Comparison of three-dimensional cell culture techniques of dedifferentiated liposarcoma and their integration with future research.

Background: Dedifferentiated liposarcoma is a formidable sarcoma subtype due to its high local recurrence rate and resistance to medical treatment. While 2D cell cultures are still commonly used, 3D cell culture systems have emerged as a promising alternative, particularly scaffold-based techniques that enable the creation of 3D models with more accurate cell-stroma interactions. Objective: To investigate how 3D structures with or without the scaffold existence would affect liposarcoma cell lines growth morphologically and biologically. Methods: Lipo246 and Lipo863 cell lines were cultured in 3D using four different methods; Matrigel® ECM scaffold method, Collagen ECM scaffold method, ULA plate method and Hanging drop method, in addition to conventional 2D cell culture methods. All samples were processed for histopathological analysis (HE, IHC and DNAscope™), Western blot, and qPCR; moreover, 3D collagen-based models were treated with different doses of SAR405838, a well-known inhibitor of MDM2, and cell viability was assessed in comparison to 2D model drug response. Results: Regarding morphology, cell lines behaved differently comparing the scaffold-based and scaffold-free methods. Lipo863 formed spheroids in Matrigel® but not in collagen, while Lipo246 did not form spheroids in either collagen or Matrigel®. On the other hand, both cell lines formed spheroids using scaffold-free methods. All samples retained liposarcoma characteristic, such as high level of MDM2 protein expression and MDM2 DNA amplification after being cultivated in 3D. 3D collagen samples showed higher cell viability after SAR40538 treatment than 2D models, while cells sensitive to the drug died by apoptosis or necrosis. Conclusion: Our results prompt us to extend our investigation by applying our 3D models to further oncological relevant applications, which may help address unresolved questions about dedifferentiated liposarcoma biology.

Gene Expression Landscape of Chronic Myeloid Leukemia K562 Cells Overexpressing the Tumor Suppressor Gene PTPRG.

This study concerns the analysis of the modulation of Chronic Myeloid Leukemia (CML) cell model K562 transcriptome following transfection with the tumor suppressor gene encoding for Protein Tyrosine Phosphatase Receptor Type G (PTPRG) and treatment with the tyrosine kinase inhibitor (TKI) Imatinib. Specifically, we aimed at identifying genes whose level of expression is altered by PTPRG modulation and Imatinib concentration. Statistical tests as differential expression analysis (DEA) supported by gene set enrichment analysis (GSEA) and modern methods of ontological term analysis are presented along with some results of current interest for forthcoming experimental research in the field of the transcriptomic landscape of CML. In particular, we present two methods that differ in the order of the analysis steps. After a gene selection based on fold-change value thresholding, we applied statistical tests to select differentially expressed genes. Therefore, we applied two different methods on the set of differentially expressed genes. With the first method (Method 1), we implemented GSEA, followed by the identification of transcription factors. With the second method (Method 2), we first selected the transcription factors from the set of differentially expressed genes and implemented GSEA on this set. Method 1 is a standard method commonly used in this type of analysis, while Method 2 is unconventional and is motivated by the intention to identify transcription factors more specifically involved in biological processes relevant to the CML condition. Both methods have been equipped in ontological knowledge mining and word cloud analysis, as elements of novelty in our analytical procedure. Data analysis identified RARG and CD36 as a potential PTPRG up-regulated genes, suggesting a possible induction of cell differentiation toward an erithromyeloid phenotype. The prediction was confirmed at the mRNA and protein level, further validating the approach and identifying a new molecular mechanism of tumor suppression governed by PTPRG in a CML context.

Pan-Cancer Analysis of Canonical and Modified miRNAs Enhances the Resolution of the Functional miRNAome in Cancer.

Epitranscriptomic studies of miRNAs have added a new layer of complexity to the cancer field. Although there is fast-growing interest in adenosine-to-inosine (A-to-I) miRNA editing and alternative cleavage that shifts miRNA isoforms, simultaneous evaluation of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modification types, including A-to-I miRNA editing and shifted miRNA isoforms, in >13,000 adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments data sets. The differences between canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint were investigated. The combination of canonical miRNAs and modified miRNAs boosted the quality of clustering results, outlining unique clinicopathologic features among cohorts. Certain modified miRNAs showed opposite expression from their canonical counterparts in cancer, potentially impacting their targets and function. Finally, a shifted and edited miRNA isoform was experimentally validated to directly bind and suppress a unique target. These findings outline the importance of going beyond the well-established paradigm of one mature miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression. SIGNIFICANCE: Modified miRNAs may act as cancer biomarkers and function as allies or antagonists of their canonical counterparts in gene regulation, suggesting the concurrent consideration of canonical and modified miRNAs can boost patient stratification.

Successful Preservation of Native BCR::ABL1 in Chronic Myeloid Leukemia Primary Leukocytes Reveals a Reduced Kinase Activity.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the acquisition of t(9;22) generating the fusion tyrosine kinase BCR::ABL1. However, despite the crucial role of this protein in the dysregulation of numerous signal transduction pathways, a direct measure of BCR::ABL1 kinase activity in chronic phase (CP) CML was never accomplished due to intense degradative activity present in mature leukocytes. Therefore, we developed a procedure suitable to preserve BCR::ABL1 protein under non-denaturing, neutral pH conditions in primary, chronic phase (CP)-CML samples. As a result, specific kinase activity was detected utilizing a biotinylated peptide substrate highly selective for c-ABL1. Furthermore, through this approach, BCR::ABL1 kinase activity was barely detectable in CP-CML compared to Ph+ acute lymphoblastic leukemia primary samples, where kinase activity is comparable to those measured in Ph+ cell lines. These in vitro findings provide the first direct measure of BCR::ABL1 kinase activity in primary CP-CML and reveal the presence of a still uncharacterized inhibitory mechanism that maintains BCR::ABL1 in a low activity state in CP-CML despite its overexpression.

Clinicopathological Variables and Outcome in Chronic Myeloid Leukemia Associated With BCR-ABL1 Transcript Type and Body Weight: An Outcome of European LeukemiaNet Project.

OBJECTIVE: It is debatable whether BCR-ABL1 transcript type has an impact on outcome of treatment of patients with CML, and it is not widely studied whether body weight influences response to treatment. In this study, we tried to find out if any of these factors has an impact on response to treatment and outcome. METHODOLOGY: We conducted a retrospective analysis of the files of 79 patients being treated in our center for CML with known BCR-ABL1 breakpoints, and patients' management and response assessment was done based on ELN 2013 guidelines. The analysis was performed based on two main groups, obese vs. normal BMI, and then based on BCR-ABL1 transcripts: e13a2 vs. e14a2. Cumulative incidence of MMR, CCyR, and DMR were estimated using the Kaplan-Meier survival curve method, and comparisons between groups were performed by the Log-rank/Gray test methods. RESULTS/CONCLUSION: In the patient-cohort studied, there was no statistically significant difference in molecular response between patients with CML based on body weight or transcript type although patients in the obesity group achieved higher and faster MMR with no statistical significance.

MiREDiBase, a manually curated database of validated and putative editing events in microRNAs.

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that function as translational repressors. MiRNAs are involved in most cellular processes, and their expression and function are presided by several factors. Amongst, miRNA editing is an epitranscriptional modification that alters the original nucleotide sequence of selected miRNAs, possibly influencing their biogenesis and target-binding ability. A-to-I and C-to-U RNA editing are recognized as the canonical types, with the A-to-I type being the predominant one. Albeit some bioinformatics resources have been implemented to collect RNA editing data, it still lacks a comprehensive resource explicitly dedicated to miRNA editing. Here, we present MiREDiBase, a manually curated catalog of editing events in miRNAs. The current version includes 3,059 unique validated and putative editing sites from 626 pre-miRNAs in humans and three primates. Editing events in mature human miRNAs are supplied with miRNA-target predictions and enrichment analysis, while minimum free energy structures are inferred for edited pre-miRNAs. MiREDiBase represents a valuable tool for cell biology and biomedical research and will be continuously updated and expanded at https://ncrnaome.osumc.edu/miredibase .

The MicroRNA Family Gets Wider: The IsomiRs Classification and Role.

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases.

Detecting and Characterizing A-To-I microRNA Editing in Cancer.

Adenosine to inosine (A-to-I) editing consists of an RNA modification where single adenosines along the RNA sequence are converted into inosines. Such a biochemical transformation is catalyzed by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs) and occurs either co- or post-transcriptionally. The employment of powerful, high-throughput detection methods has recently revealed that A-to-I editing widely occurs in non-coding RNAs, including microRNAs (miRNAs). MiRNAs are a class of small regulatory non-coding RNAs (ncRNAs) acting as translation inhibitors, known to exert relevant roles in controlling cell cycle, proliferation, and cancer development. Indeed, a growing number of recent researches have evidenced the importance of miRNA editing in cancer biology by exploiting various detection and validation methods. Herein, we briefly overview early and currently available A-to-I miRNA editing detection and validation methods and discuss the significance of A-to-I miRNA editing in human cancer.

Aberrant DNA methylation of PTPRG as one possible mechanism of its under-expression in CML patients in the State of Qatar.

BACKGROUND: Several studies showed that aberrant DNA methylation is involved in leukemia and cancer pathogenesis. Protein tyrosine phosphatase receptor gamma (PTPRG) expression is a natural inhibitory mechanism that is downregulated in chronic myeloid leukemia (CML) disease. The mechanism behind its downregulation has not been fully elucidated yet. AIM: This study aimed to investigate the CpG methylation status at the PTPRG locus in CML patients. METHODS: Peripheral blood samples from CML patients at time of diagnosis [no tyrosine kinase inhibitors (TKIs)] (n = 13), failure to (TKIs) treatment (n = 13) and healthy controls (n = 6) were collected. DNA was extracted and treated with bisulfite treatment, followed by PCR, sequencing of 25 CpG sites in the promoter region and 26 CpG sites in intron-1 region of PTPRG. The bisulfite sequencing technique was employed as a high-resolution method. RESULTS: CML groups (new diagnosed and failed treatment) showed significantly higher methylation levels in the promoter and intron-1 regions of PTPRG compared to the healthy group. There were also significant differences in methylation levels of CpG sites in the promoter and intron-1 regions amongst the groups. CONCLUSION: Aberrant methylation of PTPRG is potentially one of the possible mechanisms of PTPRG downregulation detected in CML.

Combined loss of function of two different loci of miR-15/16 drives the pathogenesis of acute myeloid leukemia.

Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS-AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS-AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease.

Regulative Loop between ß-catenin and Protein Tyrosine Receptor Type γ in Chronic Myeloid Leukemia.

Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. ß-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between ß-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene ß-Catenin. PTPRG was found to be capable of dephosphorylating ß-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of ß-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and ß-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and ß-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.

Dysregulation of different classes of tRNA fragments in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and dysregulation of tRNA-derived short noncoding RNA (tsRNA) (tRF-1) expression is an accompanying event in the development of this disease. tsRNAs are fragments originating from the 3' end of tRNA precursors and do not contain mature tRNA sequences. In contrast to tsRNAs, mature tRFs (tRF-3s, tRF-5s, and internal tRFs) are produced from mature tRNA sequences and are redundant fragments. We investigated tsRNA expression in CLL and determined tsRNA signatures in indolent CLL and aggressive CLL vs. normal B cells. We noticed that both ts-43 and ts-44 are derived from distinct genes of pre-tRNAHis, and are down-regulated in CLL 3- to 5-fold vs. normal B cells. Thus, we investigated expression levels of tRF-5 fragments from tRNAHis in CLL samples and healthy controls, and determined that such fragments are down-regulated by 5-fold in CLLs vs. normal controls. Given these results, we investigated the expression of all mature tRFs in CLLs vs. normal controls. We found a drastic dysregulation of the expression of mature tRFs in CLL. In aggressive CLL, for the top 15 up-regulated fragments, linear fold change varied from 2,053- to 622-fold. For the top 15 down-regulated fragments in CLL, linear fold change varied from 314- to 52-fold. In addition, 964 mature tRFs were up-regulated at least 2-fold in CLL, while 701 fragments were down-regulated at least 2-fold. Similar results were obtained for indolent CLL. Our results suggest that mature tRFs may have oncogenic and/or tumor suppressor function in CLL.

Experimental Validation of MicroRNA Targets: Luciferase Reporter Assay.

MicroRNAs (miRNAs) are small noncoding RNAs of 22-25 nucleotides that control gene expression at the posttranscriptional level through the degradation of mRNAs or translational repression. In the last 15 years, the study of these small molecules helped elucidate their role in the regulation of many cellular processes and the onset and development of several diseases. Therefore, many computational tools based on algorithms for target prediction have been developed to identify potential miRNA-target interactions. The improvement of experimental approaches to more easily and quickly confirm in silico predictions has become essential for the study of these small RNAs and their molecular functions. In this chapter, we summarized the principal steps of one of the most used techniques for the validation of microRNA targets, the Luciferase assay, thus explaining the underlying principles and the procedures to apply it best.

Experimental Validation of MicroRNA Targets: Mutagenesis of Binding Regions.

MicroRNAs (miRNAs) are small noncoding RNAs that are 22-25 nucleotides in length and control gene expression posttranscriptionally by degrading mRNAs or by translational repression. Many computational tools based on algorithms for target prediction have already been developed to find potential miRNA-target interactions. Since it is essential to confirm in silico predictions, experimental approaches have been improved to validate computationally predicted targets. One of the most widely used techniques is the luciferase assay which allows for the confirmation of specific binding between microRNA and the mRNA target using a reporter plasmid containing the 3' UTR of the target. Through the mutagenesis of this region it is possible to provide indirect evidence of the specific microRNA-mRNA interaction demonstrated using this assay. In this chapter we review the main experimental steps of the 3' UTR mutagenesis and the best way to apply this method to support and complete the luciferase assay procedure.

Experimental Validation of MicroRNA Targets: Analysis of MicroRNA Targets Through Western Blotting.

MicroRNAs are a class of small noncoding RNA involved in the mechanism of RNA silencing and regulation of gene expression at a posttranscriptional level. Recently, the discovery of their targets led to the understanding of the molecular role of these small molecules and their involvement in the pathogenesis of numerous diseases, including cancer. Not long ago, the improvement of several informatics tools for microRNA target prediction has supported the experimental research through the selection of potential mRNA as microRNA target candidates. Since the regulation mediated by microRNA affects gene expression at a posttranscriptional level, the analysis of the proteins encoded by the gene targets is essential in understanding the involvement of these small molecules in biological processes and their role in several diseases. In this chapter, we describe the experimental procedure of Western blotting applied to the validation of microRNA targets. Western blotting is one of the most common and largest know technique for protein analysis. This method, coupled with the luciferase assay, represents the standard procedure for the experimental confirmation of microRNA targeting.

ncRNA Editing: Functional Characterization and Computational Resources.

Noncoding RNAs (ncRNAs) have received much attention due to their central role in gene expression and translational regulation as well as due to their involvement in several biological processes and disease development. Small noncoding RNAs (sncRNAs), such as microRNAs and piwiRNAs, have been thoroughly investigated and functionally characterized. Long noncoding RNAs (lncRNAs), known to play an important role in chromatin-interacting transcription regulation, posttranscriptional regulation, cell-to-cell signaling, and protein regulation, are also being investigated to further elucidate their functional roles.Next-generation sequencing (NGS) technologies have greatly aided in characterizing the ncRNAome. Moreover, the coupling of NGS technology together with bioinformatics tools has been essential to the genome-wide detection of RNA modifications in ncRNAs. RNA editing, a common human co-transcriptional and posttranscriptional modification, is a dynamic biological phenomenon able to alter the sequence and the structure of primary transcripts (both coding and noncoding RNAs) during the maturation process, consequently influencing the biogenesis, as well as the function, of ncRNAs. In particular, the dysregulation of the RNA editing machineries have been associated with the onset of human diseases.In this chapter we discuss the potential functions of ncRNA editing and describe the knowledge base and bioinformatics resources available to investigate such phenomenon.

miR-125a and miR-34a expression predicts Richter syndrome in chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia. It is characterized by the accumulation of CD19+/CD5+ lymphocytes and can have variable outcomes. Richter syndrome (RS) is a lethal complication in CLL patients that results in aggressive B-cell lymphomas, and there are no tests to predict its occurrence. Because alterations in microRNA expression can predict the development and progression of several cancers, we investigated whether dysregulation of specific microRNAs can predict RS in CLL patients. Thus, we compared microRNA expression levels in samples from 49 CLL patients who later developed RS with samples from 59 CLL patients who did not. We found that high expression of miR-125a-5p or low expression of miR -34a-5p can predict ∼50% of RS with a false positive rate of ∼9%. We found that CLL patients predicted to develop RS show either an increase of miR-125a-5p expression (∼20-fold) or a decrease of miR-34a-5p expression (∼21-fold) compared with CLL patients that are not predicted to develop RS. Thus, miR-125a-5p and miR-34a-5p can be valuable predictor markers of RS and have the potential to provide physicians with information that can indicate the best therapeutic strategy for CLL patients.

MicroRNA dysregulation to identify therapeutic target combinations for chronic lymphocytic leukemia.

Loss of miR-15/16 is the most common genetic lesion in chronic lymphocytic leukemia (CLL), promoting overexpression of BCL2, which factors in leukemia pathogenesis. Indeed, an inhibitor of Bcl2, venetoclcax, is highly active in the treatment of patients with CLL. However, single-agent venetoclcax fails to eradicate minimal residual disease in most patients. Accordingly, we were interested in other genes that may be regulated by miR-15/16, which may target other drivers in CLL. We found that miR-15/16 targets ROR1, which encodes an onco-embryonic surface protein expressed on the CLL cells of over 90% of patients, but not on virtually all normal postpartum tissues. CLL with high-level expression of ROR1 also have high-level expression of Bcl2, but low-to-negligible miR-15/16 Moreover, CLL cases with high-level ROR1 have deletion(s) at the chromosomal location of the genes encoding miR-15/16 (13q14) more frequently than cases with low-to-negligible ROR1, implying that deletion of miR-15/16 may promote overexpression of ROR1, in addition to BCL2 ROR1 is a receptor for Wnt5a, which can promote leukemia-cell proliferation and survival, and can be targeted by cirmtuzumab, a humanized anti-ROR1 mAb. We find that this mAb can enhance the in vitro cytotoxic activity of venetoclcax for CLL cells with high-level expression of ROR1, indicating that combining these agents, which target ROR1 and Bcl2, may have additive, if not synergistic, activity in patients with this disease.