Targeting cancer through tumor-selective mRNA stabilization

The success of cancer gene therapy is hindered by various physiological barriers to therapeutic vector transport from the site of injection to the nucleus of all the tumor cells. The application of replicating viruses for the treatment of cancers can overcome this problem. But this approach is limited by normal tissue tolerance of toxicity determined by local concentration of transgene products and viral proteins. Major improvements in vector targeting technology are required before any clinical success. On this basis, this thesis tests the hypothesis to target transformed tumor cells by using a novel posttranslational mRNA stabilizing mechanism, which is occasionally deregulated in cancer. The overexpression of various proteins associated with rapid responses to inflammation and/or proliferation can be controlled at the level of mRNA stability. Since tumor cells continually recapitulate intracellular programs of proliferation, we hypothesize that we can use the tumor cell selective stabilization of mRNA as a novel means to target different malignant diseases. Cyclooxygenase (COX) is the key enzyme in the conversion of arachidonic acid to prostaglandin during inflammation and many studies have linked elevated expression of COX-2 to the pathology of breast, colorectal, head and neck and other types of cancer. It has been shown that the up-regulation of COX-2 is a downstream effect of RAS-mediated transformation. Although the COX-2 over-expression in cancer is associated with increased transcription of the COX-2 gene, a large component of RAS-induced upregulation is also mediated by a selective stabilization of the mRNA of the COX-2 gene in RAS-transformed cells. In this project, we show tumor selective mRNA stability via COX-2 3’ UTR by fusing it with the adenovirus early essential gene E1A, thereby obtaining a conditionally replicating adenovirus vector which will preferentially replicate in the RAS transformed cells. There are wide range of genes reported in the literature with 3'UTR, which confers destabilized activity on their cognate mRNA, but whose actions are reversed under certain physiological conditions. These include hypoxia responsive 3'UTR, radiation responsive elements and 3' UTR, which mediate increased mRNA stability in proliferating cells. Therefore, the linkage to 3’UTRs is a general strategy that could be used to confer tumor cell specificity to expression of therapeutic and/or replicative genes in a wide variety of vectors and to target specific physiological situations within tumors

Targeting cancer through tumor-selective mRNA stabilization

The success of cancer gene therapy is hindered by various physiological barriers to therapeutic vector transport from the site of injection to the nucleus of all the tumor cells. The application of replicating viruses for the treatment of cancers can overcome this problem. But this approach is limited by normal tissue tolerance of toxicity determined by local concentration of transgene products and viral proteins. Major improvements in vector targeting technology are required before any clinical success. On this basis, this thesis tests the hypothesis to target transformed tumor cells by using a novel posttranslational mRNA stabilizing mechanism, which is occasionally deregulated in cancer. The overexpression of various proteins associated with rapid responses to inflammation and/or proliferation can be controlled at the level of mRNA stability. Since tumor cells continually recapitulate intracellular programs of proliferation, we hypothesize that we can use the tumor cell selective stabilization of mRNA as a novel means to target different malignant diseases. Cyclooxygenase (COX) is the key enzyme in the conversion of arachidonic acid to prostaglandin during inflammation and many studies have linked elevated expression of COX-2 to the pathology of breast, colorectal, head and neck and other types of cancer. It has been shown that the up-regulation of COX-2 is a downstream effect of RAS-mediated transformation. Although the COX-2 over-expression in cancer is associated with increased transcription of the COX-2 gene, a large component of RAS-induced upregulation is also mediated by a selective stabilization of the mRNA of the COX-2 gene in RAS-transformed cells. In this project, we show tumor selective mRNA stability via COX-2 3’ UTR by fusing it with the adenovirus early essential gene E1A, thereby obtaining a conditionally replicating adenovirus vector which will preferentially replicate in the RAS transformed cells. There are wide range of genes reported in the literature with 3'UTR, which confers destabilized activity on their cognate mRNA, but whose actions are reversed under certain physiological conditions. These include hypoxia responsive 3'UTR, radiation responsive elements and 3' UTR, which mediate increased mRNA stability in proliferating cells. Therefore, the linkage to 3’UTRs is a general strategy that could be used to confer tumor cell specificity to expression of therapeutic and/or replicative genes in a wide variety of vectors and to target specific physiological situations within tumors

A genetically modified adenoviral vector with a phage display-derived peptide incorporated into fiber fibritin chimera prolongs survival in experimental glioma

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as “GliomaFF.” We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood–brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity. Keywords: siRNA; lipopolymeric nanoparticle; glioblastoma transcription factor; brain tumor-initiating; cells; convection-enhanced deliver

Ribonucleotide reductase regulatory subunit M2 drives glioblastoma TMZ resistance through modulation of dNTP production

During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 (RRM2), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients’ tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production

CCL2 produced by the glioma microenvironment is essential for the recruitment of regulatory T cells and myeloid-derived suppressor cells

In many aggressive cancers, such as glioblastoma multiforme (GBM), progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Treg and MDSC are recruited in various tumors is not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSC in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2 deficient mice failed to maximally accrue Treg and monocytic MDSC. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSC were defective in glioma accumulation. Further, administration of a small molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of GBM, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Lastly, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in GBM patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression

Plasmodium knowlesi Genome Sequences from Clinical Isolates Reveal Extensive Genomic Dimorphism.

Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology

Enhanced Transduction and Replication of RGD-Fiber Modified Adenovirus in Primary T Cells

Background: Adenoviruses are often used as vehicles to mediate gene delivery for therapeutic purposes, but their research scope in hematological cells remains limited due to a narrow choice of host cells that express the adenoviral receptor (CAR). T cells, which are attractive targets for gene therapy of numerous diseases, remain resistant to adenoviral infection because of the absence of CAR expression. Here, we demonstrate that this resistance can be overcome when murine or human T cells are transduced with an adenovirus incorporating the RGD-fiber modification (Ad-RGD). Methodology/Principal Finding: A luciferase-expressing replication-deficient Ad-RGD infected 3-fold higher number of activated primary T cells than an adenovirus lacking the RGD-fiber modification in vitro. Infection with replicationcompetent Ad-RGD virus also caused increased cell cycling, higher E1A copy number and enriched hexon antigen expression in both human and murine T cells. Transduction with oncolytic Ad-RGD also resulted in higher titers of progeny virus and enhanced the killing of T cells. In vivo, 35–45 % of splenic T cells were transduced by Ad-RGD. Conclusions: Collectively, our results prove that a fiber modified Ad-RGD successfully transduces and replicates in primary

Erratum: Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980–2017: a systematic analysis for the Global Burden of Disease Study 2017 (The Lancet (2018) 392(10159) (1736–1788)(S0140673618322037)(10.1016/S0140-6736(18)32203-7))

© 2018 Elsevier Ltd GBD 2017 Causes of Death Collaborators. Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 2018; 392: 1736–88—The bottom row in figure 7 was cut off. This correction has been made to the online version as of Nov 9, 2018, and has been made to the printed Article