κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion.

Extracellular vesicles-mediated exchange of miRNA cargos between diverse types of mammalian cells is a major mechanism of controlling cellular miRNA levels and activity, thus regulating the expression of miRNA-target genes in both donor and recipient cells. Despite tremendous excitement related to extracellular vesicles-associated miRNAs as biomarkers or having therapeutic potential, the mechanism of selective packaging of miRNAs into endosomes and multivesicular bodies for subsequent extracellular export is poorly studied due to the lack of an in vitro assay system. Here, we have developed an in vitro assay with endosomes isolated from mammalian macrophage cells to follow miRNA packaging into endocytic organelles. The synthetic miRNAs, used in the assay, get imported inside the isolated endosomes during the in vitro reaction and become protected from RNase in a time- and concentration-dependent manner. The selective miRNA accumulation inside endosomes requires both ATP and GTP hydrolysis and the miRNA-binding protein HuR. The HuR-miRNA complex binds and stimulates the endosomal RalA GTPase to facilitate the import of miRNAs into endosomes and their subsequent export as part of the extracellular vesicles. The endosomal targeting of miRNAs is also very much dependent on the endosome maturation process that is controlled by Rab5 protein and ATP. In summary, we provide an in vitro method to aid in the investigation of the mechanism of miRNA packaging process for its export from mammalian macrophage cells.

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.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.

UNASSIGNED: Osteosarcoma (OS) is the most common primary aggressive sarcoma of bone, with massive aberrant expression of oncogenes related to the development of OS. RALA, a kind of small Ras-like guanosine triphosphatases, has been identified as a potential therapeutic target in several types of tumor, but its role in OS remains largely unknown.
UNASSIGNED: Abnormal expression of RALA was proven in the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and RNA-sequence of samples and cell lines. The role of RALA in OS was analyzed in terms of DNA methylation, immune cell infiltration, and patient survival. The cancer-promoting effect of RALA was demonstrated in cell lines and xenograft osteosarcoma models. A prognostic scoring model incorporating RALA as an indicator was established with the clinical samples that we collected.
UNASSIGNED: The results showed that RALA was highly expressed in human OS tissues and cell lines. Survival analysis demonstrated that RALA was the sole independent risk factor for poor overall survival and disease-free survival in OS patients and impacted the proportion of infiltrating immune cells and DNA methylation in the OS tumor microenvironment. By gene-gene interaction analysis, we found that the expression of RALA was highly correlated to the expression of ABCE1. Similar to RALA, upregulated ABCE1 is correlated with poor survival outcome of OS patients. In addition, the functional experiment demonstrated that higher expression of RALA promoted the proliferation, migration, and invasion of OS cells. In vivo results were similar with the in vitro results. We examined m6a methylation-related genes and found that m6A methylation is responsible for the abnormal expression of RALA. Finally, the prognostic prediction model of RALA could be used to predict the long-term outcome of OS patients.
UNASSIGNED: We identified RALA as an oncogene in OS, and RALA upregulation in a concerted manner with ABCE1 was significantly associated with worse outcomes of OS patients. Targeting RALA may prove to be a novel target for OS immunotherapy in future clinical practice.

Surface levels of membrane proteins are determined by a dynamic balance between exocytosis-mediated surface delivery and endocytosis-dependent retrieval from the cell surface. Imbalances in surface protein levels perturb surface protein homeostasis and cause major forms of human disease such as type 2 diabetes and neurological disorders. Here, we found a Reps1-Ralbp1-RalA module in the exocytic pathway broadly regulating surface protein levels. Reps1 and Ralbp1 form a binary complex that recognizes RalA, a vesicle-bound small guanosine triphosphatases (GTPase) promoting exocytosis through interacting with the exocyst complex. RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex. Ralbp1 selectively recognizes GTP-bound RalA but is not a RalA effector. Instead, Ralbp1 binding maintains RalA in an active GTP-bound state. These studies uncovered a segment in the exocytic pathway and, more broadly, revealed a previously unrecognized regulatory mechanism for small GTPases, GTP state stabilization.

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.

BACKGROUND: Early metastasis is a key factor contributing to poor breast cancer (BC) prognosis. Circulating tumor cells (CTCs) are regarded as the precursor cells of metastasis, which are ultimately responsible for the main cause of death in BC. However, to date molecular mechanisms underlying CTC formation in BC have been insufficiently defined.
METHODS: RNA-seq was carried out in primary tissues from early-stage BC patients (with CTCs≥5 and CTCs = 0, respectively) and the validation study was conducted in untreated 80 BC patients. Multiple in vitro and in vivo models were used in functional studies. Luciferase reporter, ChIP-seq, CUT&Tag-seq, and GST-pulldown, etc. were utilized in mechanistic studies. CTCs were counted by the CanPatrol™ CTC classification system or LiquidBiospy™ microfluidic chips. ERK1/2 inhibitor SCH772984 was applied to in vivo treatment.
RESULTS: Highly expressed FOXD1 of primary BC tissues was observed to be significantly associated with increased CTCs in BC patients, particularly in early BC patients. Overexpressing FOXD1 enhanced the migration capability of BC cells, CTC formation and BC metastasis, via facilitating epithelial-mesenchymal transition of tumor cells. Mechanistically, FOXD1 was discovered to induce RalA expression by directly bound to RalA promotor. Then, RalA formed a complex with ANXA2 and Src, promoting the interaction between ANXA2 and Src, thus increasing the phosphorylation (Tyr23) of ANXA2. Inhibiting RalA-GTP form attenuated the interaction between ANXA2 and Src. This cascade culminated in the activation of ERK1/2 signal that enhanced metastatic ability of BC cells. In addition, in vivo treatment with SCH772984, a specific inhibitor of ERK1/2, was used to dramatically inhibit the CTC formation and BC metastasis.
CONCLUSIONS: Here, we report a FOXD1-dependent RalA-ANXA2-Src complex that promotes CTC formation via activating ERK1/2 signal in BC. FOXD1 may serve as a prognostic factor in evaluation of BC metastasis risks. This signaling cascade is druggable and effective for overcoming CTC formation from the early stages of BC.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

Sarcoplasmic/endoplasmic reticulum calcium ATPase SERCA2 mediates calcium re-uptake from the cytosol into sarcoplasmic reticulum, and its dysfunction is a hallmark of heart failure. Multiple factors have been identified to modulate SERCA2 activity, however, its regulation is still not fully understood. Here we identify a Ral-GTPase activating protein RalGAPα1 as a critical regulator of SERCA2 in cardiomyocytes through its downstream target RalA. RalGAPα1 is induced by pressure overload, and its deficiency causes cardiac dysfunction and exacerbates pressure overload-induced heart failure. Mechanistically, RalGAPα1 regulates SERCA2 through direct interaction and its target RalA. Deletion of RalGAPα1 decreases SERCA2 activity and prolongs calcium re-uptake into sarcoplasmic reticulum. GDP-bound RalA, but not GTP-bound RalA, binds to SERCA2 and activates the pump for sarcoplasmic reticulum calcium re-uptake. Overexpression of a GDP-bound RalAS28N mutant in the heart preserves cardiac function in a mouse model of heart failure. Our findings have therapeutic implications for treatment of heart failure.