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We disclose novel amphiphilic ruthenium and osmium complexes that auto-assemble into nanomedicines with potent antiproliferative activity by inhibition of mitochondrial respiration. The self-assembling units were rationally designed from the [M(p-cymene)(1,10-phenanthroline)Cl]PF(6) motif (where M is either Ru(II) or Os(II)) with an appended C(16) fatty chain to achieve high cellular activity, nano-assembling and mitochondrial targeting. These amphiphilic complexes block cell proliferation at the sub-micromolar range and are particularly potent towards glioblastoma neurospheres made from patient-derived cancer stem cells. A subcutaneous mouse model using these glioblastoma stem cells highlights one of our C(16) Os(II) nanomedicines as highly successful in vivo. Mechanistically, we show that they act as metabolic poisons, strongly impairing mitochondrial respiration, corroborated by morphological changes and damage to the mitochondria. A genetic strategy based on RNAi gave further insight on the potential involvement of microtubules as part of the induced cell death. In parallel, we examined the structural properties of these new amphiphilic metal-based constructs, their reactivity and mechanism.

PDF file - 154K, FIGURE S1 related to FIGURE2. Effect of miR-1 overexpression on GBM cells in vivo.

Supplementary Figures 1-4 from BCR/ABL Stimulates WRN to Promote Survival and Genomic Instability

PDF file - 282K, SUPPLEMENTARY TABLE 1. List of EV proteins identified by mass spectrometry.

PDF file - 149K

Supplementary Methods from BCR/ABL Stimulates WRN to Promote Survival and Genomic Instability

PDF file - 98K, FIGURE S4 related to FIGURE 5. Visualization of GBM-derived EVs uptake.

PDF file - 56K, SUPPLEMENTARY TABLE 2. Functional analysis of EV proteins identified by mass spectrometry.

Supplementary Figure Legends 1-3 from Indirubins Decrease Glioma Invasion by Blocking Migratory Phenotypes in Both the Tumor and Stromal Endothelial Cell Compartments

Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, including by conveying tumor-promoting microRNAs between cells, but their regulation is poorly understood. In this study, we report the findings of a comparative microRNA profiling and functional analysis in human glioblastoma that identifies miR-1 as an orchestrator of extracellular vesicle function and glioblastoma growth and invasion. Ectopic expression of miR-1 in glioblastoma cells blocked in vivo growth, neovascularization, and invasiveness. These effects were associated with a role for miR-1 in intercellular communication in the microenvironment mediated by extracellular vesicles released by cancer stem–like glioblastoma cells. An extracellular vesicle–dependent phenotype defined by glioblastoma invasion, neurosphere growth, and endothelial tube formation was mitigated by loading miR-1 into glioblastoma-derived extracellular vesicles. Protein cargo in extracellular vesicles was characterized to learn how miR-1 directed extracellular vesicle function. The mRNA encoding Annexin A2 (ANXA2), one of the most abundant proteins in glioblastoma-derived extracellular vesicles, was found to be a direct target of miR-1 control. In addition, extracellular vesicle–derived miR-1 along with other ANXA2 extracellular vesicle networking partners targeted multiple pro-oncogenic signals in cells within the glioblastoma microenvironment. Together, our results showed how extracellular vesicle signaling promotes the malignant character of glioblastoma and how ectopic expression of miR-1 can mitigate this character, with possible implications for how to develop a unique miRNA-based therapy for glioblastoma management. Cancer Res; 74(3); 738–50. ©2013 AACR.

Supplementary Figures 1-3 from Indirubins Decrease Glioma Invasion by Blocking Migratory Phenotypes in Both the Tumor and Stromal Endothelial Cell Compartments

PDF file - 636KB, Supplemental Table S1: information of the GBM lines used in this paper. Supplemental Table S2: information of the patient clinical data for Figure 1. Supplemental Table S3: Percentage of cells positive for staining with PRMT5 in spontaneous high grade astrocytoma from Mut3 strain (GFAP-cre; cisNf1-/+; P53-/-) and normal brain tissue from wild-type control mouse stained by PRMT5 and ki-67 antibody. Supplemental table S4. GBM cell line (U251) was transfected by scramble control or si-PRMT5. Affymetrix microarray genechip was performed in three independent experiments to evaluate genes up-regulated with PRMT5 silencing.

PDF file - 1690KB, Supplemental Figure S1. PRMT5 is over-expressed in human GBM cell lines. Supplemental Figure S2: High grade astrocytomas Supplemental Figure S3: efficacy and specificity of PRMT5 knock-down by lead siRNA. Supplemental Figure S4: PRMT5 Knockdown promotes cell death of human GBM cells Supplemental Figure S5: si-PRMT5 induced cell death is P53-independent Supplemental Figure S6: Decreased ST7 expression correlates with worse survival in all glioma patients (grade I-IV). Supplemental Figure S7: Over-expression of ST7 in three GBM cell lines was confirmed by real-time PCR and western blot (top panels).

Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease. Cancer Res; 74(6); 1752–65. ©2014 AACR.

PDF file - 168K, FIGURE S2 related to FIGURE3. Effect of miR-1 overexpression on GBM cells in vitro.

PDF file - 133K, FIGURE S5 related to FIGURE 6. Effect of miR-1 on GBM-derived EVs protein cargo.

PDF file - 154K, FIGURE S3 related to FIGURE 4. Characterization of GBM-derived EVs.

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The cGAS-STING cytoplasmic double stranded DNA (dsDNA) sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties.Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature. We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils and NK populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100-loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants the further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, CAR T cells, NK therapies or immune checkpoint blockade.Significance statementModulation of the immune microenvironment is critical for immunosuppressive and therapy refractory tumors like glioblastoma. Activation of the STING pathway deeply remodels the brain tumor environment and attracts innate immune cells and natural killer cell populations, producing a robust antitumor effect with long-term immune memory. We further show that human glioblastoma tissue can respond to the therapy and lay the foundations for combined intracranial immunotherapies by using crosslinked biodegradable brain implants.

We disclose novel amphiphilic ruthenium and osmium complexes that auto-assemble into nanomedicines with potent antiproliferative activity by inhibition of mitochondrial respiration. The self-assembling units were rationally designed from the [M(p-cymene)(1,10-phenanthroline)Cl]PF6 motif (where M is either RuII or OsII) with an appended C16 fatty chain to achieve high cellular activity, nano-assembling and mitochondrial targeting. These amphiphilic complexes block cell proliferation at the sub-micromolar range and are particularly potent towards glioblastoma neurospheres made from patient-derived cancer stem cells. A subcutaneous mouse model using these glioblastoma stem cells highlights one of our C16 OsII nanomedicines as highly successful in vivo. Mechanistically, we show that they act as metabolic poisons, strongly impairing mitochondrial respiration, corroborated by morphological changes and damage to the mitochondria. A genetic strategy based on RNAi gave further insight on the potential involvement of microtubules as part of the induced cell death. In parallel, we present a careful examination of the structural properties of these new amphiphilic metal-based constructs, their reactivity and mechanism.

BackgroundIntratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase to cancer cells, resulting in local conversion of ganciclovir or valacyclovir into a toxic metabolite. This leads to highly immunogenic cell death, followed by a local immune response against a variety of cancer neoantigens and, next, a systemic immune response against the injected tumor and uninjected distant metastases. CAN-2409 treatment has shown promising results in clinical studies in glioblastoma (GBM). Patients with GBM are usually given the corticosteroid dexamethasone to manage edema. Previous work has suggested that concurrent dexamethasone therapy may have a negative effect in patients treated with immune checkpoint inhibitors in patients with GBM. However, the effects of dexamethasone on the efficacy of CAN-2409 treatment have not been explored.MethodsIn vitro experiments included cell viability and neurosphere T-cell killing assays. Effects of dexamethasone on CAN-2409 in vivo were examined using a syngeneic murine GBM model; survival was assessed according to Kaplan-Meier; analyses of tumor-infiltrating lymphocytes were performed with mass cytometry (CyTOF - cytometry by time-of-flight). Data were analyzed using a general linear model, with one-way analysis of variance followed by Dunnett’s multiple comparison test, Kruskal-Wallis test, Dunn’s multiple comparison test or statistical significance analysis of microarrays.ResultsIn a mouse model of GBM, we found that high doses of dexamethasone combined with CAN-2409 led to significantly reduced median survival (29.0 days) compared with CAN-2409 treatment alone (39.5 days). CyTOF analyses of tumor-infiltrating immune cells demonstrated potent immune stimulation induced by CAN-2409 treatment. These effects were diminished when high-dose dexamethasone was used. Functional immune cell characterization suggested increased immune cell exhaustion and tumor promoting profiles after dexamethasone treatment.ConclusionOur data suggest that concurrent high-dose dexamethasone treatment may impair the efficacy of oncolytic viral immunotherapy of GBM, supporting the notion that dexamethasone use should be balanced between symptom control and impact on the therapeutic outcome.

Background: Oncolytic viruses (OV) have been a topic of great interest as therapeutic agents for indications such as glioblastoma multiforme (GBM), where current treatment options are poor and limited. Alongside engineering these viruses, finding useful pre-clinical models to elucidate the efficacy of the OV has been challenging. In particular, these viruses have been developed to overcome immune resistance, allowing for the reinvigoration of the immune contexture in the tumor microenvironment (TME). Current pre-clinical models, such as mouse models, are limited in their ability to recapitulate the TME fully. Thus, there is a need for more relevant pre-clinical models to study the efficacy of OV and downstream effects in the TME. Methods: rQNestin34.5v.1 herpes simplex virus (rQNestin) has previously been investigated in both in vitro and in vivo studies of GBM. To assimilate the effects of rQNestin on the TME, a more humanized system was employed; CANscriptTM. CANscript is an ex vivo human tumor model, that recapitulates the native, patient-autologous TME, incorporating autologous patient-derived peripheral blood mononucleated cells. We treated GBM tissue (n=10) with rQNestin, capable of expressing GFP, and profiled GFP expression as a proxy for replication efficiency. We coupled this analysis with gene expression of immune-related pathways to gauge the modulation of the immune contexture by rQNestin. In addition, we performed multiplex cytokine analysis and multiplex immunohistochemistry to investigate the impact of rQNestin on the spatial context and activity of the immune compartment in the TME, ex vivo. Results: Immunohistochemistry established that viral replication and tissue penetration was observed, ex vivo. Furthermore, RNA transcriptional profiling and cytokine analysis revealed that the oHSV-1 was capable of dynamically altering the tumor microenvironment, and dysregulating immune subsets within the tumor. Further stratification of tumor samples based on the pharmacodynamic profile identified subsets of patient tumors that induce adaptive immunity. Concluding remarks: Here, we report that CANscript, an ex vivo human tumor model, can be used to evaluate the effects of OV in the TME - not only viral replication but also the direct effect on the immune compartment. CANscript will be an invaluable tool to investigate the response and resistance to OV. This platform has the potential to enable better pre-clinical modeling of OV development. Citation Format: Munisha Smalley, Vidushi Kapoor, Douglas Best, Carmela Passaro, Michal O. Nowicki, Suniti K. Saha, Komal Prasad, E. Antonio Chiocca, Sean E. Lawler, Aaron Goldman. Translational efficacy of oncolytic HSV-1 in glioblastoma using a human autologous ex vivo platform, CANscript™ [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 5009.

Glioblastoma, the most common and aggressive primary brain tumor, is rapidly growing and highly infiltrative. Incomplete knowledge of the molecular biology, genetics, causes and cellular origin of these tumors may limit the development of improved therapeutics. A major and fundamental advance in recent years has been the identification of microRNAs as highly conserved regulators of gene expression. Here we will discuss further our recently published data on the role of miR-451 in the biology of glioblastoma. We initially identified miR-451 due to its downregulation in a glioma cell migration assay. We then found that by targeting the LKB1 kinase complex miR-451 suppresses the activity of downstream protein kinases including the major energy biosensor AMPK. MiR-451 levels are regulated by glucose; under conditions of abundant energy miR-451 expression is high, and the suppression of AMPK signaling allows cells to maintain elevated proliferation rates via unrestrained mTOR activation. Under conditions of glucose withdrawal, miR-451 downregulation is necessary for AMPK pathway activation, leading to suppressed proliferation rates, increased cell survival and migration. We also identified a potential feedback loop between LKB1 and miR-451, which allows a sustained and robust response to glucose deprivation. This data will be discussed in the context of potential biological significance and therapeutic implications.

The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph1)-positive leukemia cells. We find that BCR/ABL kinase - induced reactive oxygen species (ROS) cause chronic oxidative DNA damage as indicated by an enzymatic assay detecting oxidized bases. These DNA lesions result in DNA double-strand breaks (DSBs) detected by comet assay, immunofluorescent gamma-H2AX nuclear foci and linker-ligation PCR (LL-PCR). Combined analysis of the length of LL-PCR products and the sequences of two reference genes DR-GFP and Na+/K+ ATPase revealed that ROS dependent DSBs occurred in the regions containing multiple, 5–9bp long stretches of G/C, in concordance with the notion that oxidative DNA damage is predominantly detected in G/C-rich sequences. Elevated numbers of DSBs were detected in BCR/ABL cell lines, murine bone marrow cells transformed with BCR/ABL and in CML patient samples, in comparison to normal counterparts. Inhibition of the BCR/ABL kinase by STI571 and diminishion of ROS activity by the ROS scavenger PDTC reduced DSBs formation. Cell cycle analysis revealed that most of these DSBs occur during S and G2/M phases, and are probably associated with the stalled replication forks. Homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in S and G2/M cell cycle phase. Using the in vivo recombination assay consisting of the DSB-dependent reconstitution of the green fluorescent protein (GFP) gene we found that HRR is stimulated in BCR/ABL-positive cells. In addition, in vitro assay measuring the activity of NHEJ revealed that this repair process is also activated by the BCR/ABL kinase. RAD51 and Ku70 play a key role in HRR and NHEJ, respectively. The reaction sites of HRR and NHEJ in the nuclei could be visualized by double-immunofluorescence detecting co-localization of gamma-H2AX foci (DSBs sites) with RAD51 (HRR sites) or Ku70 (NHEJ sites). Equal co-localization frequency of gamma-H2AX foci with RAD51 and Ku70 was detected, suggesting that both HRR and NHEJ play an important role in reparation of ROS-dependent DSBs in BCR/ABL-transformed cells. Analysis of the DSBs repair products in the reporter DR-GFP gene in BCR/ABL cells identified ~40% of HRR and ~60% of NHEJ events. Sequencing revealed point-mutations in HRR products and large deletions in NHEJ products in BCR/ABL-positive cells, but not in non-transformed cells. We propose that the following series of events may contribute to genomic instability of Ph1-positive leukemias: BCR/ABL → ROS → oxidative DNA damage → DSBs in proliferating cells → unfaithful HRR and NHEJ repair. Since BCR/ABL share many similarities with other members of the fusion tyrosine kinases (FTKs) family, these events may contribute to genomic instability of hematological malignancies caused by FTKs.