Search

Random mutations and recombinations are the main sources for the genetic diversity in SARS-CoV-2, with mutations in the SARS-CoV-2 spike (S) receptor binding motif (RBM) representing a high potential for the emergence of new putative variants under investigation (VUI) or variants of concern (VOC). It is of importance, to measure the different circulating SARS-CoV-2 lineages in order to establish a regional SARS-CoV-2 surveillance program. We established whole genome sequencing (WGS) of circulating SARS-CoV-2 lineages in order to establish a regional SARS-CoV-2 surveillance program.

Abstract Thyroid cancer is one of the most common malignancies of the endocrine system with increasing incidence. The vast majority of thyroid carcinomas derive from thyroid hormone producing follicular cells. Carcinomas of follicular origin are classified as follicular (FTCs), papillary (PTCs), partially differentiated (PDTCs) or anaplastic (ATCs) thyroid carcinomas. While FTCs and PTCs can be managed effectively, ATCs are considered one of the most lethal human cancers. Despite the identification of various genetic alterations, pathogenic mechanisms promoting the progression of thyroid carcinomas are still largely elusive. Over the recent years, aberrant microRNA expression was revealed in all as yet analyzed human cancers, including thyroid carcinomas. In view of the rapidly evolving perception that deregulated microRNA expression serves a pivotal role in tumor progression, microRNAs provide powerful tools for the diagnosis of thyroid carcinomas as well as the identification of potential therapeutic targets. Here, we summarize recent findings on microRNA signatures in thyroid carcinomas of follicular origin and discuss how deregulated microRNA expression could promote cancer progression.

Renowned as the predominant form of kidney cancer, clear cell renal cell carcinoma (ccRCC) exhibits susceptibility to immunotherapies due to its specific expression profile as well as notable immune cell infiltration. Despite this, effectively treating metastatic ccRCC remains a significant challenge, necessitating a more profound comprehension of the underlying molecular mechanisms governing its progression. Here, we unveil that the enhanced expression of the RNA-binding protein DNA dC → dU-editing enzyme APOBEC-3C (APOBEC3C; also known as A3C) in ccRCC tissue and ccRCC-derived cell lines serves as a catalyst for tumor growth by amplifying nuclear factor-kappa B (NF-κB) activity. By employing RNA-sequencing and cell-based assays in ccRCC-derived cell lines, we determined that A3C is a stress-responsive factor and crucial for cell survival. Furthermore, we identified that A3C binds and potentially stabilizes messenger RNAs (mRNAs) encoding positive regulators of the NF-κB pathway. Upon A3C depletion, essential subunits of the NF-κB family are abnormally restrained in the cytoplasm, leading to deregulation of NF-κB target genes. Our study illuminates the pivotal role of A3C in promoting ccRCC tumor development, positioning it as a prospective target for future therapeutic strategies., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

AlphaFold is an artificial intelligence approach for predicting the three-dimensional (3D) structures of proteins with atomic accuracy. One challenge that limits the use of AlphaFold models for drug discovery is the correct prediction of folding in the absence of ligands and cofactors, which compromises their direct use. We have previously described the optimization and use of the histone deacetylase 11 (HDAC11) AlphaFold model for the docking of selective inhibitors such as FT895 and SIS17. Based on the predicted binding mode of FT895 in the optimized HDAC11 AlphaFold model, a new scaffold for HDAC11 inhibitors was designed, and the resulting compounds were tested in vitro against various HDAC isoforms. Compound 5a proved to be the most active compound with an IC 50 of 365 nM and was able to selectively inhibit HDAC11. Furthermore, docking of 5a showed a binding mode comparable to FT895 but could not adopt any reasonable poses in other HDAC isoforms. We further supported the docking results with molecular dynamics simulations that confirmed the predicted binding mode. 5a also showed promising activity with an EC 50 of 3.6 µM on neuroblastoma cells., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in-cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of vinculin (metavinculin isoform) and paxillin (extended paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell-matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed., (© The Author(s) (2024). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The oncofetal RNA-binding protein IGF2BP1 has been reported to be a driver of tumor progression in a multitude of cancer entities. Its main function is the stabilization of target transcripts by shielding these from miRNA-mediated degradation. However, there is growing evidence that several virus species recruit IGF2BP1 to promote their propagation. In particular, tumor-promoting viruses, such as hepatitis B/C and human papillomaviruses, benefit from IGF2BP1. Moreover, recent evidence suggests that non-oncogenic viruses, such as SARS-CoV-2, also take advantage of IGF2BP1. The only virus inhibited by IGF2BP1 reported to date is HIV-1. This review summarizes the current knowledge about the interactions between IGF2BP1 and different virus species. It further recapitulates several findings by presenting analyses from publicly available high-throughput datasets.

RNA-binding proteins (RBPs) are crucial players in tumorigenesis and, hence, promising targets in cancer drug discovery. However, they are largely regarded as 'undruggable', because of the often noncatalytic and complex interactions between protein and RNA, which limit the discovery of specific inhibitors. Nonetheless, over the past 10 years, drug discovery efforts have uncovered RBP inhibitors with clinical relevance, highlighting the disruption of RNA-protein networks as a promising avenue for cancer therapeutics. In this review, we discuss the role of structurally distinct RBPs in cancer, and the mechanisms of RBP-directed small-molecule inhibitors (SMOIs) focusing on drug-protein interactions, binding surfaces, potency, and translational potential. Additionally, we underline the limitations of RBP-targeting drug discovery assays and comment on future trends in the field., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment., Methods: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models., Results: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also., Conclusion: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5., (© 2023. The Author(s).)

T-prolymphocytic leukemia (T-PLL) is a rare and mature T-cell malignancy with characteristic chemotherapy-refractory behavior and a poor prognosis. Molecular concepts of disease development have been restricted to protein-coding genes. Recent global microRNA (miR) expression profiles revealed miR-141-3p and miR-200c-3p (miR-141/200c) as two of the highest differentially expressed miRs in T-PLL cells versus healthy donor-derived T cells. Furthermore, miR-141/200c expression separates T-PLL cases into two subgroups with high and low expression, respectively. Evaluating the potential pro-oncogenic function of miR-141/200c deregulation, we discovered accelerated proliferation and reduced stress-induced cell death induction upon stable miR-141/200c overexpression in mature T-cell leukemia/lymphoma lines. We further characterized a miR-141/200c-specific transcriptome involving the altered expression of genes associated with enhanced cell cycle transition, impaired DNA damage responses, and augmented survival signaling pathways. Among those genes, we identified STAT4 as a potential miR-141/200c target. Low STAT4 expression (in the absence of miR-141/200c upregulation) was associated with an immature phenotype of primary T-PLL cells as well as with a shortened overall survival of T-PLL patients. Overall, we demonstrate an aberrant miR-141/200c-STAT4 axis, showing for the first time the potential pathogenetic implications of a miR cluster, as well as of STAT4, in the leukemogenesis of this orphan disease.

Upregulation of the proto-oncogene T-cell leukemia/lymphoma 1A (TCL1A) is causally implicated in various B-cell and T-cell malignancies. High-level TCL1A correlates with aggressive disease features and inferior clinical outcomes. However, the molecular and cell biological consequences of, particularly nuclear, TCL1A are not fully elucidated. We observed here in mouse models of subcellular site-specific TCL1A-induced lymphomagenesis that TCL1A exerts a strong transforming impact via nuclear topography. In proteomic screens of TCL1A-bound molecules in chronic lymphocytic leukemia (CLL) cells and B-cell lymphoma lines, we identified regulators of cell cycle and DNA repair pathways as novel TCL1A interactors, particularly enriched under induced DNA damage and mitosis. By functional mapping and in silico modeling, we specifically identified the mitotic checkpoint protein, cell division cycle 20 (CDC20), as a direct TCL1A interactor. According to the regulatory impact of TCL1A on the activity of the CDC20-containing mitotic checkpoint and anaphase-promoting complexes during mitotic progression, TCL1A overexpression accelerated cell cycle transition in B-cell lymphoma lines, impaired apoptotic damage responses in association with pronounced chromosome missegregation, and caused cellular aneuploidy in Eμ-TCL1A mice. Among hematopoietic cancers, CDC20 levels seem particularly low in CLL. CDC20 expression negatively correlated with TCL1A and lower expression marked more aggressive and genomically instable disease and cellular phenotypes. Knockdown of Cdc20 in TCL1A-initiated murine CLL promoted aneuploidy and leukemic acceleration. Taken together, we discovered a novel cell cycle-associated effect of TCL1A abrogating controlled cell cycle transition. This adds to our concept of oncogenic TCL1A by targeting genome stability. Overall, we propose that TCL1A acts as a pleiotropic adapter molecule with a synergistic net effect of multiple hijacked pathways., (© 2023 by The American Society of Hematology.)

Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR-CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

We show the effects of the three purine derivatives, caffeine, theophylline, and istradefylline, on cAMP production by adenylyl cyclase 5 (ADCY5)-overexpressing cell lines. A comparison of cAMP levels was performed for ADCY5 wild-type and R418W mutant cells. ADCY5-catalyzed cAMP production was reduced with all three purine derivatives, while the most pronounced effects on cAMP reduction were observed for ADCY5 R418W mutant cells. The gain-of-function ADCY5 R418W mutant is characterized by an increased catalytic activity resulting in elevated cAMP levels that cause kinetic disorders or dyskinesia in patients. Based on our findings in ADCY5 cells, a slow-release formulation of theophylline was administered to a preschool-aged patient with ADCY5-related dyskinesia. A striking improvement of symptoms was observed, outperforming the effects of caffeine that had previously been administered to the same patient. We suggest considering theophylline as an alternative therapeutic option to treat ADCY5-related dyskinesia in patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tänzler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3'UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression., (© 2022. The Author(s).)

T-cell prolymphocytic leukemia (T-PLL) is a chemotherapy-refractory T-cell malignancy with limited therapeutic options and a poor prognosis. Current disease concepts implicate TCL1A oncogene-mediated enhanced T-cell receptor (TCR) signaling and aberrant DNA repair as central perturbed pathways. We discovered that recurrent gains on chromosome 8q more frequently involve the argonaute RISC catalytic component 2 (AGO2) gene than the adjacent MYC locus as the affected minimally amplified genomic region. AGO2 has been understood as a protumorigenic key regulator of miRNA (miR) processing. Here, in primary tumor material and cell line models, AGO2 overrepresentation associated (i) with higher disease burden, (ii) with enhanced in vitro viability and growth of leukemic T cells, and (iii) with miR-omes and transcriptomes that highlight altered survival signaling, abrogated cell-cycle control, and defective DNA damage responses. However, AGO2 elicited also immediate, rather non-RNA-mediated, effects in leukemic T cells. Systems of genetically modulated AGO2 revealed that it enhances TCR signaling, particularly at the level of ZAP70, PLCγ1, and LAT kinase phosphoactivation. In global mass spectrometric analyses, AGO2 interacted with a unique set of partners in a TCR-stimulated context, including the TCR kinases LCK and ZAP70, forming membranous protein complexes. Models of their three-dimensional structure also suggested that AGO2 undergoes posttranscriptional modifications by ZAP70. This novel TCR-associated noncanonical function of AGO2 represents, in addition to TCL1A-mediated TCR signal augmentation, another enhancer mechanism of this important deregulated growth pathway in T-PLL. These findings further emphasize TCR signaling intermediates as candidates for therapeutic targeting., Significance: The identification of AGO2-mediated activation of oncogenic T cells through signal amplifying protein-protein interactions advances the understanding of leukemogenic AGO2 functions and underlines the role of aberrant TCR signaling in T-PLL., (©2022 American Association for Cancer Research.)

Neuroendocrine neoplasms (NENs) represent a heterogenous class of highly vascularized neoplasms that are increasing in prevalence and are predominantly diagnosed at a metastatic state. The molecular mechanisms leading to tumor initiation, metastasis, and chemoresistance are still under investigation. Hence, identification of novel therapeutic targets is of great interest. Here, we demonstrate that the RNA-binding Protein IGF2BP1 is a post-transcriptional regulator of components of the Polycomb repressive complex 2 (PRC2), an epigenic modifier affecting transcriptional regulation and proliferation: Comprehensive in silico analyses along with in vitro experiments showed that IGF2BP1 promotes neuroendocrine tumor cell proliferation by stabilizing the mRNA of Enhancer of Zeste 2 (EZH2), the catalytic subunit of PRC2, which represses gene expression by tri-methylation of histone H3 at lysine 27 (H3K27me3). The IGF2BP1-driven stabilization and protection of EZH2 mRNA is m6A-dependent and enhances EZH2 protein levels which stimulates cell cycle progression by silencing cell cycle arrest genes through enhanced H3K27 tri-methylation. Therapeutic inhibition of IGF2BP1 destabilizes EZH2 mRNA and results in a reduced cell proliferation, paralleled by an increase in G1 and sub-G1 phases. Combined targeting of IGF2BP1, EZH2, and Myc, a transcriptional activator of EZH2 and well-known target of IGF2BP1 cooperatively induces tumor cell apoptosis. Our data identify IGF2BP1 as an important driver of tumor progression in NEN, and indicate that disruption of the IGF2BP1-Myc-EZH2 axis represents a promising approach for targeted therapy of neuroendocrine neoplasms.

T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic mature T-cell malignancy. It typically presents with exponentially rising lymphocyte counts, splenomegaly, and bone marrow infiltration. Effective treatment options are scarce and a better understanding of TPLL's pathogenesis is desirable. Activation of the TCL1 proto-oncogene and loss-of-function perturbations of the tumor suppressor ATM are TPLL's genomic hallmarks. The leukemic cell reveals a phenotype of active T-cell receptor (TCR) signaling and aberrant DNA damage responses. Regulatory networks based on the profile of microRNA (miR) have not been described for T-PLL. In a combined approach of small-RNA and transcriptome sequencing in 46 clinically and moleculary well-characterized T-PLL, we identified a global T-PLL-specific miR expression profile that involves 34 significantly deregulated miR species. This pattern strikingly resembled miR-ome signatures of TCR-activated T cells. By integrating these T-PLL miR profiles with transcriptome data, we uncovered regulatory networks associated with cell survival signaling and DNA damage response pathways. Despite a miR-ome that discerned leukemic from normal T cells, there were also robust subsets of T-PLL defined by a small set of specific miR. Most prominently, miR-141 and the miR- 200c-cluster separated cases into two major subgroups. Furthermore, increased expression of miR-223-3p as well as reduced expression of miR-21 and the miR-29 cluster were associated with more activated Tcell phenotypes and more aggressive disease presentations. Based on the implicated pathobiological role of these miR deregulations, targeting strategies around their effectors appear worth pursuing. We also established a combinatorial miR-based overall survival score for T-PLL (miROS-T-PLL), that might improve current clinical stratifications.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m 3 C) methylation at position C 32 of the mt-tRNA Ser(UCN) and mt-tRNA Thr . METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNA Ser(UCN) - and mt-tRNA Thr -dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNA Ser(UCN) - and mt-tRNA Thr -dependent codons through METTL8-mediated m 3 C 32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2021 Elsevier Inc. All rights reserved.)

Anaplastic thyroid carcinoma (ATC) is the most fatal and rapidly evolving endocrine malignancy invading the head and neck region and accounts for up to 50% of thyroid cancer-associated deaths. Deregulation of the microRNA (miRNA) expression promotes thyroid carcinoma progression by modulating the reorganization of the ATC transcriptome. Here, we applied comparative miRNA-mRNA sequencing on a cohort of 28 thyroid carcinomas to unravel the association of deregulated miRNA and mRNA expression. This identified 85 miRNAs significantly deregulated in ATC. By establishing a new analysis pipeline, we unraveled 85 prime miRNA-mRNA interactions supporting the downregulation of candidate tumor suppressors and the upregulation of bona fide oncogenes such as survivin (BIRC5) in ATC. This miRNA-dependent reprogramming of the ATC transcriptome provided an mRNA signature comprising 65 genes sharply distinguishing ATC from other thyroid carcinomas. The validation of the deregulated protein expression in an independent thyroid carcinoma cohort demonstrates that miRNA-dependent oncogenes comprised in this signature, the transferrin receptor TFRC (CD71) and the E3-ubiquitin ligase DTL, are sharply upregulated in ATC. This upregulation is sufficient to distinguish ATC even from poorly differentiated thyroid carcinomas (PDTC). In sum, these findings provide new diagnostic tools and a robust resource to explore the key miRNA-mRNA regulation underlying the progression of thyroid carcinoma.

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimized miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses were calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA N 6 -methyladenosine (m 6 A) modification occurs in approximately 25% of mRNAs at the transcriptome-wide level. RNA m 6 A is regulated by the RNA m 6 A methyltransferases methyltransferase-like 3 (METTL3), METTL14, and METTL16 (writers), demethylases FTO and ALKBH5 (erasers), and binding proteins YTHDC1-2, YTHDF1-3, IGF2BP1-3, and SND1 (readers). These RNA m 6 A modification proteins are frequently upregulated or downregulated in human cancer tissues and are often associated with poor patient prognosis. By modulating pre-mRNA splicing, mRNA nuclear export, decay, stability, and translation of oncogenic and tumor suppressive transcripts, RNA m 6 A modification proteins regulate cancer cell proliferation, survival, migration, invasion, tumor initiation, progression, metastasis, and sensitivity to anticancer therapies. Importantly, small-molecule activators of METTL3, as well as inhibitors of METTL3, FTO, ALKBH5, and IGF2BP1 have recently been identified and have shown considerable anticancer effects when administered alone or in combination with other anticancer agents, both in vitro and in mouse models of human cancers. Future compound screening and design of more potent and selective RNA m 6 A modification protein inhibitors and activators are expected to provide novel anticancer agents, appropriate for clinical trials in patients with cancer tissues harboring aberrant RNA m 6 A modification protein expression or RNA m 6 A modification protein-induced resistance to cancer therapy., (©2021 American Association for Cancer Research.)

Background: Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy., Methods: We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis., Results: We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation., Conclusion: Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

MEX3A belongs to the MEX3 (Muscle EXcess) protein family consisting of four members (MEX3A-D) in humans. Characteristic for MEX3 proteins is their domain structure with 2 HNRNPK homology (KH) domains mediating RNA binding and a C-terminal really interesting new gene (RING) domain that harbors E3 ligase function. In agreement with their domain composition, MEX3 proteins were reported to modulate both RNA fate and protein ubiquitination. MEX3 paralogs exhibit an oncofetal expression pattern, they are severely downregulated postnatally, and re-expression is observed in various malignancies. Enforced expression of MEX3 proteins in various cancers correlates with poor prognosis, emphasizing their oncogenic potential. The latter is supported by MEX3A's impact on proliferation, self-renewal as well as migration of tumor cells in vitro and tumor growth in xenograft studies.

MYCN gene amplification and upregulated expression are major hallmarks in the progression of high-risk neuroblastoma. MYCN expression and function in modulating gene synthesis in neuroblastoma is controlled at virtually every level, including poorly understood regulation at the post-transcriptional level. MYCN modulates the expression of various microRNAs including the miR-17-92 cluster. MYCN mRNA expression itself is subjected to the control by miRNAs, most prominently the miR-17-92 cluster that balances MYCN expression by feed-back regulation. This homeostasis seems disturbed in neuroblastoma where MYCN upregulation coincides with severely increased expression of the miR-17-92 cluster. In the presented study, we applied high-throughput next generation sequencing to unravel the miRNome in a cohort of 97 neuroblastomas, representing all clinical stages. Aiming to reveal the MYCN -dependent miRNome, we evaluate miRNA expression in MYCN -amplified as well as none amplified tumor samples. In correlation with survival data analysis of differentially expressed miRNAs, we present various putative oncogenic as well as tumor suppressive miRNAs in neuroblastoma. Using microRNA trapping by RNA affinity purification, we provide a comprehensive view of MYCN-regulatory miRNAs in neuroblastoma-derived cells, confirming a pivotal role of the miR-17-92 cluster and moderate association by the let-7 miRNA family. Attempting to decipher how MYCN expression escapes elevated expression of inhibitory miRNAs, we present evidence that RNA-binding proteins like the IGF2 mRNA binding protein 1 reduce miRNA-directed downregulation of MYCN in neuroblastoma. Our findings emphasize the potency of post-transcriptional regulation of MYCN in neuroblastoma and unravel new avenues to pursue inhibition of this potent oncogene., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Misiak, Hagemann, Bell, Busch, Lederer, Bley, Schulte and Hüttelmaier.)

The RNA-binding protein Musashi-1 (MSI1) promotes stemness during development and cancer. By controlling target mRNA turnover and translation, MSI1 is implicated in the regulation of cancer hallmarks such as cell cycle or Notch signaling. Thereby, the protein enhanced cancer growth and therapy resistance to standard regimes. Due to its specific expression pattern and diverse functions, MSI1 represents an interesting target for cancer therapy in the future. In this review we summarize previous findings on MSI1's implications in developmental processes of other organisms. We revisit MSI1's expression in a set of solid cancers, describe mechanistic details and implications in MSI1 associated cancer hallmark pathways and highlight current research in drug development identifying the first MSI1-directed inhibitors with anti-tumor activity.

The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) promotes tumor progression in a variety of solid tumors and its expression is associated with adverse prognosis. The main role proposed for IGF2BP1 in cancer cells is the stabilization of mRNAs encoding pro-oncogenic factors. Several IGF2BP1-RNA association studies, however, revealed a plethora of putative IGF2BP1-RNA targets. Thus, at present the main conserved target RNAs and pathways controlled by IGF2BP1 in cancer remain elusive. In this study, we present a set of genes and cancer hallmark pathways showing a conserved pattern of deregulation in dependence of IGF2BP1 expression in cancer cell lines. By the integrative analysis of these findings with publicly available cancer transcriptome and IGF2BP1-RNA association data, we compiled a set of prime candidate target mRNAs. These analyses confirm a pivotal role of IGF2BP1 in controlling cancer cell cycle progression and reveal novel cancer hallmark pathways influenced by IGF2BP1. For three novel target mRNAs identified by these studies, namely AURKA, HDLBP and YWHAZ, we confirm IGF2BP1 mRNA stabilization. In sum our findings confirm and expand previous findings on the pivotal role of IGF2BP1 in promoting oncogenic gene expression by stabilizing target mRNAs in a mainly 3'UTR, m 6 A-, miRNA-, and potentially AU-rich element dependent manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Glaß, Misiak, Bley, Müller, Hagemann, Busch, Rausch and Hüttelmaier.)

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HGSOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here, we show that the IGF2 mRNA-binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HGSOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in an RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signalling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation, only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rationale for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HGSOC.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide underlining the urgent need for new biomarkers and therapeutic targets for this disease. Long noncoding RNAs are critical players in NSCLC but the role of small RNA species is not well understood. In the present study, we investigated the role of H/ACA box small nucleolar RNAs (snoRNAs) and snoRNA-bound ribonucleoproteins (snoRNPs) in the tumorigenesis of NSCLC. H/ACA box snoRNPs including the NOP10 core protein were highly expressed in NSCLC. High levels of either NOP10 mRNA or protein were associated with poor prognosis in NSCLC patients. Loss of NOP10 and subsequent reduction of H/ACA box snoRNAs and rRNA pseudouridylation inhibited lung cancer cell growth, colony formation, migration, and invasion. A focused CRISPR/Cas9 snoRNA knockout screen revealed that genomic deletion of SNORA65, SNORA7A, and SNORA7B reduced proliferation of lung cancer cells. In line, high levels of SNORA65, SNORA7A, and SNORA7B were observed in primary lung cancer specimens with associated changes in rRNA pseudouridylation. Knockdown of either SNORA65 or SNORA7A/B inhibited growth and colony formation of NSCLC cell lines. Our data indicate that specific H/ACA box snoRNAs and snoRNA-associated proteins such as NOP10 have an oncogenic role in NSCLC providing new potential biomarkers and therapeutic targets for the disease.

Anaplastic thyroid carcinomas (ATC) are rare, but represent the most lethal malignancy of the thyroid. Selective molecular markers and drivers distinguishing ATC from other thyroid carcinomas of follicular origin remain largely unknown, limiting advances in diagnosis and treatment. In a retrospective study, we analyzed gene expression in 36 ATC, 18 poorly differentiated, 132 papillary, and 55 follicular thyroid carcinoma, as well as 124 paired and unpaired normal thyroid tissues in three independent cohorts by RNA-sequencing and immunohistochemistry. RNA-sequencing data in the test cohort suggested selective ATC protein biomarkers. Evaluation of these revealed that ATCs are characterized by the de novo expression of various testis antigens, including melanoma-associated antigen A3 (MAGEA3), but most importantly the oncofetal IGF2 mRNA binding protein 1 (IGF2BP1). Shallow whole genome sequencing essentially excluded that IGF2BP1 upregulation results from gene copy number alterations. Immunohistochemical analyses in all three tumor cohorts confirmed the selective de novo expression of IGF2BP1 protein in ATC. In sum, 75% (27/36) of all tested ATC and 0.5% (1/204) of poorly and well-differentiated thyroid carcinoma tissue samples were positive for IGF2BP1 protein. This indicates that IGF2BP1 protein expression identifies ATC with a diagnostic odds ratio of 612 (95% CI: 74.6-5021). In addition, we found that MAGEA3 is exclusively, although less consistently upregulated in ATC, presenting with an odds ratio of 411 (95% CI: 23.8-7098.7). Importantly, we provide confirmatory evidence that IGF2BP1 and MAGEA3 expression distinguishes ATC from poorly differentiated thyroid carcinoma. IGF2BP1 furthermore identified ATC foci within low-grade follicular thyroid carcinoma. In conclusion, IGF2BP1 represents the most promising single-gene marker available for ATC, followed by MAGEA3, improving on current techniques. Robust markers are essential to help distinguish this high-grade malignancy from other thyroid carcinomas, to guide surgical decision making, therapy and post-resection/therapy monitoring strategies.

The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in glioblastoma (GBM). MSI1 promotes self-renewal and impairs differentiation in cancer and non-malignant progenitor cells. However, a comprehensive understanding of its role in promoting GBM-driving networks remains to be deciphered. We demonstrate that MSI1 is highly expressed in GBM recurrences, an oncologist's major defiance. For the first time, we provide evidence that MSI1 promotes the expression of stem cell markers like CD44, co-expressed with MSI1 within recurrence-promoting cells at the migrating front of primary GBM samples. With GBM cell models of pediatric and adult origin, including isolated primary tumorspheres, we show that MSI1 promotes stem cell-like characteristics. Importantly, it impairs CD44 downregulation in a 3'UTR- and miRNA-dependent manner by controlling mRNA turnover. This regulation is disturbed by the previously reported MSI1 inhibitor luteolin, providing further evidence for a therapeutic target potential of MSI1 in GBM treatment.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.

Site-specific modification approaches have been extensively employed in the development of protein-based technologies. In this field, stability and activity integrity are the envisioned features of chemically modified proteins. These methods are especially used in the design of antibody-drug conjugates (ADCs). Nevertheless, a biochemical feature of the target protein in these reactions is often overlooked, residue specificity. Usually, in the course of developing chemical probes to modify a protein of interest (POI), specific amino acids are selected due to their reactivity. It is not critical which residue is modified as long as its modification does not compromise the POI's activity. However, no attention is paid as to why certain residues are preferentially modified over others. Physicochemical and structural constraints are often involved in the reactivity of the residue and account for the preferential modification. We propose that site-specific protein modification approaches can be applied beyond the development of ADCs or protein-drug conjugates, and used as a tool to reveal functionally relevant residues. By preferentially modifying certain side chains in the POI, chemical probes can uncover new binding motifs to investigate. Here we describe methods for protein modification, and how some pitfalls in the field can be turned into tools to reveal and exploit druggable pockets. Thus, allowing the design of innovative inhibitors against disease-relevant POIs. We discuss methodologies for site-specific modification of lysine, tryptophan, cysteine, histidine and tyrosine and comment on instances where the modified residues were used as targets for functionalization or drug design., (Copyright © 2020 Martínez, Hüttelmaier and Bertoldo.)

Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m 6 A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m 1 A, m 5 C, m 7 G, 2'-OMe, and Ψ were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use., (© 2020 Weichmann et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)