GTPase Regulator Research Articles

The burden of X-linked retinitis pigmentosa (XLRP) on patient experience and patient-reported outcomes (PROs): findings from the EXPLORE XLRP-2 study.

X-linked retinitis pigmentosa (XLRP) is considered one of the most severe forms of retinitis pigmentosa (RP), accounting for 5-15% of all RP cases and primarily affecting males. However, the real-world humanistic impacts of this disease on patients are poorly investigated, especially with respect to burdens faced by patients with varying disease severities. EXPLORE XLRP-2 was an exploratory, multicentre, non-interventional study. A retrospective chart review was conducted to collect clinical/demographic data, including XLRP clinical stage (mild, moderate or severe). Cross-sectional surveys were used to gather experiences directly from patients by validated and modified patient-reported outcomes. 176 patients with XLRP caused by retinitis pigmentosa GTPase regulator (RPGR) gene mutation were enrolled, of whom 169 were included in analyses. 81% of patients were male, mean (SD) age was 39.3 (17.61) years, and 20 adolescents were included. Mean age (SD) at genetic confirmation was 33.4 years (17.98), and the mean duration (SD) from initial symptoms to genetic diagnosis was 16.4 (15.66) years. Compared with patients with mild disease, patients with severe XLRP are more likely to experience difficulties with functioning in low luminance, depression, unemployment, productivity issues, mobility and daily activities. This is the first real-world study to collect data directly from patients on the burden of XLRP and to correlate that burden with disease stage. As a result, several areas of significant burden, especially for patients with severe disease, have been identified that should provide focus for future public policies and therapeutic prospects.

Model supports asymmetric regulation across the intercellular junction for collective cell polarization.

Symmetry breaking, which is ubiquitous in biological cells, functionally enables directed cell movement and organized embryogenesis. Prior to movement, cells break symmetry to form a well-defined cell front and rear in a process called polarization. In developing and regenerating tissues, collective cell movement requires the coordination of the polarity of the migration machineries of neighboring cells. Though several works shed light on the molecular basis of polarity, fewer studies have focused on the regulation across the cell-cell junction required for collective polarization, thus limiting our ability to connect tissue-level dynamics to subcellular interactions. Here, we investigated how polarity signals are communicated from one cell to its neighbor to ensure coordinated front-to-rear symmetry breaking with the same orientation across the group. In a theoretical setting, we systematically searched a variety of intercellular interactions and identified that co-alignment arrangement of the polarity axes in groups of two and four cells can only be achieved with strong asymmetric regulation of Rho GTPases or enhanced assembly of complementary F-actin structures across the junction. Our results held if we further assumed the presence of an external stimulus, intrinsic cell-to-cell variability, or larger groups. The results underline the potential of using quantitative models to probe the molecular interactions required for macroscopic biological phenomena. Lastly, we posit that asymmetric regulation is achieved through junction proteins and predict that in the absence of cytoplasmic tails of such linker proteins, the likeliness of doublet co-polarity is greatly diminished.

Long-Term Visual Field Progression in X-Linked Retinitis Pigmentosa Patients.

We present an image that illustrates long-term visual field progression in patients with X-linked retinitis pigmentosa (XLRP) due to the retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 protein (RP2) gene variants. Longitudinal data from 84 genetically confirmed XLRP patients were collected from the Danish Retinitis Pigmentosa Registry, spanning the years 1948 to 2014. A visual field summation image revealed the characteristic pattern of retinal degeneration and visual field preservation in XLRP.

Single-Cell Transcriptomic Dataset of RPGR-associated Retinitis Pigmentosa Patient-Derived Retinal Organoids

X-linked retinitis pigmentosa (XLRP) is a severe hereditary retinal disorder marked by progressive vision loss due to photoreceptor dysfunction. The retinitis pigmentosa GTPase regulator (RPGR) gene, responsible for most XLRP cases, encodes a protein crucial for the transport of visual signal proteins between the photoreceptor inner and outer segments. However, the mechanism of RPGR mutation causing photoreceptor disorder is not clear and effective treatments remain elusive. This study utilized retinal organoids (ROs) derived from normal and RPGR-mutant human induced pluripotent stem cells (hiPSC) at four developmental stages (40, 90, 150, and 200 days). Single-cell RNA sequencing (scRNA-seq) was conducted on 71,096 cells, including 33,839 cells from the control group and 37,257 cells from the RPGR group. Key retinal cell types were identified and the obtained scRNAseq dataset was validated reliable and high -quality. This study has provided data resources and references for exploring the mechanism of RPGR-related retinal degeneration and support the development of targeted therapies.

Gut microbiome's causal role in head and neck cancer: findings from mendelian randomization.

The gut microbiome (GM) has been implicated in cancer pathogenesis and treatment, including head and neck cancers (HNC). However, the specific microbial compositions influencing HNC and the underlying mechanisms remain largely unknown. This study utilized published genome-wide association studies (GWAS) summary data-based two-sample Mendelian randomization (MR) to uncover the GM compositions that exert significant causal effects on HNC. Functional annotation and enrichment analysis were conducted to better understand the significant genetic variables and their connection with HNC. The HNC dataset included 2,281 cases and 314,193 controls. The GM GWAS data of 211 gut taxa (35 families, 20 orders, 16 classes, 9 phyla, and 131 genera) were obtained from the MibioGen consortium, involving 18,340 participants. MR analysis revealed four GM compositions exerting causal effects on HNC. Specifically, family Peptococcaceae.id.2024 was significantly associated with a 35% reduced risk of HNC (OR=0.65; 95%CI=0.48-0.90; P=0.0080). In contrast, genus DefluviitaleaceaeUCG-011.id.11287 (OR=1.54; 95%CI=1.13-2.09; P=0.0060), genus Gordonibacter.id.821 (OR=1.23; 95%CI=1.05-1.45; P=0.012), and genus Methanobrevibacter.id.123 (OR=1.28; 95%CI=1.01-1.62; P=0.040) showed a significant association with an increased risk of HNC. These GMs interact with genes and genetic variants involved in signaling pathways, such as GTPase regulation, influencing tumor progression and disease prognosis. Our study demonstrates, for the first time, the causal influence of specific gut microbiome compositions on HNC, offering significant insights for advancing clinical research and personalized treatments. The identified GMs may serve as potential biomarkers or therapeutic targets, paving the way for innovative approaches in HNC diagnosis, prevention, and therapy.

Role of Rab35 in modulating lipid metabolism and viral entry during pseudorabies virus infection

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease in swine, is a significant pathogen in veterinary medicine. Rab35 is a key regulatory GTPase involved in diverse cellular functions, including endocytic recycling, cytokinesis, and the regulation of the actin cytoskeleton. Although Rab35's roles in these processes are well-documented, its contribution to PRV replication dynamics had not been previously elucidated. Our study demonstrated that PRV infection led to an increase in Rab35 expression at both the mRNA and protein levels in both in vitro cell culture and in vivo models. Elevated Rab35 expression was associated with an acceleration of PRV replication, whereas knocking down Rab35 expression significantly impeded viral proliferation. Further investigation revealed that while Rab35 depletion did not impact the initial attachment of PRV to host cells, it critically suppressed subsequent viral entry and effectively obstructed the transcription of early PRV genes. The downregulation of Rab35 disrupted the expression of enzymes critical to lipid synthesis, which are upregulated during PRV infection. Moreover, Rab35 knockdown disrupted lipid dynamics necessary for the virus to integrate into clathrin-coated pits, a pivotal mechanism for PRV cellular entry. These findings collectively suggest that Rab35 plays a facilitatory role in PRV infection by modulating lipid metabolism and the viral entry process, thereby offering new insights into the complex intracellular mechanisms underlying PRV replication.

P01.08.A HYOXIA-INDUCED EXPRESSION OF ARHGAP FAMILY MEMBERS PROMOTES GLIOBLASTOMA DISSEMINATION

Abstract BACKGROUND The Rho GTPase activating proteins (ARHGAPs) perform vital roles in the regulation of the Rho GTPases with direct effect on cellular plasticity and cell migration/invasion. We recently reported on the role of five ARHGAPs, identified by a microarray screen, in morphological plasticity in established and patient-derived Glioblastoma (GBM) cell lines and associated clinical relevance in terms of tumour recurrence after treatment. We established that the ARHGAPs either promoted or targeted cell migration/invasion by allowing cells to undergo morphological changes in preparation for dissemination. We also showed that co-expression of two ARHGAPs, 12 and 29, is associated with reduced time to recurrence after initial treatment in patients. ARHGAP activity has been also reported in metastatic cancers including breast and colorectal cancer highlighting the biological relevance of these, as yet understudied, proteins. MATERIAL AND METHODS Here, we explored the GBM microenvironment and the effect of hypoxia on ARHGAP expression and localisation in glioma cell lines. RESULTS Using immunocytological and in vitro migration and invasion assays we were able to ascertain that ARHGAP4, 12, 22, 25 and 29 acquired subcellular distributions different to those recorded under normoxia and expression also decreased or increased under hypoxia. We also found that the effect of the migrastatic inhibitor 6-bromo-indirubin-3′-oxime (BIO) targeting GSK-3 signalling was potentiated when cells were challenged with the drug under hypoxic conditions. CONCLUSION Our results confirm our initial findings of biological relevance of the ARHGAPs in cellular activities and highlight important roles for the ARHGAPs for promoting migratory/invasive activity and behaviour in GBM cells. We also conclude that migrastatic inhibitors will exert enhanced activity on GBM cells under hypoxic conditions making them excellent candidates for complement treatment with cytotoxic drugs or radiotherapy for an improved management of GBM.

ARL13B controls male reproductive tract physiology through primary and Motile Cilia

ARL13B is a small regulatory GTPase that controls ciliary membrane composition in both motile cilia and non-motile primary cilia. In this study, we investigated the role of ARL13B in the efferent ductules, tubules of the male reproductive tract essential to male fertility in which primary and motile cilia co-exist. We used a genetically engineered mouse model to delete Arl13b in efferent ductule epithelial cells, resulting in compromised primary and motile cilia architecture and functions. This deletion led to disturbances in reabsorptive/secretory processes and triggered an inflammatory response. The observed male reproductive phenotype showed significant variability linked to partial infertility, highlighting the importance of ARL13B in maintaining a proper physiological balance in these small ducts. These results emphasize the dual role of both motile and primary cilia functions in regulating efferent duct homeostasis, offering deeper insights into how cilia related diseases affect the male reproductive system.

Establishing Clinical Trial Endpoints in Selecting Patients for RPGR Retinal Gene Therapy.

Clinical trials for X-linked retinitis pigmentosa (RP) often assess retinal structure using optical coherence tomography (OCT) and function using microperimetry to evaluate initial eligibility and endpoints. Therefore, we seek to determine which parameters might be most sensitive in screening new patients for enrollment. Thirty-one patients (62 eyes) with confirmed retinitis pigmentosa GTPase regulator (RPGR) mutations attending Oxford Eye Hospital were included in this retrospective analysis. Outer retinal structure was investigated by measuring the remaining ellipsoid zone (EZ) and external limiting membrane (ELM) on OCT. Visual function was evaluated by using 10-2 microperimetry mean sensitivity. The median age of patients with RPGR was 31 years (interquartile range [IQR] = 22-39 years). For the right and left eyes, respectively, the median EZ length through the foveal section was 921 µm (IQR = 607-1570) and 865 µm (IQR = 508-1442) and median ELM length was 2056 µm (IQR = 1336-2764) and 1860 µm (IQR = 1152-2680). Similarly, the median microperimetry sensitivity (MS) was 2.0 decibel (dB; IQR = 0.4-5.4) and 1.1 dB (IQR = 0.1-5.4). Linear mixed model regression analysis showed that EZ was significantly positively correlated to ELM (P < 0.001, R² = 0.931). EZ and ELM were significantly correlated with the microperimetry sensitivity with exponential relationship (P < 0.001, R² = 0.71 and 0.72, respectively). Using the exponential equation of regression line, EZ below approximately 600 µm (RE = 637 µm, 95% confidence interval [CI] = 397-877, LE = 586 µm, 95% CI = 356-817) results in microperimetry sensitivity of approximately 0 dB. There was high degree of inter-eye symmetry for progression of EZ, ELM, and microperimetry sensitivity. Age was significantly correlated with the analyzed parameters (P < 0.001), although with low R² for each of them. EZ may comprise a surrogate biomarker for prediction of visual function in X-linked RP caused by mutations in RPGR and, in turn, identification of appropriate patients for enrollment in clinical trials. As expected, age plays a role in predicting potential biomarkers and visual function, however, it should not be used to preclude patients for gene therapy due to the poor correlation and heterogeneity of disease onset. Biomarkers of visual function in RPGR-associated RP may lead to identification of appropriate patients for enrollment in clinical trials.

ARHGEF3 Regulates Hair Follicle Morphogenesis.

During embryogenesis, cells arrange into precise patterns that enable tissues and organs to develop specialized functions. Despite its critical importance, the molecular choreography behind these collective cellular behaviors remains elusive, posing a major challenge in developmental biology and limiting advances in regenerative medicine. By using the mouse hair follicle as a mini-organ system to study the formation of bud-like structures during embryonic development, our work uncovers a crucial role for the Rho GTPase regulator ARHGEF3 in hair follicle morphogenesis. We demonstrate that Arhgef3 expression is upregulated at the onset of hair follicle placode formation. In Arhgef3 knockout animals, we observed defects in placode compaction, leading to impaired hair follicle downgrowth. Through cell culture models, we show that ARHGEF3 promotes F-actin accumulation at the cell cortex and P-cadherin enrichment at cell-cell junctions. Collectively, our study identifies ARHGEF3 as a new regulator of cell shape rearrangements during hair placode morphogenesis, warranting further exploration of its role in other epithelial appendages that arise from similar developmental processes.

XOLARIS: A 24-Month, Prospective, Natural History Study of 201 Participants with Retinitis Pigmentosa GTPase Regulator-Associated X-Linked Retinitis Pigmentosa

ObjectiveTo improve the understanding of the natural disease progression of retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). DesignA multicenter, prospective, observational natural history study over 24 months. ParticipantsMale participants aged ≥7 years with a pathogenic variant in the RPGR gene, a best-corrected visual acuity (BCVA) score of ≥34 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, and a mean 68-loci retinal sensitivity (assessed by microperimetry) of 0.1–20 decibels (dB). MethodsParticipants were divided into subgroups based on their BCVA score at baseline: 34–73 (lower BCVA) or ≥74 (higher BCVA) ETDRS letters. There were 7 visits over 24 months. Main Outcome MeasuresChange from baseline in BCVA, retinal sensitivity, low luminance visual acuity (LLVA), fixation stability, contrast sensitivity, visual field, anatomical measures, 25-item Visual Function Questionnaire (VFQ-25), intraocular pressure, and adverse events (AEs). ResultsOverall, 201 participants were included. The mean (standard deviation [SD]) age was 30.3 (11.9) years in the lower BCVA subgroup (n=170) and 27.7 (10.1) years in the higher BCVA subgroup (n=31). The study eye baseline mean (SD) BCVA scores were 59.4 (10.30) and 77.3 (3.95) in the lower and higher BCVA subgroups, respectively; the lower BCVA subgroup had lower retinal sensitivity in the study eye at baseline than the higher BCVA subgroup. Over 24 months, there were small observed changes in BCVA, retinal sensitivity, LLVA, fixation, contrast sensitivity, and fundus photography findings. There were observed mean (SD) changes at 24 months in the lower and higher BCVA subgroups of −1.01 (4.67) and 0.03 (5.83) dB-steradians in the volume of full-field hill of vision, −330.6 (869.51) and −122.7 (22.01) μm in distance from foveal center to the nearest border of preserved fundus autofluorescence, −104.3 (277.80) and −207.1 (171.01) μm in central ellipsoid width, and −2.8 (9.7) and −0.6 (7.6) in VFQ-25 composite score, respectively. There was 1 death from completed suicide. There were no ocular serious AEs, and most AEs were mild/moderate. ConclusionsThis study provides evidence of the slow natural progression of XLRP over 24 months in both subgroups and provides important functional, anatomical, and safety data.

BCAR3 and BCAR3-related competing endogenous RNA expression in hepatocellular carcinoma and their prognostic value.

Hepatocellular carcinoma (HCC) is a malignant tumor that has a high incidence and mortality worldwide. Despite extensive studies, the detailed molecular mechanism of HCC development remains unclear. Studies have shown that the occurrence and development of HCC are closely related to abnormal gene expression. BCAR3 has been shown to be overexpressed in a variety of malignant tumors. However, the role of BCAR3 in HCC remains unclear. To investigate the expression of BCAR3 and BCAR3-related competing endogenous RNAs (ceRNAs) in HCC and their clinical significance, in order to provide new ideas for the diagnosis and treatment of HCC. The data of HCC were obtained from the Cancer Genome Atlas database and The Genotype Tissue Expression, including transcriptome data and clinical information. Multiple common databases, including UALCAN, Timer 2.0, cBioPortal, LinkedOmics, starBase, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, were used to analyse the expression of BCAR3, prognostic value, genetic alteration, co-expressed genes, differentially expressed genes, BCAR3 gene-related ceRNAs and functional enrichment analysis in HCC patients. Kaplan-Meier analysis, univariate and multivariate Cox regression analysis were used to analyze survival prognosis and the Spearman test was used to measure correlations between BCAR3 and immune functions. And R language package was used to analyze the correlation between BCAR3 and immune invasion of HCC. Our study indicated that BCAR3 was differentially expressed in various tumor tissues. The over-expression of BCAR3 gene was an unfavorable prognostic indicator for HCC patients, and associated with unfavorable cytogenetic risk and gene mutations. Moreover, most immune cells were positively correlated with BCAR3 (P < 0.05). According to the results of functional enrichment analysis, BCAR3 was involved in the positive regulation of epidermal growth factor receptor signaling pathway and ERBB signaling pathway, and was related to DNA replication and GTPase regulator activity. Finally, our study found that based on RAB30-DT and miR-19b-3p pathways, targeting BCAR3 might promote the occurrence and development of HCC. Collectively, this study indicated that the BCAR3 gene was involved in the occurrence and development of HCC, and it might be a new biomarker and therapeutic target for HCC, but the specific mechanism remains to be further verified.

Investigating the impact of asymmetric macular sensitivity on visual acuity chart reading in choroideremia.

Degeneration in choroideremia, unlike typical centripetal photoreceptor degenerations, is centred temporal to the fovea. Once the fovea is affected, the nasal visual field (temporal retina) is relatively spared, and the preferred retinal locus shifts temporally. Therefore, when reading left to right, only the right eye reads into a scotoma. We investigate how this unique property affects the ability to read an eye chart. Standard- and low-luminance visual acuity (VA) for right and left eyes were measured with the Early Treatment of Diabetic Retinopathy Study (ETDRS) chart. Letters in each line were labelled by column position. The numbers of letter errors for each position across the whole chart were summed to produce total column error scores for each participant. Macular sensitivity was assessed using microperimetry. Central sensitivity asymmetry was determined by the temporal-versus-nasal central macular difference and subsequently correlated to a weighted ETDRS column error score. Healthy volunteers and participants with X-linked retinitis pigmentosa GTPase regulator associated retinitis pigmentosa (RPGR-RP) were used as controls. Thirty-nine choroideremia participants (median age 44.9 years [IQR 35.7-53.5]), 23 RPGR-RP participants (median age 30.8 years [IQR 26.5-40.5]) and 35 healthy controls (median age 23.8 years [IQR 20.3-29.0]) were examined. In choroideremia, standard VA in the right eye showed significantly greater ETDRS column errors on the temporal side compared with the nasal side (p = 0.002). This significantly correlated with greater asymmetry in temporal-versus-nasal central macular sensitivity (p = 0.04). No significant patterns in ETDRS column errors or central macular sensitivity were seen in the choroideremia left eyes, nor in RPGR-RP and control eyes. Difficulty in tracking across lines during ETDRS VA testing may cause excess errors independent of true VA. VA assessment with single-letter optotype systems may be more suitable, particularly for patients with choroideremia, and potentially other retinal diseases with asymmetric central macular sensitivity or large central scotomas including geographic atrophy.

Validation of Nanopore long-read sequencing to resolve RPGR ORF15 genotypes in individuals with X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is characterized by progressive vision loss leading to legal blindness in males and a broad severity spectrum in carrier females. Pathogenic alterations of the retinitis pigmentosa GTPase regulator gene (RPGR) are responsible for over 70% of XLRP cases. In the retina, the RPGRORF15 transcript includes a terminal exon, called ORF15, that is altered in the large majority of RPGR-XLRP cases. Unfortunately, due to its highly repetitive sequence, ORF15 represents a considerable challenge in terms of sequencing for molecular diagnostic laboratories. However, in a recent preliminary work Yahya et al. reported a long-read sequencing approach seeming promising. Here, the aim of the study was to validate and integrate this new sequencing strategy in a routine screening workflow. For that purpose, we performed a masked test on 52 genomic DNA samples from male and female individuals carrying 32 different pathogenic ORF15 variations including 20 located in the highly repetitive region of the exon. For the latter, we have obtained a detection rate of 80-85% in males and 60-80% in females after bioinformatic analyses. These numbers raised to 100% for both status after adding a complementary visual inspection of ORF15 long-reads. In accordance with these results, and considering the frequency of ORF15 pathogenic variations in XLRP, we suggest that a long-read screening of ORF15 should be systematically considered before any other sequencing approach in subjects with a diagnosis compatible with XLRP.

A rho-type GTPase activating protein affects the growth and development of Cordyceps cicadae.

Cordyceps cicadae is recognized for its medicinal properties, attributed to bioactive constituents like polysaccharides and adenosine, which have been shown to improve kidney and liver functions and possess anti-tumor properties. Rho GTPase activating proteins (Rho GAPs) serve as inhibitory regulators of Rho GTPases in eukaryotic cells by accelerating the GTP hydrolysis of Rho GTPases, leading to their inactivation. In this study, we explored the function of the CcRga8 gene in C. cicadae, which encodes a Rho-type GTPase activating protein. Our study found that the knockout of CcRga8 resulted in a decrease in polysaccharide levels and an increase in adenosine concentration. Furthermore, the mutants exhibited altered spore yield and morphology, fruiting body development, decreased infectivity, reduced resistance to hyperosmotic stress, oxidative conditions, and cell wall inhibitors. These findings suggest that CcRga8 plays a crucial role in the development, stress response, and bioactive compound production of C. cicadae.

Exploring self-reported visual function and vision-related anxiety in patients with RPGR-associated retinal degeneration

Variants in the retinitis pigmentosa GTPase regulator (RPGR) gene are responsible for the majority of X-linked retinitis pigmentosa cases, which not only affects male patients but also some heterozygous females. Vision-related disability and anxiety of patients with RPGR-associated retinal degeneration have never been explored before. This study aimed to evaluate self-reported visual function and vision-related anxiety in a Portuguese cohort of male and female patients with RPGR-associated retinal degeneration using two validated patient-reported outcome measures. Cross-sectional data of thirty-two genetically-tested patients was examined, including scores of the Michigan retinal degeneration questionnaire (MRDQ) and Michigan vision-related anxiety questionnaire. Patients were classified according to retinal phenotypes in males (M), females with male phenotype (FM), and females with radial or focal pattern. Both M and FM revealed higher rod-function and cone-function anxiety scores (p < 0.017). Most MRDQ disability scores were higher in M and FM (p < 0.004). Overall, positive correlations (p < 0.004) were found between every MRDQ domain and both anxiety scores. In RPGR-associated retinal degeneration, males and females with male phenotype show similar levels of increased vision-related anxiety and disability. Every MRDQ visual function domain showed a strong correlation with anxiety scores.

RhoGDI in RBL-2H3 cells acts as a negative regulator of Rho GTPase signaling to inhibit granule exocytosis.

Mast cells are hematopoietic-derived immune cells that possess numerous cytoplasmic granules containing immune mediators such as cytokines and histamine. Antigen stimulation triggers mast cell granule exocytosis, releasing granule contents in a process known as degranulation. We have shown that Rho GTPase signaling is an essential component of granule exocytosis, however, the proteins that regulate Rho GTPases during this process are not well defined. Here we examined the role of Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) in regulating Rho GTPase signaling using RBL-2H3 cells as a mast cell model. We found that RBL-2H3 cells express two RhoGDI isoforms which are primarily localized to the cytosol. Knockdown of RhoGDI1 and RhoGDI2 greatly reduced the levels of all Rho GTPases tested: RhoA, RhoG, Rac1, Rac2, and Cdc42. The reduction in Rho GTPase levels was accompanied by an increase in their membrane-localized fraction and an elevation in the levels of active Rho GTPases. All RhoGDI knockdown strains had altered resting cell morphology, although each strain was activation competent when stimulated. Live cell imaging revealed that the RhoGDI1/2 double knockdown (DKD) strain maintained its activated state for prolonged periods of time compared to the other strains. Only the RhoGDI1/2 DKD strain showed a significant increase in granule exocytosis. Conversely, RhoGDI overexpression in RBL-2H3 cells did not noticeably affect Rho GTPases or degranulation. Based on these results, RhoGDIs act as negative regulators of Rho GTPases during mast cell degranulation, and inhibit exocytosis by sequestering Rho GTPases in the cytosol.

Phase 1/2 AAV5-hRKp.RPGR (Botaretigene Sparoparvovec) Gene Therapy: Safety and Efficacy in RPGR-Associated X-Linked Retinitis Pigmentosa

To assess the safety and efficacy of AAV5-hRKp.RPGR in participants with retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). Open-label, phase 1/2 dose escalation/expansion study (ClinicalTrials.gov Identifier: NCT03252847). Males (≥5 years old) with XLRP-RPGR were evaluated. In the dose escalation phase, subretinal AAV5-hRKp.RPGR (low: 1.0 × 1011 vg/ml; intermediate: 2.0 × 1011 vg/ml; high: 4.0 × 1011 vg/ml) was administered to the poorer-seeing eye (n = 10). Dose confirmation (intermediate dose) was carried out in 3 pediatric participants. In the dose expansion phase, 36 participants were randomized 1:1:1 to immediate (low or intermediate dose) or deferred (control) treatment. The primary outcome was safety. Secondary efficacy outcomes included static perimetry, microperimetry, vision-guided mobility, best corrected visual acuity, and contrast sensitivity. Safety and efficacy outcomes were assessed for 52 weeks for immediate treatment participants and 26 weeks for control participants. AAV5-hRKp.RPGR was safe and well tolerated, with no reported dose-limiting events. Most adverse events (AEs) were transient and related to the surgical procedure, resolving without intervention. Two serious AEs were reported with immediate treatment (retinal detachment, uveitis). A third serious AE (increased intraocular pressure) was reported outside the reporting period. All ocular inflammation-related AEs responded to corticosteroids. Treatment with AAV5-hRKp.RPGR resulted in improvements in retinal sensitivity and functional vision compared with the deferred group at Week 26; similar trends were observed at Week 52. AAV5-hRKp.RPGR demonstrated an anticipated and manageable AE profile through 52 weeks. Safety and efficacy findings support investigation in a phase 3 trial.

RPGRIP1L as a new biomarker for prognosis and tumor immune of breast cancer.

The Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) gene encodes an important protein that performs various physiological functions. Variants of RPGRIP1L are related to a number of diseases. However, it is currently unknown whether RPGRIP1L is correlated with breast invasive carcinoma (BRCA). In BRCA tissue specimens, the expression of RPGRIP1L was found to be elevated in comparison to its levels in normal breast tissue. A notable decline in survival rates was associated with patients exhibiting heightened RPGRIP1L gene expression. Consistent with these findings, our data also show the above results. Furthermore, elevated expression of RPGRIP1L corresponded with a spectrum of unfavorable clinicopathological features, including the presence of human epidermal growth factor receptor 2 (HER2) positive, estrogen receptor (ER) positive, over 60 years old, T2, N0, and N3. At the same time, our research indicated that 50 genes and 15 proteins were positively related to RPGRIP1L, and that these proteins and genes were mostly involved in T cell proliferation, immune response, cytokine activity, and metabolic regulation. In addition, in the present study, there was a significant correlation between RPGRIP1L expression and immune cell infiltration. Finally, we found that four Chemicals could downregulate the expression of RPGRIP1L. Altogether, our results strongly indicated that RPGRIP1L might serve as a new prognostic biomarker for BRCA.

Abstract PO4-05-13: Expression of DLC1/STARD12 and variants in clinical breast cancer and implication in skeletal metastasis

Abstract Introduction: Deleted in Liver Cancer 1 Protein, DLC1, also known as StAR Related Lipid Transfer Domain Containing 12 (StarD12), is a known negative regulator of the Rho GTPases and at the cellular level regulates cell migration. DLC1 gene transcription produces multiple isoforms of the DLC1 protein. The present study examined the clinical and prognostic implications of DLC1 isoforms, DLC1/V1/V2/V3, in relationship with Rho GTPases in human breast cancer. Methods: Expression of three DLC1 isoforms was evaluated in a cohort of breast cancer tissues and against the clinical, pathological and clinical outcome of patients. DLC1 expression was also assessed and compared against Rho GTPases, including Rho and Rho GTP Dissociation Inhibitor (Rho GDI), in the same cohort. Results: DLC1/V3 had a significant impact on the overall survival of patients and high levels were linked to shorter survival (Hazard Ratio=3.551 (95%CI 1.332-9.471, p=0.011), a prediction that was independent of other factors in multivariate analysis. RUC analysis demonstrated a significant predictive value for bone metastasis with DLC1/V3 (RUC=0.714, p=0.020), showing a Hazard ratio of 9.0 (1.8-44.5, p=0.007). Similarly, high DLC1/V2 expression was also linked to a shorter overall survival (p=0.035). DLC1/V2 did not show a significant correlation with bone metastasis. In contrast to V3 and V2 variants, DLC1 had a marginal overall survival benefit for the patients, in that patients with high DLC1 had a longer survival (p=0.056). DLC1/V3 had a contrasting correlation with Rho-A (r=-0.203, p=0.015) and Rho-GDI-G (r=0.194, p=0.019) and the combined expression of DLC1/V3 and RhoA/Rho-GDI-G further identified patients with poorer clinical outcome. Conclusions: DLC1 and its variants have differing impacts on breast cancer; whereas DLC1 appears to be a tumour suppressive molecule, its variant isoform-3 was more oncogenic and a significant negative prognostic factor, together with its regulators Rho and Rho GDI. Citation Format: Binbin Cong, Jane Lane, Xiaoshan Cao, Tracey Martin, Robert E Mansel, Eleri Davies, Wen Jiang. Expression of DLC1/STARD12 and variants in clinical breast cancer and implication in skeletal metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-05-13.