Roundabout Guidance Receptor Research Articles

Investigation of Association Between Expression of DYX1C1, KIAA0319, and ROBO1 Genes and Specific Learning Disorder in Children and Adolescents.

Specific learning disorder (SLD) is prevalent worldwide and is a complex disorder with variable symptoms and significant differences among individuals. Epigenetic markers may alter susceptibility to neurodevelopmental disorders (NDDs). Aberrant expression of protein-coding (mRNA) genes in this pathology shows that the detection of epigenetic molecular biomarkers is of increasing importance in the diagnosis and treatment of individuals with SLD. We compared gene expression level of dyslexia susceptibility 1 candidate gene 1 (DYX1C1), dyslexia-associated protein KIAA0319 (KIAA0319), and roundabout guidance receptor 1 (ROBO1) between children with SLD and healthy children by performing quantitative polymerase chain reaction (qPCR). In addition, we evaluated these gene expressions of severe children with SLD compared to non-severe and male SLD children compared to females. The expression of the DYX1C1, KIAA0319, and ROBO1 genes was statistically significantly upregulated in children with SLD (P < 0.05*). DYX1C1 was also upregulated in severe SLD children (P = 0.03*). In addition, KIAA0319 and ROBO1 genes were differentially expressed in male SLD children compared to females (P < 0.05*). Furthermore, we found that DYX1C1 and ROBO1 genes significantly affect the likelihood of the SLD (respectively, P < 0.001** and P = 0.007*). We expect that the findings provided from this study may contribute to the determination expression level of the relevant genes in the diagnosis, prognosis, and treatment of SLD. In addition, our findings could be a guide for future epigenetics studies on the use of the DYX1C1, KIAA0319, and ROBO1 in therapeutic applications in the SLD.

Cells of the adult mouse cardiac conduction system in wild type and Nkx2-5 heterozygous loss-of-function mutation

Abstract Background Specialised cardiomyocytes in the cardiac conduction system (CCS) generate and spread the electrical impulse that initiates synchronised heartbeats. Disrupted signal transduction in the atrioventricular (AV) node, a key electrical connection between atria and ventricles, causes AV block. AV block potentially leads to electromechanical uncoupling and sudden cardiac death. Nkx2-5 encodes a cardiac transcription factor that is critical for embryonic CCS formation. Heterozygous loss-of-function mutation of Nkx2-5 (Nkx2-5+/–) results in a hypoplastic CCS in humans and mice and progressive AV block. Purpose We aimed to create a cell atlas of the adult mouse CCS and define the consequences of Nkx2-5 haploinsufficiency on the structural organisation and transcript expression of CCS cells. Methods We performed single-nucleus RNA sequencing on the sinoatrial nodes, AV nodes, His bundles, right bundle branches (RBB) and moderator bands of 17 wild type and 14 Nkx2-5+/– mice (aged 13 to 35 weeks, 5159 CCS cells). Employing single-molecule fluorescent RNA in situ hybridisation (smFISH), we validated selected transcripts and examined the spatial organisation of identified cell populations. Results In the AV node, we identified two subpopulations of pacemaker cells that were spatially separated in the proximal and distal pacemaker region (Fig1). Within these subpopulations, expressed transcripts indicated an atrial-to-ventricular gradient of electrical activity, corresponding to their respective topography. We determined two subpopulations in the His bundle and RBB that were enriched for either slow or fast conduction markers. Spatially, the slow-conducting populations seemed to surround the fast-conducting ones and might act as electrical isolation (Fig1). In Nkx2-5+/- cells of the pacemaker AV node, His bundle, RBB and Purkinje fibres, genes involved in Slit-Robo signalling were dysregulated. Slit-Robo signalling was previously reported in neuronal differentiation and embryonic heart lumen formation. Slit ligand 3 was downregulated in Nkx2-5+/- pacemaker AV node cells (Slit3, log2FC = -2.22, p = 7.0e-42), as was the roundabout guidance receptor 1 (Robo1, log2FC = -0.96, p = 1.6e-4). By contrast, there was upregulation of neural EGF-like 1 (Nell1, log2FC = 3.18, p = 5.3e-6), an alternative ligand of roundabout guidance receptors. Altered Nell1 and Slit3 expressions were confirmed by smFISH (Fig2). Conclusions Our cell atlas defines new CCS cell populations with informative transcriptomic profiles and spatial organisation, providing insights into subpopulations in the AV junction where electrical signals switch from slow to fast conduction. We identified disrupted Slit-Robo signalling in Nkx2-5+/-, possibly impairing differentiation and maturation of the CCS and altering electrophysiologic functions. Our normative anatomical and transcription data on the CCS can inform future studies of cardiac electrophysiology and arrhythmias.Fig1 - Subpopulations in AV junctionFig2 - Nell1 smFISH in pacemaker AV node

Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood-brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50-100% of mice. Our study identifies a promising multi-targetable PTP4A-ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

Role of Csdc2 in Regulating Secondary Hair Follicle Growth in Cashmere Goats.

Cashmere goats possess two types of hair follicles, with the secondary hair follicles producing valuable cashmere fiber used for textiles. The growth of cashmere exhibits a seasonal pattern arising from photoperiod change. Transcription factors play crucial roles during this process. The transcription factor, cold-shock domain, containing C2 (Csdc2) plays a crucial role in modulating cell proliferation and differentiation. Our preceding research indicated that the expression of Csdc2 changes periodically during anagen to telogen. However, the mechanisms of Csdc2 in regulating SHF growth remain unclear. Here, we found that the knockdown of Csdc2 inhibits the proliferation of dermal papilla cells. ChIP-Seq analysis showed that Csdc2 had a unique DNA binding motif in SHFs. Through conjoint analysis of ChIP-Seq and RNA-Seq, we revealed a total of 25 candidate target genes of Csdc2. Notably, we discovered a putative Csdc2 binding site within roundabout guidance receptor 2 (Robo2) on chromosome 1 of the goat genome. Furthermore, qRT-PCR and dual-luciferase reporter assay confirmed Csdc2's positive regulatory influence on Robo2. These findings expand the research field of hair follicle transcriptional regulatory networks, offering insights into molecular breeding strategies to enhance cashmere production in goats.

Abstract 60: Functional mapping of glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in highly invasive brain cancers

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic effects of PTP4A2 inhibition. Since a pan-PTP4A inhibitor was limited by poor penetrance across the blood brain barrier (BBB) in vivo, a second-generation chimeric antigen receptor (CAR)-T cell therapy was engineered against ROBO1 that elicits specific and potent anti-tumor responses in vivo. A single dose of anti-ROBO1 CAR-T cells doubles median survival in patient-derived xenograft (PDX) models of recurrent glioblastoma, and also eradicates tumors in around 50% of mice engrafted with PDX models of lung-to-brain metastases and pediatric relapsed medulloblastoma. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A-ROBO1 signaling axis at GBM recurrence, with potential in other malignant brain tumors. Citation Format: Sheila Kumari Singh, Chirayu Chokshi, Vaseem Shaikh, Benjamin Brakel, Alisha Anand, Chitra Venugopal, Thomas Kislinger, Kevin Henry, Martin Rossotti, Kevin Brown, John Lazo, Jason Moffat. Functional mapping of glioblastoma recurrence reveals targetable dependencies in an axonal guidance pathway in highly invasive brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 60.

Endothelial Robo4 suppresses endothelial-to-mesenchymal transition induced by irradiation and improves hematopoietic reconstitution

Bone marrow ablation is routinely performed before hematopoietic stem cell transplantation (HSCT). Hematopoietic stem and progenitor cells (HSPCs) require a stable bone marrow microenvironment to expand and refill the peripheral blood cell pool after ablation. Roundabout guidance receptor 4 (Robo4) is a transmembrane protein exclusive to endothelial cells and is vital in preserving vascular integrity. Hence, the hypothesis is that Robo4 maintains the integrity of bone marrow endothelial cells following radiotherapy. We created an endothelial cell injury model with γ-radiation before Robo4 gene manipulation using lentiviral-mediated RNAi and gene overexpression techniques. We demonstrate that Robo4 and specific mesenchymal proteins (Fibronectin, Vimentin, αSma, and S100A4) are upregulated in endothelial cells exposed to irradiation (IR). We found that Robo4 depletion increases the expression of endoglin (CD105), an auxiliary receptor for the transforming growth factor (TGF-β) family of proteins, and promotes endothelial-to-mesenchymal transition (End-MT) through activation of both the canonical (Smad) and non-canonical (AKT/NF-κB) signaling pathways to facilitate Snail1 activation and its nuclear translocation. Endothelial Robo4 overexpression stimulates the expression of immunoglobulin-like adhesion molecules (ICAM-1 and VCAM-1) and alleviates irradiation-induced End-MT. Our coculture model showed that transcriptional downregulation of endothelial Robo4 reduces HSPC proliferation and increases HSC quiescence and apoptosis. However, Robo4 overexpression mitigated the damaged endothelium’s suppressive effects on HSC proliferation and differentiation. These findings indicate that by controlling End-MT, Robo4 preserves microvascular integrity after radiation preconditioning, protects endothelial function, and lessens the inhibitory effect of damaged endothelium on hematopoietic reconstitution.

Downregulation of Roundabout guidance receptor 2 suppresses hepatocellular carcinoma progression by interacting with Y-box binding protein 1

Roundabout guidance receptor 2 (Robo2) is closely related to malignant tumors such as pancreatic cancer and liver fibrosis, but there is no relevant research on the role of Robo2 in HCC. The study will further explore the function and mechanism of Robo2 and its downstream target genes in HCC. Firstly, Robo2 protein levels in human HCC tissues and paired adjacent normal liver tissues were detected. Then we established HepG2 and Huh7 hepatoma cell lines with knock-down Robo2 by transfection with lentiviral vectors, and examined the occurrence of EMT, proliferation and apoptosis abilities in HCC cells by western blot, flow cytometry, wound healing assay and TUNEL staining. Then we verified the interaction between Robo2 and its target gene by Co-IP and immunofluorescence co-staining, and further explored the mechanism of Robo2 and YB-1 by rescue study. The protein expression level of Robo2 in HCC was considerably higher than that in the normal liver tissues. After successfully constructing hepatoma cells with knock-down Robo2, it was confirmed that down-regulated Robo2 suppressed EMT and proliferation of hepatoma cells, and accelerated the cell apoptosis. High-throughput sequencing and validation experiments verified that YB-1 was the downstream target gene of Robo2, and over-expression of YB-1 could reverse the apoptosis induced by Robo2 down-regulation and its inhibitory effect on EMT and proliferation. Robo2 deficiency inhibits EMT and proliferation of hepatoma cells and augments the cell apoptosis by regulating YB-1, thus inhibits the occurrence of HCC and provides a new strategy for the treatment of HCC.

Recombinant Slit2 attenuates tracheal fibroblast activation in benign central airway obstruction by inhibiting the TGF-β1/Smad3 signaling pathway

Airway fibrosis is among the pathological manifestations of benign central airway obstruction noted in the absence of effective treatments and requires new drug targets to be developed. Slit guidance ligand 2–roundabout guidance receptor 1 (Slit2–Robo1) is involved in fibrosis and organ development. However, its significance in airway fibrosis has not yet been reported. The study explored how the recombinant protein Slit2 functions in transforming growth factor-β1 (TGF-β1)-mediated airway fibrosis in vivo and in vitro. In this study, Slit2 expression initially increased in the tracheal granulation tissues of patients with tracheobronchial stenosis but decreased in the fibrotic tissue. In primary rat tracheal fibroblasts (RTFs), recombinant Slit2 inhibited the expression of extracellular matrices such as Timp1, α-SMA, and COL1A2, whereas recombinant TGF-β1 promoted the expression of Robo1, α-SMA, and COL1A2. Slit2 and TGF-β1 played a mutual inhibitory role in RTFs. Slit2 supplementation and Robo1 downregulation inhibited excessive extracellular matrix (ECM) deposition induced by TGF-β1 in RTFs via the TGF-β1/Smad3 pathway. Ultimately, exogenous Slit2 and Robo1 knockdown-mediated attenuation of airway fibrosis were validated in a trauma-induced rat airway obstruction model. These findings demonstrate that recombinant Slit2 alleviated pathologic tracheobronchial healing by attenuating excessive ECM deposition. Slit2–Robo1 is an attractive target for further exploring the mechanisms and treatment of benign central airway obstruction.

ROBO1 deficiency impairs HSPC homeostasis and erythropoiesis via CDC42 and predicts poor prognosis in MDS.

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic neoplasms originating from hematopoietic stem progenitor cells (HSPCs). We previously identified frequent roundabout guidance receptor 1 (ROBO1) mutations in patients with MDS, while the exact role of ROBO1 in hematopoiesis remains poorly delineated. Here, we report that ROBO1 deficiency confers MDS-like disease with anemia and multilineage dysplasia in mice and predicts poor prognosis in patients with MDS. More specifically, Robo1 deficiency impairs HSPC homeostasis and disrupts HSPC pool, especially the reduction of megakaryocyte erythroid progenitors, which causes a blockage in the early stages of erythropoiesis in mice. Mechanistically, transcriptional profiling indicates that Cdc42, a member of the Rho-guanosine triphosphatase family, acts as a downstream target gene for Robo1 in HSPCs. Overexpression of Cdc42 partially restores the self-renewal and erythropoiesis of HSPCs in Robo1-deficient mice. Collectively, our result implicates the essential role of ROBO1 in maintaining HSPC homeostasis and erythropoiesis via CDC42.

Gene Signature Associated with Nervous System in an Experimental Radiation- and Estrogen-Induced Breast Cancer Model.

Breast cancer is frequently the most diagnosed female cancer in the world. The experimental studies on cancer seldom focus on the relationship between the central nervous system and cancer. Despite extensive research into the treatment of breast cancer, chemotherapy resistance is an important issue limiting the efficacy of treatment. Novel biomarkers to predict prognosis or sensitivity to chemotherapy are urgently needed. This study examined nervous-system-related genes. The profiling of differentially expressed genes indicated that high-LET radiation, such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of tumorigenicity in human breast epithelial cells. Bioinformatic tools allowed us to analyze the genes involved in breast cancer and associated with the nervous system. The results indicated that the gene expression of the Ephrin A1 gene (EFNA1), the roundabout guidance receptor 1 (ROBO1), and the kallikrein-related peptidase 6 (KLK6) was greater in T2 and A5 than in the A3 cell line; the LIM domain kinase 2 gene (LIMK2) was greater in T2 than A3 and A5; the kallikrein-related peptidase 7 (KLK7), the neuroligin 4 X-linked gene (NLGN4X), and myelin basic protein (MBP) were greater than A3 only in T2; and the neural precursor cell expressed, developmentally down-regulated 9 gene (NEDD9) was greater in A5 than in the A3 and E cell lines. Concerning the correlation, it was found a positive correlation between ESR1 and EFNA1 in BRCA-LumA patients; with ROBO1 in BRCA-Basal patients, but this correlation was negative with the kallikrein-related peptidase 6 (KLK6) in BRCA-LumA and -LumB, as well as with LIMK2 and ROBO1 in all BRCA. It was also positive with neuroligin 4 X-linked (NLGN4X) in BRCA-Her2 and BRCA-LumB, and with MBP in BRCA-LumA and -LumB, but negative with KLK7 in all BRCA and BRCA-LumA and NEDD9 in BRCA-Her2. The differential gene expression levels between the tumor and adjacent tissue indicated that the ROBO1, KLK6, LIMK2, KLK7, NLGN4X, MBP, and NEDD9 gene expression levels were higher in normal tissues than in tumors; however, EFNA1 was higher in the tumor than the normal ones. EFNA1, LIMK2, ROBO1, KLK6, KLK7, and MBP gene expression had a negative ER status, whereas NEDD9 and NLGN4X were not significant concerning ER status. In conclusion, important markers have been analyzed concerning genes related to the nervous system, opening up a new avenue of studies in breast cancer therapy.

Abstract 13857: Slit2/ROBO4 Ligand-Receptor Pair as Novel Biomarkers for Survival in Pulmonary Arterial Hypertension

Background: Slit ligand guidance 2 (Slit2) is a ligand of the Roundabout guidance receptor (ROBO4), a transmembrane endothelial-specific receptor expressed in lung tissue. Pulmonary vascular remodeling contributes to PAH development and ROBO4 has been shown to play a role in inhibiting VEGF related angiogenesis. No studies have evaluated ROBO4 and Slit2 levels in PAH. Research Question: This study sought to assess if ROBO4 and/or Slit2 levels are detectable, altered in and associated with survival outcomes and hemodynamic measurements in patients with PAH. Methods: Serum ROBO4 levels were measured in 139 adults with PAH and 90 controls. Serum Slit2 levels were measured in 210 adults with PAH and 40 controls. Mann-Whitney U tests were used to compare biomarker levels in PAH and controls. Biomarker levels in the PAH cohort were compared to hemodynamic measurements and survival analyses were conducted for a composite outcome (death or transplant) using Cox proportional hazard models, and Kaplan Meier analysis with biomarkers dichotomized at the median. Results: ROBO4 and Slit2 levels were higher in PAH vs control patients (p=0.0005, p=0.002). In Kaplan-Meier analysis, ROBO4 and Slit2 levels above the median were associated with time to death or transplant (Logrank test p=0.016, p=0.03). ROBO4 and Slit2 levels were positively associated with death or transplant using Cox proportional regression, adjusted for age with a hazard ratio of 1.8 (95% CI 1.07-2.97, p=0.003) and 1.49 (95% CI 1.11-1.99, p=0.008), respectively. There were no statistically significant associations between ROBO4 and Slit2 levels and hemodynamic data such as mPAP, mRAP and PVR. Conclusions: This pilot data demonstrates that the angiogenic protein Slit2 and its receptor ROBO4 levels are both associated with survival outcomes in PAH. Future studies should evaluate in vitro studies to localize Slit2/ROBO4 in lung tissue using immunohistochemistry.

SYST-28 FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND A NOVEL TARGETABLE PTP4A2-ROBO1 SIGNALING AXIS AT GLIOBLASTOMA RECURRENCE

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and aberrant ROBO1 signaling axis is associated with higher tumorigenicity in GBM. ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB). Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We therefore developed and validated second-generation CAR-T cells against ROBO1, which demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death compared to untransduced cells in all the three brain cancers. These findings were further validated in vivo in rGBM, MB and BM models, where ROBO1 CAR-T cells showed significant reduction in tumor burden and increase in survival of treated mice. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence.

Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9

The secretory properties of cancer-associated fibroblasts (CAFs) play predominant roles in shaping a pro-metastatic tumor microenvironment. The present study demonstrated that SLIT2, an axon guidance protein, produced by CAFs and promoted gastric cancer (GC) metastasis in two gastric cancer cell lines (AGS and MKN45) by binding to roundabout guidance receptor 1 (ROBO1). Mass-spectrometry analysis revealed that ROBO1 could interact with NEK9, a serine/threonine kinase. And their mutual binding activities were further enhanced by SLIT2. Domain analysis revealed the kinase domain of NEK9 was critical in its interaction with the intracellular domain (ICD) of ROBO1, and it also directly phosphorylated tripartite motif containing 28 (TRIM28) and cortactin (CTTN) in AGS and MKN45 cells. TRIM28 function as a transcriptional elongation factor, which directly facilitate CTTN activation. In addition, Bioinformatics analysis and experimental validation identified transcriptional regulation of STAT3 and NF-κB p100 by TRIM28, and a synergetic transcription of CTTN by STAT3 and NF-κB p100 was also observed in AGS and MKN45. Therefore, CAF-derived SLIT2 increased the expression and phosphorylation levels of CTTN, which induced cytoskeletal reorganization and GC cells metastasis. A simultaneous increase in the expression levels of NEK9, TRIM28 and CTTN was found in metastatic GC lesions compared with paired non-cancerous tissues and primary cancer lesions via IHC and Multiplex IHC. The analysis of the data from a cohort of patients with GC revealed that increased levels of NEK9, TRIM28 and CTTN were associated with a decreased overall survival rate. On the whole, these findings revealed the connections of CAFs and cancer cells through SLIT2/ROBO1 and inflammatory signaling, and the key molecules involved in this process may serve as potential biomarkers and therapeutic targets for GC.

ROBO1 CAR-T CELL THERAPY FOR TREATMENT REFRACTORY BRAIN CANCER

Abstract BTFC travel award recipient Glioblastoma (GBM) is the most common malignant adult brain tumor, with a dismal prognosis. Despite the multimodal treatment regimen, tumor recurrence is inevitable, and no standardized treatment exists for recurrent glioblastoma (rGBM). Neurodevelopment signaling pathways are often hijacked during tumor progression. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment. Our preliminary findings implicated aberrant ROBO1 signaling axis to be associated with higher tumorigenecity in GBM, making it a functionally relevant therapeutic target. Moreover, ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB), prompting the development of an anti-ROBO1 CAR-T cell therapy. Here we report the development and validation of second-generation CAR-T cells using a single-domain antibody targeting ROBO1 expressing BTICs across three different brain tumors. We validated anti-ROBO1 CAR-T cells in-vitro and in-vivo using our established patient derived tumor models. In-vitro studies demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death with ROBO1 CAR-T cells as compared to untransduced cells (UT) in all the three cancers. These findings were further validated in-vivo using 3 different BTIC lines one each from rGBM, MB and BM. ROBO1 CAR-T showed significant reduction in tumor burden and significant increase in survival of the mice treated with ROBO1 CAR-T cells in all the three cohorts. Thus, targeting ROBO1 could be a therapeutically tractable strategy for treating rGBM as well as other brain malignancies.

Coadaptation fostered by the SLIT2-ROBO1 axis facilitates liver metastasis of pancreatic ductal adenocarcinoma

To explore the mechanism of coadaptation and the potential drivers of pancreatic ductal adenocarcinoma (PDAC) metastasis to the liver, we study key molecules involved in this process and their translational value. Premetastatic niche (PMN) and macrometastatic niche (MMN) formation in a mouse model is observed via CT combined with 3D organ reconstruction bioluminescence imaging, and then we screen slit guidance ligand 2 (SLIT2) and its receptor roundabout guidance receptor 1 (ROBO1) as important factors. After we confirm the expression and distribution of SLIT2 and ROBO1 in samples from PDAC patients and several mouse models, we discover that SLIT2-ROBO1-mediated coadaptation facilitated the implantation and outgrowth of PDAC disseminated tumour cells (DTCs) in the liver. We also demonstrate the dependence receptor (DR) characteristics of ROBO1 in a follow-up mechanistic study. A neutralizing antibody targeting ROBO1 significantly attenuate liver metastasis of PDAC by preventing the coadaptation effect. Thus, we demonstrate that coadaptation is supported by the DR characteristics in the PMN and MMN.

Slit2/Robo1 signaling inhibits small-cell lung cancer by targeting β-catenin signaling in tumor cells and macrophages.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with poor patient prognosis. However, the mechanisms that regulate SCLC progression and metastasis remain undefined. Here, we show that the expression of the slit guidance ligand 2 (SLIT2) tumor suppressor gene is reduced in SCLC tumors relative to adjacent normal tissue. In addition, the expression of the SLIT2 receptor, roundabout guidance receptor 1 (ROBO1), is upregulated. We find a positive association between SLIT2 expression and the Yes1 associated transcriptional regulator (YAP1)-expressing SCLC subtype (SCLC-Y), which shows a better prognosis. Using genetically engineered SCLC cells, adenovirus gene therapy, and preclinical xenograft models, we show that SLIT2 overexpression or the deletion of ROBO1 restricts tumor growth in vitro and in vivo. Mechanistic studies revealed significant inhibition of myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (TAMs) in the SCLC tumors. In addition, SLIT2 enhances M1-like and phagocytic macrophages. Molecular analysis showed that ROBO1 knockout or SLIT2 overexpression suppresses the transforming growth factor beta 1 (TGF-β1)/β-catenin signaling pathway in both tumor cells and macrophages. Overall, we find that SLIT2 and ROBO1 have contrasting effects on SCLC tumors. SLIT2 suppresses, whereas ROBO1 promotes, SCLC growth by regulating the Tgf-β1/glycogen synthase kinase-3 beta (GSK3)/β-catenin signaling pathway in tumor cells and TAMs. These studies indicate that SLIT2 could be used as a novel therapeutic agent against aggressive SCLC.

Horizontal Gaze Palsy with Progressive Scoliosis Syndrome: A Concise Synopsis from the Ophthalmologist’s Perspective

The aim of this article is to provide a concise synopsis of Horizontal Gaze Palsy and Progressive Scoliosis syndrome and give the ophthalmologist’s perspective. Horizontal Gaze Palsy and Progressive Scoliosis is a rare autosomal recessive genetic condition. It has been described in both consanguineous and non-consanguineous families. It is caused by mutations in the round-about guidance receptor 3 (ROBO3) gene. Based on our literature review, to date, roughly 100 patients with this rare syndrome and 55 round-about guidance receptor 3 gene mutations have been reported. The age of onset of symptoms varies from 2 months up to 60 years, but usually start in early childhood. Musculoskeletal symptoms include scoliosis and difficulties with posturing. Mental retardation and global developmental delay may also ensue. Ophthalmological manifestations include convergent squint, pendular nystagmus and horizontal gaze palsy. Vertical and convergence eye movements are preserved. Lid position, pupillary reflexes and fundus examination are usually normal as well. Brain neuroimaging reveals striking radiological findings, including brainstem hypoplasia with an anterior and dorsal pontine cleft and a butterfly shaped medulla and absence of the normal promontories of the facial colliculi. To date, there is no definitive treatment of this condition. Convergent squint can be corrected with prisms or surgery. Spectacles are used to correct any significant refractive error. Patching or atropine drops can be used for the treatment of amblyopia. This rare syndrome is a systemic condition that requires a multidisciplinary team to treat the patients with a holistic approach, address their needs and alleviate the emotional burden. It should always be included in the differential diagnosis in patients who present with skeletal anomalies and ocular motility disorders.

MicroRNA-588 regulates the invasive, migratory and vasculogenic mimicry-forming abilities of hypoxic glioma cells by targeting ROBO1

BackgroundThe microenvironment of hypoxia is an important factor contributing to the development of glioblastoma (GBM). MicroRNA-588 and its potential target Roundabout-directed receptor 1 (ROBO1) have been reported to promote tumor invasion and proliferation in diseases such as gastric, pancreatic and hepatocellular carcinoma, while their function in GBM and response to hypoxic states remain elusive.MethodsA microarray was leveraged to identify differentially expressed microRNAs in U251 glioma cells cultured under normoxic and hypoxic conditions. The expression of miR-588 was assessed using quantitative real-time PCR (qRT‒PCR). Gain- and loss-of-function studies were used to evaluate the role of miR-588 under hypoxic and normoxic conditions. Cell invasion, migration, proliferation, and vasculogenic mimicry (VM) formation experiments were performed. The relationship between miR-588 and ROBO1 was confirmed using western blot and luciferase reporter assays. Intracranial xenograft tumor mouse models were used to study the function of miR-588 in vivo.ResultsThe expression of miR-588 was significantly upregulated in hypoxic glioma cells relative to normoxic glioma cells. miR-588 inhibited the invasive, migratory and VM-forming abilities of glioma cells in vitro and in vivo. Mechanistically, roundabout guidance receptor 1 (ROBO1) is a direct, functionally relevant target of miR-588 in glioma. ROBO1 knockdown suppressed the expression of matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 9 (MMP9), thereby inhibiting the invasive, migratory and VM-forming abilities of glioma.ConclusionsMiR-588 regulated the behaviors of hypoxic glioma cells by targeting ROBO1. miR-588 can be used as a prognostic marker for glioma and has potential implications in glioma gene therapy.

Clinical features and genotypes of six patients from four families with horizontal gaze palsy with progressive scoliosis

BackgroundHorizontal gaze palsy with progressive scoliosis (HGPPS) is a rare disorder mainly involved in ocular movement and spinal development. It is caused by a roundabout guidance receptor 3 (ROBO3) gene mutation. This study aimed to describe the clinical features of six patients with HGPPS and investigate the corresponding ROBO3 gene mutations.MethodsPatients underwent detailed clinical and imaging examinations. Whole-exome sequencing was performed to detect nucleotide variations in the disease-causing genes of HGPPS.ResultsSix pathogenic variants were detected in the ROBO3 gene from six patients with HGPPS, including two novel compound heterozygous mutations, c.1447C > T (p.R483X) and c.2462G > C (p.R821P); c.1033G > C (p.V345L) and c.3287G > T (p.C1096F); a novel homozygous indel mutation, c.565dupC (p.R191Pfs*61); and a known missense mutation, c.416G > T (p.G139V). Patients with HGPPS had horizontal conjugated eye movement defects and scoliosis with variable degrees, as well as flattened pontine tegmentum and uncrossed corticospinal tracts on magnetic resonance imaging.ConclusionOur genetic findings will expand the spectrum of ROBO3 mutations and help inform future research on the molecular mechanism of HGPPS.

ROBO3s: a novel ROBO3 short isoform promoting breast cancer aggressiveness

Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development.