Deregulated circRNAs in Epithelial Ovarian Cancer With Activity in Preclinical In Vivo Models: Identification of Targets and New Modalities for Therapeutic Intervention.

Epithelial ovarian cancer (EOC) is associated with a dismal prognosis due to development of resistance to chemotherapy and metastasis in the peritoneal cavity and distant organs. In order to identify new targets and treatment modalities we searched the literature for up- and and down-regulated circRNAs with efficacy in preclinical EOC-related in vivo systems. Our search yielded circRNAs falling into the following categories: cisplatin and paclitaxel resistance, transmembrane receptors, secreted factors, transcription factors, RNA splicing and processing factors, RAS pathway-related components, proteolysis and cell-cycle regulation, signaling-related proteins, and circRNAs regulating proteins in additional categories. These findings can be potentially translated by validation and manipulation of the corresponding targets, inhibition of circRNAs with antisense oligonucleotides (ASO), small interfering RNAs (siRNA) or small hairpin RNA (shRNA) or by reconstituting their activity.

Circular RNA in Non-small Cell Lung Carcinoma: Identification of Targets and New Treatment Modalities.

Despite availability of several treatment options for non-small cell lung cancer (NSCLC), such as surgery, chemotherapy, radiation, targeted therapy and immunotherapy, the survival rate of patients for five years is in the range of 22%. Therefore, identification of new targets and treatment modalities for this disease is an important issue. In this context, we screened the PubMed database for up-regulated circular RNAs (circRNAs) which promote growth of NSCLC in preclinical models in vitro as well as in vivo xenograft models in immuno-compromised mice. This approach led to potential targets for further validation and inhibition with small molecules or antibody-derived entities. In case of preclinical validation, the corresponding circRNAs can be inhibited with small interfering RNAs (siRNA) or short hairpin RNAs (shRNA). The identified circRNAs act by sponging microRNAs (miRs) preventing cleavage of the mRNA of the corresponding targets. We identified nine circRNAs up-regulating transmembrane receptors, five circRNAs increasing expression of secreted proteins, nine circRNAs promoting expression of components of signaling pathways, six circRNAs involved in regulation of splicing and RNA processing, six circRNAs up-regulating actin-related and RNA processing components, seven circRNAs increasing the steady-state levels of transcription factors, two circRNAs increasing high-mobility group proteins, four circRNAs increasing components of the epigenetic modification system and three circRNAs up-regulating protein components of additional systems.

Perspectives of data science in preclinical safety assessment.

The data landscape in preclinical safety assessment is fundamentally changing because of not only emerging new data types, such as human systems biology, or real-world data (RWD) from clinical trials, but also technological advancements in data-processing software and analytical tools based on deep learning approaches. The recent developments of data science are illustrated with use cases for the three factors: predictive safety (new in silico tools), insight generation (new data for outstanding questions); and reverse translation (extrapolating from clinical experience to resolve preclinical questions). Further advances in this field can be expected if companies focus on overcoming identified challenges related to a lack of platforms and data silos and assuring appropriate training of data scientists within the preclinical safety teams.

Triple-negative Breast Cancer: Identification of circRNAs With Efficacy in Preclinical In Vivo Models.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with insufficient options for therapy. In order to identify new targets and treatment modalities we searched the literature for circular RNAs (circRNAs) which mediate efficacy in TNBC-related in vivo preclinical models. In addition to 5 down-regulated circRNAs which modulate tumor-suppressive pathways, we identified 15 up-regulated circRNAs. Down- and up-regulated refers to expression in corresponding non-transformed cells and tissues. The up-regulated circRNAs comprise five transmembrane receptors and secreted proteins as targets, five transcription factors and transcription-associated targets, four cell-cycle related circRNAs and one involved in paclitaxel resistance. In this review article we discuss drug-discovery related aspects and modalities of therapeutic intervention. Down-regulated circRNAs can be reconstituted by re-expression of corresponding circRNAs in tumor cells or up-regulation of corresponding targets. Up-regulated circRNAs can be inhibited by small-interfering RNA (siRNA) or short hairpin RNA (shRNA)-based approaches or inhibition of the corresponding targets with small molecules or antibody-related moieties.

In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti-PD-1/PD-L1 Therapies.

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti-PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti-PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti-PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

Long Non-coding RNAs With In Vitro and In Vivo Efficacy in Preclinical Models of Esophageal Squamous Cell Carcinoma Which Act by a Non-microRNA Sponging Mechanism.

Esophageal squamous cell carcinoma is a type of cancer with dismal prognosis. Surgery, chemo- and radiation therapy, as well as immune checkpoint-blocking immunotherapy lead to limited improvement of survival of patients; therapy resistance and recurrencies hamper these treatment modalities. Therefore, the identification of new targets and treatment approaches is of paramount importance. We have searched the literature and identified 7 down-regulated and 16 up-regulated non-coding RNAs, which showed efficacy in preclinical esophageal squamous cell carcinoma-related in vitro and in vivo models, and discuss their diverse mode of actions. We excluded long non-coding RNAs, which act by sponging of microRNAs. It is presently unclear whether long non-coding RNA/protein, DNA and RNA interactions can be targeted with small molecules. We describe reconstitution therapy and inhibition of the corresponding long non-coding RNAs with small interfering RNAs and antisense oligonucleotides. Also, we discuss emerging targets for treatment of esophageal squamous cell carcinoma.

Down-regulated MicroRNAs in Gastric Carcinoma May Be Targets for Therapeutic Intervention and Replacement Therapy.

Gastric cancer is one of the leading types of cancer with an annual death toll of 700,000 worldwide. Despite the fact that several agents are approved for its treatment, high percentage of recurrence and intractability of metastatic disease remain a major problem. The identification of new targets and modalities for treatment are therefore of high priority. We have searched the literature for microRNAs down-regulated in gastric cancer with efficacy in gastric cancer-related murine xenograft models after reconstitution therapy. Among the identified miRs were 25 miRs targeting transcription factors, seven of them regulating cell-cycle and apotosis-related targets, and five of them regulating GTPase-related targets such as GAPs and GEFs. According to criteria such as prognostic impact, functional data, and tractability, miR-133 b/a (MCL1) and miR-518 (MDM2) are suggested as potentially valuable targets for further evaluation and possible treatment of gastric cancer.

Gastric Cancer: Identification of microRNAs Inhibiting Druggable Targets and Mediating Efficacy in Preclinical In Vivo Models.

In addition to chemotherapy, targeted therapies have been approved for treatment of locally advanced and metastatic gastric cancer. The therapeutic benefit is significant but more durable responses and improvement of survival should be achieved. Therefore, the identification of new targets and new approaches for clinical treatment are of paramount importance. In this review, we searched the literature for down-regulated microRNAs which interfere with druggable targets and exhibit efficacy in preclinical in vivo efficacy models. As druggable targets, we selected transmembrane receptors, secreted factors and enzymes. We identified 38 microRNAs corresponding to the criteria as outlined. A total of 13 miRs target transmembrane receptors, nine inhibit secreted proteins and 16 attenuate enzymes. These microRNAs are targets for reconstitution therapy of gastric cancer. Further target validation experiments are mandatory for all of the identified microRNAs.

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.

Bladder Cancer-related microRNAs With In Vivo Efficacy in Preclinical Models.

Progressive and metastatic bladder cancer remain difficult to treat. In this review, we critique seven up-regulated and 25 down-regulated microRNAs in order to identify new therapeutic entities and corresponding targets. These microRNAs were selected with respect to their efficacy in bladder cancer-related preclinical in vivo models. MicroRNAs and related targets interfering with chemoresistance, cell-cycle, signaling, apoptosis, autophagy, transcription factor modulation, epigenetic modification and metabolism are described. In addition, we highlight microRNAs targeting transmembrane receptors and secreted factors. We discuss druggability issues for the identified targets.

An Introduction to Machine Learning.

In the last few years, machine learning (ML) and artificial intelligence have seen a new wave of publicity fueled by the huge and ever-increasing amount of data and computational power as well as the discovery of improved learning algorithms. However, the idea of a computer learning some abstract concept from data and applying them to yet unseen situations is not new and has been around at least since the 1950s. Many of these basic principles are very familiar to the pharmacometrics and clinical pharmacology community. In this paper, we want to introduce the foundational ideas of ML to this community such that readers obtain the essential tools they need to understand publications on the topic. Although we will not go into the very details and theoretical background, we aim to point readers to relevant literature and put applications of ML in molecular biology as well as the fields of pharmacometrics and clinical pharmacology into perspective.

MicroRNAs Involved in Metastasis of Hepatocellular Carcinoma: Target Candidates, Functionality and Efficacy in Animal Models and Prognostic Relevance.

Hepatocellular carcinoma (HCC) is responsible for the second-leading cancer-related death toll worldwide. Although sorafenib and levantinib as frontline therapy and regorafenib, cabazantinib and ramicurimab have now been approved for second-line therapy, the therapeutic benefit is in the range of only a few months with respect to prolongation of survival. Aggressiveness of HCC is mediated by metastasis. Intrahepatic metastases and distant metastasis to the lungs, lymph nodes, bones, omentum, adrenal gland and brain have been observed. Therefore, the identification of metastasis-related new targets and treatment modalities is of paramount importance. In this review, we focus on metastasis-related microRNAs (miRs) as therapeutic targets for HCC. We describe miRs which mediate or repress HCC metastasis in mouse xenograft models. We discuss 18 metastasis-promoting miRs and 35 metastasis-inhibiting miRs according to the criteria as outlined. Six of the metastasis-promoting miRs (miR-29a, -219-5p, -331-3p, 425-5p, -487a and -1247-3p) are associated with unfavourable clinical prognosis. Another set of six down-regulated miRs (miR-101, -129-3p, -137, -149, -503, and -630) correlate with a worse clinical prognosis. We discuss the corresponding metastasis-related targets as well as their potential as therapeutic modalities for treatment of HCC-related metastasis. A subset of up-regulated miRs -29a, -219-5p and -425-5p and down-regulated miRs -129-3p and -630 were evaluated in orthotopic metastasis-related models which are suitable to mimic HCC-related metastasis. Those miRNAs may represent prioritized targets emerging from our survey.

The Genome Sequence of the Eastern Woodchuck (Marmota monax) - A Preclinical Animal Model for Chronic Hepatitis B.

The Eastern woodchuck (Marmota monax) has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

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.

Diphthamide affects selenoprotein expression: Diphthamide deficiency reduces selenocysteine incorporation, decreases selenite sensitivity and pre-disposes to oxidative stress.

The diphthamide modification of translation elongation factor 2 is highly conserved in eukaryotes and archaebacteria. Nevertheless, cells lacking diphthamide can carry out protein synthesis and are viable. We have analyzed the phenotypes of diphthamide deficient cells and found that diphthamide deficiency reduces selenocysteine incorporation into selenoproteins. Additional phenotypes resulting from diphthamide deficiency include altered tRNA-synthetase and selenoprotein transcript levels, hypersensitivity to oxidative stress and increased selenite tolerance. Diphthamide-eEF2 occupies the aminoacyl-tRNA translocation site at which UGA either stalls translation or decodes selenocysteine. Its position is in close proximity and mutually exclusive to the ribosomal binding site of release/recycling factor ABCE1, which harbors a redox-sensitive Fe-S cluster and, like diphthamide, is present in eukaryotes and archaea but not in eubacteria. Involvement of diphthamide in UGA-SECIS decoding may explain deregulated selenoprotein expression and as a consequence oxidative stress, NFkB activation and selenite tolerance in diphthamide deficient cells.

Interplay between reversible phosphorylation and irreversible ADP-ribosylation of eukaryotic translation elongation factor 2.

The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.

The Functional Role of Prostate Cancer Metastasis-related Micro-RNAs.

The mortality of patients with hormone-resistant prostate cancer can be ascribed to a large degree to metastasis to distant organs, predominantly to the bones. In this review, we discuss the contribution of micro-RNAs (miRs) to the metastatic process of prostate cancer. The criteria for selection of miRs for this review were the availability of preclinical in vivo metastasis-related data in conjunction with prognostic clinical data. Depending on their function in the metastatic process, the corresponding miRs are up- or down-regulated in prostate cancer tissues when compared to matching normal tissues. Up-regulated miRs preferentially target suppressors of cytokine signaling or tumor suppressor-related genes and metastasis-inhibitory transcription factors. Down-regulated miRs promote epithelial-mesenchymal transition or mesenchymal-epithelial transition and diverse pro-metastatic signaling pathways. Some of the discussed miRs exert their function by simultaneously targeting epigenetic pathways as well as cell-cycle-related, anti-apoptotic and signaling-promoting targets. Finally, we discuss potential therapeutic options for the treatment of prostate cancer-related metastases by substitution or inhibition of miRs.

Potential of Protein-based Anti-metastatic Therapy with Serpins and Inter α-Trypsin Inhibitors.

In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.

LST1: A multifunctional gene encoded in the MHC class III region.

The LST1 gene is located in the MHC class III cluster between the MHC class I and II regions. While most genes in this cluster have been sufficiently characterised, a definitive function and expression pattern for LST1 still remains elusive. In the present review we describe its promotor, gene organisation, splice variants and expression in human tissues, cell lines and cancer. We focus on LST1 expression in inflammation and discuss known correlations with autoimmune diseases and cancer. Current data on LST1 polymorphisms and their known associations with pathologies are also discussed in detail. We summarize the potential functions that have been described for the full-length LST1 protein including its function as a transmembrane adaptor protein with inhibitory signal transduction and its role as a membrane scaffold facilitating the formation of tunnelling nanotubes. We also discuss further potential functions by compiling all known LST1-interacting proteins. Furthermore, we address knowledge gaps and conflictive issues regarding disease association, non-hematopoietic expression and the discrepancy between RNA and protein expression data.