Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1-mediated nuclear export.

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110α, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor.

Comparative Effectiveness of COVID-19 Vaccines in Preventing Infections and Disease Progression from SARS-CoV-2 Omicron BA.5 and BA.2, Portugal.

We estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April-June 2022, we implemented a case-case and cohort study and classified lineages using whole-genome sequencing or spike gene target failure. For the case-case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93-1.23]) or booster (aOR 0.96 [95% CI 0.84-1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%-90%] vs. VE 93% [95% CI 86%-97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease.

mTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

CRISPR/Cas9-mediated genome editing: From basic research to translational medicine.

The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.

Unveiling Inter- and Intra-Patient Sequence Variability with a Multi-Sample Coronavirus Target Enrichment Approach.

Amid the global challenges posed by the COVID-19 pandemic, unraveling the genomic intricacies of SARS-CoV-2 became crucial. This study explores viral evolution using an innovative high-throughput next-generation sequencing (NGS) approach. By taking advantage of nasal swab and mouthwash samples from patients who tested positive for COVID-19 across different geographical regions during sequential infection waves, our study applied a targeted enrichment protocol and pooling strategy to increase detection sensitivity. The approach was extremely efficient, yielding a large number of reads and mutations distributed across 10 distinct viral gene regions. Notably, the genes Envelope, Nucleocapsid, and Open Reading Frame 8 had the highest number of unique mutations per 1000 nucleotides, with both spike and Nucleocapsid genes showing evidence for positive selection. Focusing on the spike protein gene, crucial in virus replication and immunogenicity, our findings show a dynamic SARS-CoV-2 evolution, emphasizing the virus-host interplay. Moreover, the pooling strategy facilitated subtle sequence variability detection. Our findings painted a dynamic portrait of SARS-CoV-2 evolution, emphasizing the intricate interplay between the virus and its host populations and accentuating the importance of continuous genomic surveillance to understand viral dynamics. As SARS-CoV-2 continues to evolve, this approach proves to be a powerful, versatile, fast, and cost-efficient screening tool for unraveling emerging variants, fostering understanding of the virus's genetic landscape.

Comparison of the ABC and ACMG systems for variant classification.

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as "maybe report" after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

FOXO family isoforms.

FOXO family of proteins are transcription factors involved in many physiological and pathological processes including cellular homeostasis, stem cell maintenance, cancer, metabolic, and cardiovascular diseases. Genetic evidence has been accumulating to suggest a prominent role of FOXOs in lifespan regulation in animal systems from hydra, C elegans, Drosophila, and mice. Together with the observation that FOXO3 is the second most replicated gene associated with extreme human longevity suggests that pharmacological targeting of FOXO proteins can be a promising approach to treat cancer and other age-related diseases and extend life and health span. However, due to the broad range of cellular functions of the FOXO family members FOXO1, 3, 4, and 6, isoform-specific targeting of FOXOs might lead to greater benefits and cause fewer side effects. Therefore, a deeper understanding of the common and specific features of these proteins as well as their redundant and specific functions in our cells represents the basis of specific targeting strategies. In this review, we provide an overview of the evolution, structure, function, and disease-relevance of each of the FOXO family members.

Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1.

Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate-activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)-FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1-dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis.

Abrogation of RUNX1 gene expression in de novo myelodysplastic syndrome with t(4;21)(q21;q22).

The disruption of RUNX1 function is one of the main mechanisms of disease observed in hematopoietic malignancies and the description of novel genetic events that lead to a RUNX1 loss of function has been accelerated with the development of genomic technologies. Here we describe the molecular characterization of a new t(4;21)(q21;q22) in a de novo myelodysplastic syndrome that resulted in the deletion of the RUNX1 gene. We demonstrated by quantitative real-time RT-PCR an almost complete depletion of the expression of the RUNX1 gene in our t(4;21) case compared with CD34(+) cells that was independent of mutation or DNA methylation. More importantly, we explored and confirmed the possibility that this abrogation also prevented transactivation of RUNX1 target genes, perhaps confirming the genetic origin of the thrombocytopenia and the myelodysplastic features observed in our patient, and certainly mimicking what has been observed in the presence of the RUNX1/ETO fusion protein.

Decavanadate and metformin-decavanadate effects in human melanoma cells.

Decavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive-non-competitive inhibition with an IC50 value about 6 times higher (87 µM) than the previously described for decavanadate (15 µM). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.

Screening Health-Promoting Compounds for Their Capacity to Induce the Activity of FOXO3.

Several chemical compounds including natural products have been suggested as being effective against age-related diseases or as beneficial for a healthy life. On the other hand, forkhead box O (FOXO) proteins are emerging as key cellular components associated with extreme human longevity. FOXO proteins are mainly regulated by posttranslational modifications and as these modifications are reversible, activation and inactivation of FOXO are attainable through pharmacological treatment. Here, we questioned whether a panel of compounds with known health-beneficial properties has the capacity to induce the activity of FOXO factors. We show that resveratrol, a phytoalexin present in grapes and other food products, the amide alkaloid piperlongumine found in the fruit of the long pepper, and the plant-derived ß-carboline compound harmine induced nuclear translocation of FOXO3. We also show that piperlongumine and harmine but not resveratrol activate FOXO-dependent transcription. We determined the half maximal effective concentration (EC50) values for resveratrol, piperlongumine, and harmine for FOXO translocation, and analyzed their inhibitory impact on chromosomal maintenance 1 (CRM1)-mediated nuclear export and the production of reactive oxygen species (ROS). We also used chemical biology approach and Western blot analysis to explore the underlying molecular mechanisms. We show that harmine, piperlongumine, and resveratrol activate FOXO3 independently of phosphoinositide 3-kinase (PI3K)/AKT signaling and the CRM1-mediated nuclear export. The effect of harmine on FOXO3 activity is at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylationregulated kinase 1A (DYRK1A) and can be reverted by the inhibition of sirtuins (SIRTs).

Tribbles Pseudokinases in Colorectal Cancer.

The Tribbles family of pseudokinases controls a wide number of processes during cancer on-set and progression. However, the exact contribution of each of the three family members is still to be defined. Their function appears to be context-dependent as they can act as oncogenes or tumor suppressor genes. They act as scaffolds modulating the activity of several signaling pathways involved in different cellular processes. In this review, we discuss the state-of-knowledge for TRIB1, TRIB2 and TRIB3 in the development and progression of colorectal cancer. We take a perspective look at the role of Tribbles proteins as potential biomarkers and therapeutic targets. Specifically, we chronologically systematized all available articles since 2003 until 2020, for which Tribbles were associated with colorectal cancer human samples or cell lines. Herein, we discuss: (1) Tribbles amplification and overexpression; (2) the clinical significance of Tribbles overexpression; (3) upstream Tribbles gene and protein expression regulation; (4) Tribbles pharmacological modulation; (5) genetic modulation of Tribbles; and (6) downstream mechanisms regulated by Tribbles; establishing a comprehensive timeline, essential to better consolidate the current knowledge of Tribbles' role in colorectal cancer.

Small Molecule Inhibitors of CRM1.

The transport through the nuclear pore complex is used by cancer cells to evade tumor-suppressive mechanisms. Several tumor-suppressors have been shown to be excluded from the cell nucleus in cancer cells by the nuclear export receptor CRM1 and abnormal expression of CRM1 is oncogenic. Inhibition of CRM1 has long been postulated as potential approach for the treatment of cancer and to overcome therapy resistance. Furthermore, the nuclear export of viral components mediated by the CRM1 is crucial in various stages of the viral lifecycle and assembly of many viruses from diverse families, including coronavirus. However, the first nuclear export inhibitors failed or never entered into clinical trials. More recently CRM1 reemerged as a cancer target and a successful proof of concept was achieved with the clinical approval of Selinexor. The chemical complexity of natural products is a promising perspective for the discovery of new nuclear export inhibitors with a favorable toxicity profile. Several screening campaigns have been performed and several natural product-based nuclear export inhibitors have been identified. With this review we give an overview over the role of CRM1-mediated nuclear export in cancer and the effort made to identify and develop nuclear export inhibitors in particular from natural sources.

Harmine and Piperlongumine Revert TRIB2-Mediated Drug Resistance.

Therapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.

Monitoring the Transcriptional Activity of FOXO Transcription Factors by Analyzing their Target Genes.

FOXO proteins represent a subfamily of transcription factors that belong to the forkhead family. The study of FOXO target genes can be performed using Real-Time PCR (RT-PCR). The RT-PCR is a sensitive method that allows the detection and quantification of minute amounts of nucleic acids. In RT-PCR the accumulation of the amplicon is detected and measured as the reaction progresses. Here, we describe the application of RT-PCR technique to monitor the transcriptional activity of FOXO transcription factors.

Image-based Identification of Chemical Compounds Capable of Trapping FOXO in the Cell Nucleus.

Forkhead box O (FOXO) factors are tumor suppressor proteins commonly inactivated in human tumors. Furthermore, genetic variation within the FOXO3a gene is consistently associated with human longevity. FOXO proteins are usually inactivated by posttranslational modifications leading to cytoplasmic mislocalization. Therefore, the pharmacological activation by promoting nuclear localization of FOXOs is considered an attractive therapeutic approach to treat cancer and age-related diseases. We developed a cell-based imaging assay to screen for chemical agents capable of inhibiting the nuclear export and in turn trapping proteins that contain a nuclear export sequence including FOXO factors in the nucleus. The fluorescent signal of untreated assay cells localizes predominantly to the cytoplasm. Upon treatment with the nuclear export inhibitors the fluorescent-tagged reporter proteins appear as speckles in the nucleus. In a personalized medicine context, drugs capable of reactivating FOXO factors might be of enormous clinical value in human tumors in which these proteins are inactivated. Here, we describe the procedures for monitoring nuclear export which is suitable for high-throughput screening of compound collections.

Comparative genome profiling across subtypes of low-grade B-cell lymphoma identifies type-specific and common aberrations that target genes with a role in B-cell neoplasia.

BACKGROUND: Low-grade B-cell lymphomas are a very heterogeneous group of tumors, whose differential diagnosis is frequently compromised by the lack of specific cytogenetic or molecular features. Our objective was to search for genomic features that allow a better molecular identification of the different types of lymphoma studied. DESIGN AND METHODS: We selected a panel of 87 low-grade B-cell lymphoma tumor samples that were unambiguously diagnosed (clinically and cytogenetically) as: follicular, splenic marginal zone, nodal marginal zone, lymphoplasmacytic, mantle cell, extranodal marginal zone MALT-type lymphoma or B-cell chronic lymphocytic leukemia. All samples were subjected to the same high-resolution genomic DNA analysis (array-based comparative genomic hybridization): a whole genome platform that contained 44000 probes distributed across the genome. Genomic imbalances were recorded, compiled and analyzed. RESULTS: Eighty percent of analyzed cases showed genomic imbalances (deletions and gain/amplifications) but the frequency of these imbalances ranged from 100% in mantle cell lymphomas to 33% in MALT lymphomas. A total of 95 new genomic imbalances affecting all lymphoma subtypes, were defined. We evaluated the extension of the genomic instability, detecting distinct patterns of genomic instability within subtypes. Specific pathways, such as nuclear factor kB (gains of REL and BCL11A, and losses of COMMD3, BIRC1, IKK1 and NFKB2), Polycomb group proteins (gain of BMI1 and deletion of PCGF6), DNA repair checkpoint pathways (deletion of 16q24 involving CDT1), or miRNA with a role in B-cell lymphoma pathogenesis (MIRN15A, MIRN16-1), were targeted by this genomic instability. CONCLUSIONS: Although all subtypes of lymphomas showed gains and losses of DNA, the analysis of their genomic profiles indicated that there are specific aberrations in almost every subtype as well as frequent aberrations that are common to a large number of lymphoma types. These common aberrations target genes that are important in B-cell lymphomagenesis.

The Emerging Therapeutic Landscape of Advanced Melanoma.

Melanoma is the deadliest form of skin cancer being responsible for 80% of skin cancer deaths. Furthermore, the incidence of metastatic melanoma has increased over the past three decades with a mortality rate that continues to rise faster than most of all other cancers. The last few years have witnessed an unparalleled change in treatment options for patients with metastatic melanoma by the development of new therapeutic strategies like targeted therapies and immunotherapies that highly improved the patient's prognosis. Despite the paradigm- shifting success of these novel treatments, their effectiveness is still limited by intrinsic or acquired resistance. The objective of this review is to provide an overview of the new available treatment modalities, criteria to select patients who might benefit from a specific therapy, mechanisms of innate and acquired resistance to these treatments and to discuss strategies to overcome drug resistance.

Adaptive mechanisms of resistance to anti-neoplastic agents.

Intrinsic and acquired resistance to conventional and targeted therapeutics is a fundamental reason for treatment failure in many cancer patients. Targeted approaches to overcome chemoresistance as well as resistance to targeted approaches require in depth understanding of the underlying molecular mechanisms. The anti-cancer activity of a drug can be limited by a broad variety of molecular events at different levels of drug action in a cell-autonomous and non-cell-autonomous manner. This review summarizes recent insights into the adaptive mechanisms used by tumours to resist therapy including cellular phenotypic plasticity, dynamic alterations of the tumour microenvironment, activation of redundant signal transduction pathways, modulation of drug target expression levels, and exploitation of pro-survival responses.