Glioblastoma (GBM) is a devastating brain cancer for which new effective therapies are urgently needed. GBM, after an initial response to current treatment regimens, develops therapeutic resistance, leading to rapid patient demise. Cancer cells exhibit an inherent elevation of endoplasmic reticulum (ER) stress due to uncontrolled growth and an unfavorable microenvironment, including hypoxia and nutrient deprivation. Cancer cells utilize the unfolded protein response (UPR) to maintain ER homeostasis, and failure of this response promotes cell death. In this study, as integrins are upregulated in cancer, we have evaluated the therapeutic potential of individually targeting all αβ1 integrin subunits using RNA interference. We found that GBM cells are uniquely susceptible to silencing of integrin α3. Knockdown of α3-induced proapoptotic markers such as PARP cleavage and caspase 3 and 8 activation. Remarkably, we discovered a non-canonical function for α3 in mediating the maturation of integrin β1. In its absence, generation of full length β1 was reduced, immature β1 accumulated, and the cells underwent elevated ER stress with upregulation of death receptor 5 (DR5) expression. Targeting α3 sensitized TRAIL-resistant GBM cancer cells to TRAIL-mediated apoptosis and led to growth inhibition. Our findings offer key new insights into integrin α3’s role in GBM survival via the regulation of ER homeostasis and its value as a therapeutic target.

PDF file, 591K, Supplementary Figure 4. KCN1 inhibits the growth of pre-established human glioblastoma s.c. xenografts. Supplementary Figure 5. Effect of KCN1 treatment on mice weight. Supplementary Figure 6. Chronic systemic KCN1 administration shows little effect on major organs, except liver. Supplementary Figure 7. Effect of KCN1 on the survival of mice with orthotopic (intracranial) brain tumor models. Supplementary Figure 8. Permeability coefficients (PC) of KCN1 calculated from the transfer across monolayers of brain endothelial cells.

Supplementary Figure 1 from Vasculostatin Inhibits Intracranial Glioma Growth and Negatively Regulates In vivo Angiogenesis through a CD36-Dependent Mechanism

Suppl. Figure 1. Determination of sensitivity, specificity and accuracy using ROC curves. Suppl. Figure 2. CSF levels of D-2HG and WHO grade of IDH mutant gliomas Suppl. Figure 3. CSF D-2HG levels and gender/age. Suppl. Figure 4. D- and L-2HG levels in CSF drawn at different locations in WT and mutant IDH glioma patients. Suppl. Figure 5. Comparison of NMR and MS for the detection of D-2HG in tumor tissue and CSF.

Table of contents: 1. Abbreviations 2. Author Contributions 3. Extended Material and Methods 4. Supplementary Figure Legends 5. Supplementary References

Suppl. Table 2. CSF and Matching Tissues Available for the Study Suppl. Table 3. Metabolite concentrations in the three cohorts (t-test Statistics) Suppl. Table 4. Site-specific thresholds for D-/L-2HG Suppl. Table 5. Information about site-specific accuracy for D-2HG Suppl. Table 6. Tumor tissue sample demographics tested in parallel by MS and NMR

Suppl. Table 1. Histology, age, gender, diagnosis, site of CSF draws, longitudinal # and IDH1/2 mutation status of 84 tumor samples analyzed. Highlighted in 'yellow' are CSF samples that tested positive for IDH1 mutant staining, while their DNA indicated WT IDH.

Supplementary Figure Legends 1-2 from Vasculostatin Inhibits Intracranial Glioma Growth and Negatively Regulates In vivo Angiogenesis through a CD36-Dependent Mechanism

PDF file, 285K, Supplementary Figure 1. Synthetic scheme and purity of KCN1 3,4-dimethoxy-N-(2,2-dimethyl-2H-chromen-6-yl)methyl-N-phenylbenzenesulfonamide. Supplementary Figure 2. KCN1 is chemically stable in cell culture medium under normoxic or hypoxic conditions. Supplementary Figure 3. Effect of KCN1 in the NCI-60 Tumor Cell Line Screen.

Supplementary Note from Vasculostatin Inhibits Intracranial Glioma Growth and Negatively Regulates In vivo Angiogenesis through a CD36-Dependent Mechanism

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. GBM cells spread extensively in the brain and disseminate into the cerebrospinal fluid space, strongly restricting multimodal therapies. Acquiring a better knowledge of molecular defects underlying GBM invasion is essential for developing effective treatments.Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in the normal brain, but its expression is lost in the majority of GBM through epigenetic silencing and restoration of its expression can inhibit glioma growth. However, whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in GBM has not been investigated.Microarray analysis of the GBM TCGA dataset (restricted to IDHwt GBM; WHO 2021) revealed that low BAI1 mRNA expression correlates with elevated expression of many mesenchymal genes. Restoration of BAI1 expression in human GBM cells suppresses mesenchymal gene expression in culture and dramatically decreases brain tumor invasion in mice xenografts. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in GBM cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically suppresses cell invasion in the brain and reduces leptomeningeal dissemination of GBM cells to the spine in mouse xenografts.These experiments demonstrate that epigenetic silencing of BAI1 is important for activating the GBM invasive phenotype through TGFβ1 pathway activation. This new tumor-suppressive pathway can be epigenetically targeted to reactivate BAI1 expression in GBM patients.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive <1 year, highlighting the urgent need for new therapies. TCGA has recently revealed that a hypoxia gene signature is associated with poor UM patient prognosis. Here we show that expression of hypoxia-regulated collagen prolyl-4-hydroxylase genes P4HA1 and P4HA2 is significantly upregulated in UM patients with metastatic disease and correlates with poor prognosis, suggesting these enzymes might be key tumor drivers. We targeted hypoxia-induced expression of P4HA1/2 in UM with KCN1, a hypoxia inducible factor-1 (HIF-1) pathway inhibitor and found potent inhibition of primary and metastatic disease and extension of animal survival, without overt side effects. At the molecular level, KCN1 antagonized hypoxia-induced expression of P4HA1 and P4HA2, which regulate collagen maturation and deposition in the extracellular matrix. The treatment decreased prolyl hydroxylation, induced proteolytic cleavage and rendered a disordered structure to collagen VI, the main collagen produced by UM, and reduced UM cell invasion. Together, these data demonstrate that extracellular collagen matrix formation can be targeted in UM by inhibiting hypoxia-induced P4HA1 and P4HA2 expression, warranting further development of this strategy in patients with uveal melanoma.

ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Abstract Purpose: Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide–Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. Experimental Design: sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. Results: We found sGal-3 preferentially binds to β1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-β1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. Conclusions: We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin β1–dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Medulloblastoma (MB), is the most aggressive primary malignant brain tumor in children and are classified into four molecular subgroups. While some subtypes of MB show a favorable prognosis with treatment, still one third of patients succumb to this disease and the children who survive after therapy suffer from long-term neurocognitive and endocrine side effects of the conventional treatments. ADGRB3 (formerly called BAI3) is a member of the ADGRB1-3 subfamily of adhesion GPCR transmembrane proteins, which are highly expressed in the brain specially in cerebellum and hippocampal neurons. Our recent analysis of RNA-seq data from a published panel of medulloblastoma tumor samples and RT-PCR experiments with MB tumor samples showed that ADGRB3 mRNA expression was selectively repressed in WNT-MB tumor tissue compared to other three molecular subgroups and normal human cerebellar tissue. Using bisulfite sequencing and MS-PCR we have detected hypermethylation of the ADGRB3 promoter region exclusively in WNT-MB subgroup of MB tissues but not in the other three molecular subgroups and normal human cerebellar tissue. ChIP assays revealed enrichment of repressive methyl CpG binding protein MBD2 and the trimethylated histone H3K9me3 in the ADGRB3 promoter region of UW288-1 cells. Collectively, these indicates epigenetic silencing of ADGRB3 in WNT-MB via promoter hypermethylation and repressive histone modifications. We found that knockdown of ATP dependent chromatin remodeler protein Brg1 in MB cells can silence ADGRB3 expression through epigenetic reprogramming at the gene promoter. Lentiviral reconstitution of ADGRB3 in silent MB cells (UW288-1, PFSK-1) inhibited growth of these cells in culture and inhibited WNT signaling targets. ADGRB3 reconstituted UW288-1 MB cells when xenografted yielded significantly reduced tumor in immunocompromised mice compared to the parental cells. Pharmacological reactivation of ADGRB3 expression in silent WNT MB cells using our recently established MBD2 antagonist and an EZH2 inhibitor significantly reduced cell growth in vitro and inhibits some specific WNT signaling targets. We further identified a novel mechanism underlying ADGRB3 mediated regulation of WNT signaling by performing co-immunoprecipitation experiments. Altogether, our findings define an epigenetic mechanism for ADGRB3 silencing in WNT-MB and demonstrates a mechanism through which ADGRB3 restrains activation of WNT signaling involved in cerebellar transformation. Our findings highlight the potential of epigenetic reactivation of ADGRB3 as a less toxic therapeutic intervention for the children suffering from WNT-MB. Citation Format: Debanjan Bhattacharya, Dan Zhu, Satoru Osuka, Saroja Narra Devi, Erwin G. Van Meir. ADGRB3 is epigenetically silenced in WNT-medulloblastoma and inhibits WNT signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3477.