of small GTPases. Rho family proteins are involved in cytoskeletal organization, cell mobility, and polarity, and are implicated in cancer morphogenesis. Although Rho homologs from higher-order mammalian organisms are well studied, there are few studies examining Rho proteins in lower-level single-celled organisms. Here, we report on the crystal structure of Rho1 from Schizosaccharomyces pombe (SpRho1) in complex with GDP in the presence of Mg2+ at a 2.78 Å resolution. The overall structure is similar to that of known Rho homologs, including human RhoA, human RhoC, and Aspergillus fumigatus Rho1 (AfRho1), with some exceptions. We observed subtle differences at the Switch I and II regions, in β2 and β3, and in the Rho insert domain and loop from Phe107 to Pro112. Our analysis suggests that SpRho is evolutionarily closer to HsRhoC than HsRhoA, as previously believed.

Non-canonical Small GTPase RBJ Promotes NSCLC Progression Through the Canonical MEK/ERK Signaling Pathway.

Although the majority of members belonging to the small GTPase Ras superfamily have been studied in several malignancies, the function of RBJ has remained unclear, particularly in non-small cell lung cancer (NSCLC). The research aims to determine the function of RBJ in NSCLC. The levels of RBJ protein in tumor tissue and para-carcinoma normal tissue were ascertained via immunohistochemistry (IHC). The growth, migration, and invasion of NSCLC cells were assessed by 5- ethynyl-2-deoxyuridine (EdU) assay, colony formation, cell counting kit-8 (CCK8), transwell and wound healing assays. Furthermore, a nude mouse xenograft model was established to study the function of RBJ in tumorigenesis in vivo. The IHC analysis revealed that the protein levels of RBJ were notably increased in tumor tissue and positively associated with the clinical stage. In addition, the knockdown of RBJ restrained the growth, invasion, and migration of NSCLC cell lines by inhibiting the epithelial-mesenchymal transition (EMT) through the MEK/ERK signaling pathway. Accordingly, opposite results were observed when RBJ was overexpressed. In addition, the overexpression of RBJ accelerated tumor formation by A549 cells in nude mice. RBJ promoted cancer progression in NSCLC by activating EMT via the MEK/ERK signaling. Thus, RBJ could be used as a potential therapeutic against NSCLC.

RAB6B is a potential prognostic marker and correlated with the remolding of tumor immune microenvironment in hepatocellular carcinoma.

Backgrounds: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the second leading cause of death among all cancers. The Ras-associated binding (Rab) proteins constitute the largest family of the Ras superfamily of small GTPases, which mainly mediate membrane trafficking processes. RAB6B is a member of Rab GTPases, and it has been found to be dysregulated in various tumors. However, the clinical significance, correlations with immune cells, and stroma infiltration of RAB6B in HCC remain unclear. Methods: RAB6B mRNA and protein expression in HCC were examined using the TIMER, HCCDB, UALCAN, and HPA databases. The genetic alterations of RAB6B were analyzed by cBioPortal and COSMIC databases. The correlations between RAB6B and tumor-infiltrating immune cells and cancer-associated fibroblasts were explored by using TIMER, TISIDB, and GEPIA databases. Co-expression networks of RAB6B were investigated based on LinkedOmics. Drug sensitivity was analyzed through the GDSC and CTRP databases. RAB6B was knocked down with siRNA in HCC cell lines. EdU assay was performed to detect the cell proliferation ability, flow cytometry was used to compare the differences in the ability of apoptosis, and MTT was used to evaluate the drug sensitivity in vitro. Results: RAB6B mRNA and protein expression were upregulated in the HCC tissues. Kaplan–Meier and Cox regression analyses suggested that highly expressed RAB6B was an independent prognostic factor for poor survival in HCC patients. Moreover, we found that RAB6B expression was positively correlated with the infiltration of immune cells in HCC, including some immunosuppressive cells, chemokines, and receptors, meanwhile RAB6B expression was associated with CD8+T cells exhaustion, resulting in an immunosuppressive microenvironment. Additionally, functional enrichment analysis indicated that RAB6B may be involved in ECM remodeling in the TME, and RAB6B expression was positively associated with CAFs infiltration. Furthermore, RAB6B presented a positive association with sensitivity to GDSC and CTRP drugs. RAB6B knockdown inhibited the cell proliferation and promoted apoptosis and sensitivity to cisplatin of HCC cells in vitro. Conclusion: Our study revealed that RAB6B is a potential biomarker for poor prognosis in HCC patients and correlates with the formation of the immunosuppressive microenvironment in HCC.

Abstract 1864: Baicalein attenuates endometrial cancer growth by suppressing the ARF6

Abstract Using mass spectrometry-based proteomics, we have previously shown enhanced expression of the Adenosine diphosphate-Ribosylation Factor-6 (ARF6) in endometrial cancer cell lines compared to immortalized EM-E6/E7-TERT endometrial epithelial cells. ARFs belong to the RAS superfamily of small GTPases. ARF family proteins are activated by Guanine nucleotide Exchange Factors (GEFs) and inactivated by GTPase-Activating Proteins (GAPs). When active, they bind effectors, which mediate downstream functions. ARF6 localized on the plasma membrane plays a role in actin cytoskeleton dynamics and endocytic recycling. Dysregulation of expression and/or activity of ARF6 has been associated with enhanced cell migration, invasion, and proliferation in several types of cancer. Knockdown of ARF6 significantly inhibited the invasiveness of the cancer cells. There is a critical need for novel promising anticancer agents with lesser side effects and higher efficacy. Baicalein, extracted from the roots of the Chinese herb Huang Qin (Scutellaria baicalensis), showed a significant anticancer impact on many human cancers. However, the specific molecular mechanisms are still nebulous. Endometrial cancer cell lines were exposed to different concentrations of baicalein for 24 to 120 h. The effect on cell proliferation and invasion was determined utilizing MTS and Boyden chamber assays, respectively. The baicalein induced a dose and time-dependent reduction in cell viability. The invasiveness of cells was attenuated by baicalein. Furthermore, the anticancer effects were mediated by decreased ARF6, Rac1, and PAK1 expression in baicalein-treated cancer cells. In summary, the data reveal that suppression of ARF6 could reduce the invasive potential of endometrial cancer by modulating the major downstream effectors Rac1 and PAK1. Citation Format: Latoya Mcglorthan, Viqar Syed. Baicalein attenuates endometrial cancer growth by suppressing the ARF6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1864.

Determining the role of the Ras family GTPase Rap1 in regulating mTORC2 activity and function in cell migration

Directed cell migration is an important biological process that is necessary for the embryonic development, maintenance of multicellular organisms and immune response to external stimuli. Dysregulation of cell migration is implicated in the onset and progression of diseases including cardiovascular diseases and cancer metastasis. Many studies have identified genes and proteins that are important for directed cell migration, but the process remains incompletely understood. The mechanistic Target of Rapamycin Complex 2 (mTORC2) is a complex of proteins that have been identified as important players in cell migration. mTORC2 is known to have a role in regulating F‐actin organization and actomyosin (MyoII). Moreover, there is increasing evidence that shows a correlation between mTORC2 and cancer cell migration and invasion. Despite this knowledge, the role and regulation of mTORC2 is not fully understood. Recently, we found that Rap1, a member of the Ras superfamily of small GTPases with conserved roles in cytoskeleton remodeling and cell adhesion, is a conserved binding partner of mTORC2 component RIP3/SIN1. We previously showed that Rap1 positively regulates mTORC2 activation in response to the chemoattractant cAMP in the model organism Dictyostelium discoideum. Moreover, we recently found that expression of a constitutively active Rap1 mutant (Rap1CA) potentiates the insulin‐induced activation of mTORC2 in human HEK293 cells. We then undertook a study to investigate if Rap1 regulation of mTORC2 is part of a mechanism involved in regulating human cell migration. We observed that basal mTORC2 activity, as well as that induced by several potential promigratory signals, including insulin, epidermal growth factor (EGF), insulin‐like growth factor‐1 (IGF‐1), and lysophosphatidic acid (LPA), was increased by the overexpression of wild‐type Rap1 or Rap1CA. Therefore, these observations suggest that Rap1 plays a role in positively regulating mTORC2 activation in HEK293 cells in response to stimulation of promigratory signals, similar to our previous observations made in Dictyostelium. Ongoing studies now aim at determining how Rap1 regulates the function of mTORC2 in cell migration.

Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling.

Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.

FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.

Presence of serum RalA and serum p53 autoantibodies in 1833 patients with various types of cancers.

RalA is a member of the Ras superfamily of small GTPases. The Anti-RalA autoantibodies (s-RalA-Abs) act as tumor markers in various types of cancer and are negatively associated with the p53 autoantibodies (s-p53-Abs). This study aimed to evaluate the relationship between s-RalA-Abs and s-p53-Abs in various types of cancer. A total of 1833 cancer patients (esophageal cancer, 172; hepatocellular carcinoma, 91; lung cancer, 269; gastric cancer, 317; colon cancer, 262; breast cancer, 364; and prostate cancer, 358) and 73 healthy subjects were enrolled in the study. The levels of s-RalA-Abs and s-p53-Abs were analyzed using enzyme-linked immunosorbent assay, and the positivity rates and relations between the two autoantibodies were evaluated. The cutoff values for s-RalA abs and s-p53 abs were set as mean + 2 standard deviation and the values higher than the cutoff values were defined as positive. The titers in all cancer types were significantly higher than those in the controls (P < 0.01). The positivity rates for s-RalA-Abs ranged between 11.7 and 21.5%, and those for s-p53-Abs ranged between 12 and 28.5%. A combined assay of the two antibodies revealed positivity rates of 20.9 and 44.2%. In Stage 0/I/II tumors, the positivity rates of the combination of the two antibodies ranged between 21.5 and 42.3%. The two autoantibodies were complementary to each other in the prostate and breast cancers, but independent in other carcinomas. The combined use of s-RalA-Abs and s-p53-Abs tended to increase the positivity rate in all cancers, including Stage 0/I/II cancers.

Targeting small GTPases and their downstream pathways with intracellular macromolecule binders to define alternative therapeutic strategies in cancer.

The RAS superfamily of small GTPases regulates major physiological cellular processes. Mutation or deregulation of these small GTPases, their regulators and/or their effectors are associated with many diseases including cancer. Hence, targeting these classes of proteins is an important therapeutic strategy in cancer. This has been recently achieved with the approval of the first KRASG12C covalent inhibitors for the clinic. However, many other mutants and small GTPases are still considered as 'undruggable' with small molecule inhibitors because of a lack of well-defined pocket(s) at their surface. Therefore, alternative therapeutic strategies have been developed to target these proteins. In this review, we discuss the use of intracellular antibodies and derivatives - reagents that bind their antigen inside the cells - for the discovery of novel inhibitory mechanisms, targetable features and therapeutic strategies to inhibit small GTPases and their downstream pathways. These reagents are also versatile tools used to better understand the biological mechanisms regulated by small GTPases and to accelerate the drug discovery process.

Protein farnesylation is upregulated in Alzheimer\u2019s human brains and neuron-specific suppression of farnesyltransferase mitigates pathogenic processes in Alzheimer\u2019s model mice

The pathogenic mechanisms underlying the development of Alzheimer’s disease (AD) remain elusive and to date there are no effective prevention or treatment for AD. Farnesyltransferase (FT) catalyzes a key posttranslational modification process called farnesylation, in which the isoprenoid farnesyl pyrophosphate is attached to target proteins, facilitating their membrane localization and their interactions with downstream effectors. Farnesylated proteins, including the Ras superfamily of small GTPases, are involved in regulating diverse physiological and pathological processes. Emerging evidence suggests that isoprenoids and farnesylated proteins may play an important role in the pathogenesis of AD. However, the dynamics of FT and protein farnesylation in human brains and the specific role of neuronal FT in the pathogenic progression of AD are not known. Here, using postmortem brain tissue from individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimer’s dementia, we found that the levels of FT and membrane-associated H-Ras, an exclusively farnesylated protein, and its downstream effector ERK were markedly increased in AD and MCI compared with NCI. To elucidate the specific role of neuronal FT in AD pathogenesis, we generated the transgenic AD model APP/PS1 mice with forebrain neuron-specific FT knockout, followed by a battery of behavioral assessments, biochemical assays, and unbiased transcriptomic analysis. Our results showed that the neuronal FT deletion mitigates memory impairment and amyloid neuropathology in APP/PS1 mice through suppressing amyloid generation and reversing the pathogenic hyperactivation of mTORC1 signaling. These findings suggest that aberrant upregulation of protein farnesylation is an early driving force in the pathogenic cascade of AD and that targeting FT or its downstream signaling pathways presents a viable therapeutic strategy against AD.

Targeting Small GTPases and Their Prenylation in Diabetes Mellitus.

A fundamental role of pancreatic β-cells to maintain proper blood glucose level is controlled by the Ras superfamily of small GTPases that undergo post-translational modifications, including prenylation. This covalent attachment with either a farnesyl or a geranylgeranyl group controls their localization, activity, and protein–protein interactions. Small GTPases are critical in maintaining glucose homeostasis acting in the pancreas and metabolically active tissues such as skeletal muscles, liver, or adipocytes. Hyperglycemia-induced upregulation of small GTPases suggests that inhibition of these pathways deserves to be considered as a potential therapeutic approach in treating T2D. This Perspective presents how inhibition of various points in the mevalonate pathway might affect protein prenylation and functioning of diabetes-affected tissues and contribute to chronic inflammation involved in diabetes mellitus (T2D) development. We also demonstrate the currently available molecular tools to decipher the mechanisms linking the mevalonate pathway’s enzymes and GTPases with diabetes.

Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and β1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles’ heel for a significant and unique subset of GBM patients.