Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans. Here, we describe the application of virus-like vesicles (VLV) for delivery of three immunomodulators alone and in combination, as a promising approach for cancer immunotherapy. VLV vectors were designed to deliver single chain interleukin (IL)-12, short-hairpin RNA (shRNA) targeting programmed death ligand 1 (PD-L1), and a dominant-negative form of IL-17 receptor A (dn-IL17RA) as a single payload or as a combination payload. Intralesional delivery of the VLV vector expressing IL-12 alone, as well as the trivalent vector (designated CARG-2020) eradicated large established tumors. However, only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice, indicating a benefit of the combined immunomodulation. The abscopal effects of CARG-2020 on the non-injected contralateral tumors, as well as protection from the tumor cell re-challenge, suggest immune-mediated mechanism of protection and establishment of immunological memory. Mechanistically, CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation. The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.

We aimed to study mechanisms controlling the transition from dormant to clinically relevant brain metastases, a critical process responsible for the majority of melanoma deaths. To this end, we developed in vivo models that accurately mimic this switch and identified novel molecular events that can be ascribed to their distinct phenotypes, latent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.

We aimed to study mechanisms controlling metastatic outgrowth of melanoma into clinically relevant lesions, a critical process responsible for the majority of melanoma deaths. To this end, we developed novel in vivo models and identified molecular events that can be ascribed to their distinct phenotypes, indolent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required the upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.

e14560 Background: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in humans. Treatment of late-stage CRC remains ineffective, even with the use of latest immunotherapies. Oncolytic viruses have shown limited use for the treatment of cancers, and further improvement of these agents with immune-modulating activities may prove crucial for patients with CRC and other malignancies. To this end, we developed CARG-2020 as an artificial virus for infectious diseases and immuno-oncology (AVIDIO) that employs virus-like vesicles (VLV). VLVs, which are membrane-encapsulated RNA replicons, are oncolytic and can deliver multiple genes resulting in the modulation of several independent immune pathways. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki Forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Unarmed or empty vector (VLV), VLV armed with IL-12 (VLV-IL-12) and a VLV that simultaneously expresses IL-12, a dominant-negative form of IL-17 receptor A (dn-1L17RA) and shRNAs targeting PD-L1 (CARG-2020), were given intratumorally to test their therapeutic potential against established (500-600mm 3 ) MC38 tumors in mice. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with these agents to monitor their antitumor and immunomodulatory activities, respectively. Results: Both VLV-IL-12 and CARG-2020 regressed the tumors to undetectable levels in most mice harboring syngeneic MC38 tumors when given intratumorally. CARG-2020, carrying IL-12, dn-IL17RA and PD-L1 shRNA, exerts broader spectrum of immuno-responses and higher number of complete response rates compared with VLV-IL-12. Treatment of primary tumors with VLV-IL-12 or CARG-2020 also significantly repressed the growth of secondary tumors on the other flank of mouse’s body, suggesting an effective systemic immunity elicited by these two agents against the same type of tumors. In addition to the marked local and systemic activation of Th1 cells and CD8 + T cells by both vectors, CARG-2020 also downregulated PD-L1 expression in tumors, and suppressed expression of IL-17A-activated chemokines CXCL1 and CXCL2 that are known to promote cancer development and therapy resistance. Conclusions: As an oncolytic RNA replicon, AVIDIO platform-derived CARG-2020 encodes IL-12, dn-IL-17RA and PD-L1 shRNAs which are expressed concurrently within the same vector. CARG-2020 modulates IL-12, IL-17RA and PD-L1 signaling, and exerts broad immune modulation in tumor microenvironment. Treatment with CARG-2020 eliminates the majority of grafted large tumors in mice, and is effective against both primary and distal tumors. Based on this impressive efficacy results, further development of CARG-2020 in colorectal patients is warranted.

e15210 Background: Colorectal cancer is the third deadliest of all cancers causing more than 50,000 deaths per year in the U.S. Oncolytic viruses have seen limited use for the treatment of cancers, and further improvement of these methods with immune-modulating activities may prove crucial for the effectiveness of these agents in the treatment of human malignancies. To this end, we developed an artificial virus for infectious diseases and immuno-oncology (AVIDIO) platform that employs virus-like vesicles (VLV) for both the delivery of immunomodulatory agents to tumors and oncolytic activity. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Both unarmed VLVs and VLVs armed with the p35 subunit of IL-12 (VLV-IL12p35), an immunomodulatory cytokine that can induce Th1-mediated immunity, were tested for oncolytic activity against various cancer cell lines, including MC38 colorectal cancer cells, in vitro. Using the MC38 syngeneic murine tumor model, we evaluated the antitumor activity of VLV-IL-12p35 in vivo. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with VLV-IL-12p35 to monitor its antitumor and immunomodulatory activities, respectively. Results: VLV-IL-12p35 showed robust oncolytic activity against MC38 cells in vitro, killing over 80% of cells within 24 h. Treatment of intradermal MC38 tumors by intra-tumoral delivery of VLV-IL-12p35 resulted in more than 65% suppression of tumor growth within 2 weeks ( p< 0.05). VLV-IL-12p35-treated tumors also harbored significantly more CD8+ T cells, IFN-gamma-producing CD4+ T cells, and reduced numbers of Foxp3+ regulatory T cells. Conclusions: Our results show that VLV-IL-12p35 derived from the AVIDIO platform has oncolytic activity in vitro and antitumor and immunomodulatory activities in vivo. Therefore, AVIDIO is a promising platform for the delivery of immunomodulatory agents to tumors. Further optimization of the platform, including the addition of other immunomodulatory agents, is in progress to advance the AVIDIO platform to clinical applications for colorectal cancer.

Infections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.

The Polycomb repressive complex 2 (PRC2), which contains three core proteins EZH2, EED and SUZ12, controls chromatin compaction and transcription repression through trimethylation of lysine 27 on histone 3. The (7;17)(p15;q21) chromosomal translocation present in most cases of endometrial stromal sarcomas (ESSs) results in the in-frame fusion of the JAZF1 and SUZ12 genes. We have investigated whether and how the fusion protein JAZF1-SUZ12 functionally alters PRC2. We found that the fusion protein exists at high levels in ESS containing the t(7;17). Co-transient transfection assay indicated JAZF1-SUZ12 destabilized PRC2 components EZH2 and EED, resulting in decreased histone methyl transferase (HMT) activity, which was confirmed by in vitro studies using reconstituted PRC2 and nucleosome array substrates. We also demonstrated the PRC2 containing the fusion protein decreased the binding affinity to target chromatin loci. In addition, we found that trimethylation of H3K27 was decreased in ESS samples with the t(7;17), but there was no detectable change in H3K9 in these tissues. Moreover, re-expression of SUZ12 in Suz12 (-/-) ES cells rescued the neuronal differentiation while the fusion protein failed to restore this function and enhanced cell proliferation. In summary, our studies reveal that JAZF1-SUZ12 fusion protein disrupts the PRC2 complex, abolishes HMT activity and subsequently activates chromatin/genes normally repressed by PRC2. Such dyesfunction of PRC2 inhibits normal neural differentiation of ES cell and increases cell proliferation. Related changes induced by the JAZF-SUZ12 protein in endometrial stromal cells may explain the oncogenic effect of the t(7;17) in ESS.

The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a highly conserved long non-coding RNA (lncRNA) gene. Previous studies showed that Malat1 is abundantly expressed in many tissues and involves in promoting tumor growth and metastasis by modulating gene expression and target protein activities. However, little is known about the biological function and regulation mechanism of Malat1 in normal cell proliferation.

The Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is one of the most significant molecules of long noncoding RNA (lncRNA), also known as Nuclear Enriched Transcript2 (NEAT2). MALAT1 was first discovered as a prognostic marker for non-small cell lung carcinoma [1]. Soon after, researchers found MALAT1 is linked to other cancers such as endometrial cancer [2], breast cancer [3], cervical cancer [4], colorectal cancer [5], hepatocellular carcinoma [6], liver cancer [7], neuroblastoma [8], osteosarcoma [9], pancreatic cancer [10], prostate cancer [11], bladder cancer [12], gastric cancer [13] and etc. In addition to its role as a biomarker for many human tumors, MALAT1 was also identified as a critical regulatory molecule to control target gene expression, modify RNA and protein (enzyme) activity, as well as affect cellular distribution [14-16], consequently it is intimately associated with the regulation of cell growth and proliferation. Dysregulations of MALAT1 result in multiple tissue carcinogenesis as well as many other human disease processes [17-20]. As such the critical regulatory role and potential clinical implications of MALAT1 have attracted more and more attention recently [10,17]. This review will focus on the regulatory role and molecular mechanisms of MALAT1 on gene expression and biochemical function of proteins. In addition, the paper will put forward the concept that the regulatory mechanisms of lncRNA on the gene expression and target protein function are in multiple layer and flexible manner.

PGRP (peptidoglycan recognition protein) is a conserved protein family that recognizes the peptidoglycan in bacterial cell wall and causes the activation of various innate immune responses. Previous studies have reported that PGRP-SCs in Drosophila dampen the activation of Immune Deficiency (Imd) pathway to microbial infection, and participate in the lifespan extension of the insects. To facilitate understanding the function of PGRP-SCs from an evolutionary angle, we identified and functionally characterized the PGRP-SC gene in the housefly Musca domestica, a species that has adapted to a septic environment much harsher than the natural habitat of Drosophila. The gene designated as MdPGRP-SC was found most abundantly expressed in the 3rd instar larvae, and is expressed at this developmental stage predominantly in the gut. MdPGRP-SC was virtually unchanged in whole larvae after a septic injury at the second larval instar, while two antimicrobial peptides (AMPs), diptericin and attacin, were upregulated in the first 24h but not later. Through dsRNA microinjection, MdPGRP-SC was knocked down by RNA interference (RNAi), and caused the significant increased expression of diptericin and attacin. The pupation of MdPGRP-SC-depleted larvae was severely suppressed compared to controls. Opposite to the expression trend of MdPGRP-SC, a spontaneous active expression of diptericin and attacin was found in pre-pupae but not in third instar larvae. Taken together, our study reveals that downregulation of MdPGRP-SC leads to the overexpression of the AMPs, and is involved in the larvae-to-pupa transition of housefly.

The DNA methylation is catalyzed by DNA methyltransferases, which consist of three members of DNMT1, DNMT3A and DNMT3B. DNMT1 is the most abundant DNA methyltransferase among these enzymes. DNMT1 catalyzes the methylation of the 5’-cytosine in the CpG dinucleotide sequence, and plays an important role in maintaining the DNA methylation patterns during cell division [7]. The DNMT3A/3B catalyzes de novo methylation of DNA [8]. These three enzymes cooperatively catalyze the methylation reactions of CpG islands, which are often located in promoter regions of target genes [9]. Hypermethylation means the methylation exceeds physiological level of target DNAs (Figure 1), the hypermethylation of promoters leads to inactivate the expression of tumor suppressor genes and loss of corresponding proteins to repress carcinogenesis, thereby promoting carcinogenesis and enhancing the metastases of cancer cells. A number of tumor suppressor genes have been determined with frequent hypermethylation on promoter regions during endometrial carcinogenesis (See Table 1). The development of new assay methods for DNA methylation such as MLPA (methylationIntroduction

The Endometrial Cancer (EC) is the most common gynecologic malignancy that starts in the endometrium of women. Carcinogenesis of EC is associated with several critical regulatory molecules, which involve in different signaling pathways. A number of signaling pathways have been identified to be involved in the multiple-step development of EC, including PI3K/AKT/mTOR signaling pathway, WNT/β-catenin signal transduction cascades (including APC/β-catenin pathway), MAPK/ERK pathway, VEGF/VEGFR ligand receptor signaling pathway, ErbB signaling pathway, P53/P21 and P16INK4a/pRB signaling pathways. This review mainly focuses on the molecular signaling pathways relevant to human endometrial cancer and discusses those critical capabilities of transforming endometrial cells, including evading apoptosis; enhancing cell proliferation; blocking differentiation; and inducing angiogenesis.

Mint is a newly identified molecule that mediates signal transduction and modulates chromatin repression. Mint family members contain a highly conserved C-terminus SPOC domain (SpenParalog and OrthologsC-terminal domain) commonly associated with proliferation and related diseases (for example: cancer) due to its role in cell differentiation and apoptosis. In this study, we addressed the SPOC function using a tetracycline-inducible system to express the target domain in Ain V15 embryonic ES cells and bone marrow stem cells from SPOC transenic mice. In vitro differentiation of Ain V15 ES cells as a model of early hematopoietic development, we found expression of SPOC domain induces hematopoietic differentiation via up-regulation of transcription factors Bmp4 and Smad5, which induce the expression of hematopoietic factors Eklf1 and hematopoietic proliferation associated factor Gata2, the SPOC domain also plays the regulation function in the differentiation of hematopoitic progenitor by colony forming Unit (CFU) assays. Further, we determined SPOC expression enhances erythrocyte and granulocyte maturationusing bone marrow cells derived from tiSPOC chimeric mice. Finally, we identified that overexpression of full length Mint in ES cells drive Smad5 and Bmp4 up-regulation under culture conditions, and up-regulation of endogenous Mint when induceshematopoitic differentiation of EML, M1 and WT18 cells. In summary, our study reveals the conserved SPOC domain of Mint protein induces differentiation both in the stages of embryonic stem cells and hematopoietic progenitor cells.

Chimeric gene products, most often resulting from chromosome translocations, have been considered unique features of cancer, or at least of cells at high risk for becoming cancerous. Chimeric JAZF1-JJAZ1 mRNA transcribed from DNA spanning the site of recombination in the (7;17)(p15;q21) chromosomal translocation found in half of endometrial stromal sarcomas and most cases of benign stromal nodules is one such example. The recent finding that chimeric JAZF1-JJAZ1 mRNA can also be detected in normal endometrial stromal cells suggests that chimeric gene products are not limited to cancer or pre-cancerous cells. The JAZF1-JJAZ1 mRNA and the protein encoded by it appear to be identical to that synthesized from the gene fusion in neoplastic cells. In cultured cells, the chimeric protein has anti-apoptotic properties and is pro-proliferative when unrearranged JJAZ1 alleles are silenced, as they are in endometrial stromal sarcomas but not in the stromal nodules. These observations are consistent with the conclusion that chromosomal rearrangements and gene fusions in neoplastic cells may represent mechanisms for the deregulated expression of chimeric gene products that are generated at specific stages in cell development and have physiologic functions in normal cells. Furthermore, it may be possible that other means for abnormal production of chimeric gene products, such as hyperactive trans-splicing of RNA, may be another mechanism underlying the neoplastic properties of tumor cells.

Polycomb group genes (PcGs) have been implicated in cancer based on altered levels of expression observed in certain tumors and the behavior of cultured cells containing inserted PcG transgenes. Endometrial stromal tumors provide evidence for a direct causal relationship because they contain several chromosomal translocations and resultant gene fusions involving PcGs, the most common of which joins portions of the JAZF1 gene to the PcGJJAZ1/SUZ12. We show here that both benign and malignant forms of this tumor have the JAZF1-JJAZ1 fusion but only the malignant form also exhibits exclusion of the unrearranged JJAZ1 allele. To evaluate the effects of both the JJAZ1/SUZ12 fusion and allelic exclusion on functions related to cell growth, we studied HEK293 cells that were modified with respect to JJAZ1 expression. We found that the JAZF1-JJAZ1 fusion restored levels of the polycomb protein EZH2 and histone 3 lysine 27 trimethylation, which were reduced by knockdown of endogenous JJAZ1. At the same time, the presence of JAZF1-JJAZ1 markedly inhibited apoptosis and induced above normal proliferation rates, although the latter effect occurred only when normal JJAZ1 was suppressed. Our findings suggest a genetic pathway for progression of a benign precursor to a sarcoma involving increased cell survival associated with acquisition of a PcG rearrangement, followed by accelerated cellular proliferation upon allelic exclusion of the unrearranged copy of that gene. Furthermore, these results indicate the likely functional importance of allelic exclusion of genes disrupted by chromosomal translocations, as seen in a variety of other cancers.

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJ kappa, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJ kappa.