CircRNAs in Pancreatic Cancer: New Tools for Target Identification and Therapeutic Intervention.

We have reviewed the literature for circular RNAs (circRNAs) with efficacy in preclinical pancreatic-cancer related in vivo models. The identified circRNAs target chemoresistance mechanisms (n=5), secreted proteins and transmembrane receptors (n=15), transcription factors (n=9), components of the signaling- (n=11), ubiquitination- (n=2), autophagy-system (n=2), and others (n=9). In addition to identifying targets for therapeutic intervention, circRNAs are potential new entities for treatment of pancreatic cancer. Up-regulated circRNAs can be inhibited by antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) or clustered regularly interspaced short-palindromic repeats-CRISPR associated protein (CRISPR-CAS)-based intervention. The function of down-regulated circRNAs can be reconstituted by replacement therapy using plasmids or virus-based vector systems. Target validation experiments and the development of improved delivery systems for corresponding agents were examined.

CircRNAs as New Therapeutic Entities and Tools for Target Identification in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a genetically extremely heterogeneous disease. Drug resistance after induction therapy is a very frequent event resulting in poor medium survival times. Therefore, the identification of new targets and treatment modalities is a medical high priority issue. We addressed our attention to circular RNAs (circRNAs), which can act as oncogenes or tumor suppressors in AML. We searched the literature (PubMed) and identified eight up-regulated and two down-regulated circ-RNAs with activity in preclinical in vivo models. In addition, we identified twenty-two up-regulated and four down-regulated circRNAs with activity in preclinical in vitro systems, but pending in vivo activity. Up-regulated RNAs are potential targets for si- or shRNA-based approaches, and down-regulated circRNAs can be reconstituted by replacement therapy to achieve a therapeutic benefit in preclinical systems. The up-regulated targets can be tackled with small molecules, antibody-based entities, or other modes of intervention. For down-regulated targets, up-regulators must be identified. The ranking of the identified circRNAs with respect to therapy of AML will depend on further target validation experiments.

Hepatocellular Carcinoma: Up-regulated Circular RNAs Which Mediate Efficacy in Preclinical In Vivo Models.

Hepatocellular carcinoma (HCC) ranges as number two with respect to the incidence of tumors and is associated with a dismal prognosis. The therapeutic efficacy of approved multi-tyrosine kinase inhibitors and checkpoint inhibitors is modest. Therefore, the identification of new therapeutic targets and entities is of paramount importance. We searched the literature for up-regulated circular RNAs (circRNAs) which mediate efficacy in preclinical in vivo models of HCC. Our search resulted in 14 circRNAs which up-regulate plasma membrane transmembrane receptors, while 5 circRNAs induced secreted proteins. Two circRNAs facilitated replication of Hepatitis B or C viruses. Three circRNAs up-regulated high mobility group proteins. Six circRNAs regulated components of the ubiquitin system. Seven circRNAs induced GTPases of the family of ras-associated binding proteins (RABs). Three circRNAs induced redox-related proteins, eight of them up-regulated metabolic enzymes and nine circRNAs induced signaling-related proteins. The identified circRNAs up-regulate the corresponding targets by sponging microRNAs. Identified circRNAs and their targets have to be validated by standard criteria of preclinical drug development. Identified targets can potentially be inhibited by small molecules or antibody-based moieties and circRNAs can be inhibited by small-interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) for therapeutic purposes.

Up-regulated Circular RNAs in Colorectal Cancer: New Entities for Therapy and Tools for Identification of Therapeutic Targets.

Patients with disseminated colorectal cancer have a dismal prognosis with a 5-year survival rate of only 13%. In order to identify new treatment modalities and new targets, we searched the literature for up-regulated circular RNAs in colorectal cancer which induce tumor growth in corresponding preclinical in vivo models. We identified nine circular RNAs that mediate resistance against chemotherapeutic agents, seven that up-regulate transmembrane receptors, five that induce secreted factors, nine that activate signaling components, five which up-regulate enzymes, six which activate actin-related proteins, six which induce transcription factors and two which up-regulate the MUSASHI family of RNA binding proteins. All of the circular RNAs discussed in this paper induce the corresponding targets by sponging microRNAs (miRs) and can be inhibited by RNAi or shRNA in vitro and in xenograft models. We have focused on circular RNAs with demonstrated activity in preclinical in vivo models because the latter is an important milestone in drug development. All circular RNAs with in vitro activity only data are not referenced in this review. The translational impact of inhibition of these circular RNAs and of the identified targets for treatment of colorectal cancer (CRC) are discussed.

Long Non-coding RNAs Sponging MicroRNAs With Efficacy in Preclinical In Vivo Models of Esophageal Squamous Cell Cancer.

Esophageal cancer is of two subtypes: Esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC). Both are associated with a dismal prognosis. Therefore, the identification of new targets and treatment modalities is an issue of paramount importance. In this review, we focus on long non-coding RNAs (lncRNAs) which have been shown to mediate efficacy in preclinical in vivo models of ESCC by sponging microRNAs. Searching the literature, we identified four lncRNAs which were down-regulated and 23 which were up-regulated in comparison to corresponding normal tissues. The down-regulated lncRNAs lead to up-regulation of oncogenic pathways and down-regulation of tumor suppressors. The up-regulated lncRNAs target transcription factors, transmembrane receptors, cell-cycle related proteins, actin-binding proteins, signaling pathways, enzymes including epigenetic modification factors, cellular transport proteins and other categories. We describe reconstitution and inhibition of function of the corresponding lncRNAs and comment on validation and druggability of the identified targets.

MicroRNAs and Corresponding Targets in Esophageal Cancer as Shown In Vitro and In Vivo in Preclinical Models.

Squamous cell carcinoma of the esophagus is associated with a dismal prognosis. Therefore, identification of new targets and implementation of new treatment modalities are issues of paramount importance. Based on a survey of the literature, we identified microRNAs conferring antitumoral activity in preclinical in vivo experiments. In the category of miRs targeting secreted factors and transmembrane receptors, four miRs were up-regulated and 10 were down-regulated compared with five out of nine in the category transcription factors, and six miRs were down-regulated in the category enzymes, including metabolic enzymes. The down-regulated miRs have targets which can be inhibited by small molecules or antibody-related entities, or re-expressed by reconstitution therapy. Up-regulated miRs have targets which can be reconstituted with small molecules or inhibited with antagomirs.

MicroRNAs Involved in Small-cell Lung Cancer as Possible Agents for Treatment and Identification of New Targets.

Small-cell lung cancer, a neuro-endocrine type of lung cancers, responds very well to chemotherapy-based agents. However, a high frequency of relapse due to adaptive resistance is observed. Immunotherapy-based treatments with checkpoint inhibitors has resulted in improvement of treatment but the responses are not as impressive as in other types of tumor. Therefore, identification of new targets and treatment modalities is an important issue. After searching the literature, we identified eight down-regulated microRNAs involved in radiation- and chemotherapy-induced resistance, as well as three up-regulated and four down-regulated miRNAs with impacts on proliferation, invasion and apoptosis of small-cell lung cancer cells in vitro. Furthermore, one up-regulated and four down-regulated microRNAs with in vivo activity in SCLC cell xenografts were identified. The identified microRNAs are candidates for inhibition or reconstitution therapy. The corresponding targets are candidates for inhibition or functional reconstitution with antibody-based moieties or small molecules.

Clear Cell Renal Carcinoma: MicroRNAs With Efficacy in Preclinical In Vivo Models.

In order to identify new targets and treatment modalities for clear cell renal carcinoma, we surveyed the literature with respect to microRNAs involved in this disease. In this review, we have focused on up- and down-regulated miRs which mediate efficacy in preclinical clear-cell renal carcinoma-related in vivo models. We have identified 10 up-regulated and 33 down-regulated micro-RNAs according to this criterion. As proof-of-concept, micro-RNAs interfering with VEGF (miR-205p) and mTOR (mir-99a) pathways, which are modulated by approved drugs for this disease, have been identified. miRs targeting hypoxia induced factor-2α (HIF-2α) (miR-145), E3 ubiquitinylases speckle-type POZ protein (SPOP) (miR 520/372/373) and casitas B-lineage lymphoma (CBL) (miR-200a-3p), interfere with druggable targets. Further identified miRs interfere with cell-cycle dependent kinases, such as CDK2 (miR-200c), CDK4, 6 (miR-1) and CDK4, 9 (206c). Transmembrane receptor Ral interacting protein of 76 kD (RLIP76), targeted by mir-137, has emerged as another important target for ccRCC. Additional miRs and their targets merrying further preclinical validation are discussed.

microRNAs Promoting Growth of Gastric Cancer Xenografts and Correlation to Clinical Prognosis.

The annual death toll for gastric cancer is in the range of 700,000 worldwide. Even in patients with early-stage gastric cancer recurrence within five years has been observed after surgical resection and following chemotherapy with therapy-resistant features. Therefore, the identification of new targets and treatment modalities for gastric cancer is of paramount importance. In this review we focus on the role of microRNAs with documented efficacy in preclinical xenograft models with respect to growth of human gastric cancer cells. We have identified 31 miRs (-10b, -19a, -19b, -20a, -23a/b, -25, -27a-3p, -92a, -93, -100, -106a, -130a, -135a, -135b-5p, -151-5p, -187, -199-3p, -215, -221-3p, -224, -340a, -382, -421, -425, -487a, -493, -532-3p, -575, -589, -664a-3p) covering 26 different targets which promote growth of gastric cancer cells in vitro and in vivo as xenografts. Five miRs (miRs -10b, 151-5p, -187, 532-3p and -589) additionally have an impact on metastasis. Thirteen of the identified miRs (-19b, -20a/b, -25, -92a, -106a, -135a, -187, -221-3p, -340a, -421, -493, -575 and -589) have clinical impact on worse prognosis in patients.

Identification of MicroRNAs With In Vivo Efficacy in Multiple Myeloma-related Xenograft Models.

BACKGROUND/AIM: Multiple myeloma is a B-cell neoplasm, which can spread within the marrow of the bones forming many small tumors. In advanced disease, multiple myeloma can spread to the blood as plasma cell leukemia. In some cases, a localized tumor known as plasmacytoma is found within a single bone. Despite the approval of several agents such as melphalan, corticosteroids, proteasome inhibitors, thalidomide-based immuno-modulatory agents, histone deacetylase inhibitors, a nuclear export inhibitor and monoclonal antibodies daratuzumab and elatuzumab, the disease presently remains uncurable. MATERIALS AND METHODS: In order to define new targets and treatment modalities we searched the literature for microRNAs, which increase or inhibit in vivo efficacy in multiple-myeloma-related xenograft models. RESULTS AND CONCLUSION: We identified six up-regulated and twelve down-regulated miRs, which deserve further preclinical validation.

Pancreatic Ductal Adenocarcinoma: MicroRNAs Affecting Tumor Growth and Metastasis in Preclinical In Vivo Models.

Patients with pancreatic ductal adenocarcinoma have a dismall prognosis because at the time of diagnosis, in the vast majority of patients the tumor has already disseminated to distant organs and the therapeutic benefit of approved agents such as gemcitabine is limited. Therefore, the identification and preclinical and clinical validation of therapeutic agents covering new targets is of paramount importance. In this review we have summarized microRNAs and corresponding targets which affect growth and metastasis of pancreatic tumors in preclinical mouse in vivo models. We identified four up-regulated and 16 down-regulated miRs in PDAC in comparison to corresponding normal tissues. Three sub-categories of miRs have emerged: miRs affecting tumor growth and miRs with an impact on both, tumor growth and metastasis or metastasis only. Finally, we discuss technical and therapeutic aspects of miR-related therapeutic agents for the treatment of pancreatic ductal adenocarcinoma.

MicroRNAs as Potential Targets for Therapeutic Intervention With Metastasis of Non-small Cell Lung Cancer.

The death toll of non-small cell lung cancer (NSCLC) patients is primarily due to metastases, which are poorly amenable to therapeutic intervention. In this review we focus on miRs associated with metastasis of NSCLC as potential new targets for anti-metastatic therapy. We discuss miRs validated as therapeutic targets by in vitro data, identification of target(s) and pathway(s) and in vivo efficacy data in at least one clinically-relevant metastasis-related model. A few of the discussed miRs correlate with the clinical status of NSCLC patients. Using miRs as therapeutic agents has the advantage that targeting a single miR can potentially interfere with several metastatic pathways. Depending on their mode of action, the corresponding miRs can be up- or down-regulated compared to normal matching tissues. Here, we describe therapeutic approaches for reconstitution therapy and miR inhibition, general principles of anti-metastatic therapy as well as current technical pitfalls.

Potential microRNA-related Targets for Therapeutic Intervention with Ovarian Cancer Metastasis.

Treatment of disseminated epithelial ovarian cancer (EOC) is an unmet medical need. Therefore, the identification along with preclinical and clinical validation of new targets is an issue of high importance. In this review we focus on microRNAs that mediate metastasis of EOC. We summarize up-regulated metastasis-promoting and down-regulated metastasis-suppressing microRNAs. We focus on preclinical in vitro and in vivo functions as well as their metastasis-related clinical correlations. Finally, we outline modalities for therapeutic intervention and critical issues of microRNA-based therapeutics in the context of metastatic EOC.

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients.

PURPOSE: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform expression impacts antitumoral responses in vivo to treatment with RG7356, a humanized anti-CD44 antibody inhibiting CD44-HA interaction. EXPERIMENTAL DESIGN: CD44 isoform expression on various tumor cell lines was analyzed by RNASeq while data on patients with different tumor types were obtained from the publicly available TCGA RNASeq dataset as well as a phase I clinical study (NCT01358903). We analyzed the link between HA production and CD44 isoform expression as well as the consequences of blocking the CD44-mediated cell adhesion to HA using RG7356. The correlation between CD44 isoform expression and antitumor response to RG7356 treatment was investigated in the corresponding murine xenograft in vivo models as well as in a subset of patients treated with RG7356 from a recently completed phase I clinical trial. RESULTS: CD44 isoform expression, in particular expression of CD44s, is associated with HA production and predicts response to treatment with RG7356 in tumor xenograft models. Furthermore, patient data suggest that CD44 isoform status is a potential predictive biomarker for clinical response to treatment with RG7356. CONCLUSIONS: We provide new insights into the close interplay between CD44 and HA and a potential biomarker to enrich patient responses to RG7356 in the clinic.

Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist.

The cell surface glycoprotein CD44 plays an important role in the development and progression of various tumor types. RG7356 is a humanized antibody targeting the constant region of CD44 that shows antitumor efficacy in mice implanted with CD44-expressing tumors such as MDA-MB-231 breast cancer cells. CD44 receptor seems to function as the main receptor for hyaluronic acid and osteopontin, serving as coreceptor for growth factor pathways like cMet, EGFR, HER-2, and VEGFR and by cytoskeletal modulation via ERM and Rho kinase signaling. To assess the direct impact of RG7356 binding to the CD44 receptor, a global mass spectrometry-based phosphoproteomics approach was applied to freshly isolated MDA-MB-231 tumor xenografts. Results from a global phosphoproteomics screen were further corroborated by Western blot and ELISA analyses of tumor lysates from CD44-expressing tumors. Short-term treatment of tumor-bearing mice with RG7356 resulted in modifications of the MAPK pathway in the responsive model, although no effects on downstream phosphorylation were observed in a nonresponsive xenograft model. Taken together, our approach augments the value of other high throughput techniques to identify biomarkers for clinical development of targeted agents.

Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity.

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Bcl-2 controls dendritic cell longevity in vivo.

Dendritic cells (DC) were found to down-regulate Bcl-2 protein upon maturation in vivo. Because Bcl-2 has been shown to exert anti-apoptotic functions, down-regulation of Bcl-2 could be a mechanism by which DC longevity is controlled. To dysregulate this potential control system and to study the role of Bcl-2 in DC, we expressed human Bcl-2 under control of the murine CD11c-promoter as a transgene specifically in DC and show that DC frequencies and numbers increase in transgenic mice. In vivo bromodeoxyuridin, as well as adoptive, DC transfer studies show that the relative turnover/survival of mature Bcl-2 transgenic DC is increased. This had a direct impact on CD4+ T cell, as well as humoral immune, responses, which were elevated in transgenic animals. When Bcl-2 transgenic DC were used as DC vaccines, they induced 2- to 3-fold greater expansion of Ag-specific CTL, and stronger in vivo cytotoxicity. Overall, these data indicate that down-regulation of Bcl-2 controls DC longevity, which in turn directly regulates immune responses and the efficacy of DC when used as vaccines.