A first-in-class Wiskott-Aldrich syndrome protein activator with anti-tumor activity in hematologic cancers.

Hematological cancers are among the most common cancers in adults and children. Despite significant improvements in therapies, many patients still succumb to the disease. Therefore, novel therapies are needed. The Wiskott-Aldrich syndrome protein (WASp) family regulates actin assembly in conjunction with the Arp2/3 complex, a ubiquitous nucleation factor. WASp is expressed exclusively in hematopoietic cells and exists in two allosteric conformations: autoinhibited or activated. Here, we describe the development of EG-011, a first-in-class small molecule activator of the WASp auto-inhibited form. EG-011 possesses in vitro and in vivo anti-tumor activity as a single agent in lymphoma, leukemia, and multiple myeloma, including models of secondary resistance to PI3K, BTK, and proteasome inhibitors. The in vitro activity was confirmed in a lymphoma xenograft. Actin polymerization and WASp binding was demonstrated using multiple techniques. Transcriptome analysis highlighted homology with drugs-inducing actin polymerization.

Metabolic drives affecting Th17/Treg gene expression changes and differentiation: impact on immune-microenvironment regulation.

The CD4+ T-cell population plays a vital role in the adaptive immune system by coordinating the immune response against different pathogens. A significant transformation occurs in CD4+ cells during an immune response, as they shift from a dormant state to an active state. This transformation leads to extensive proliferation, differentiation, and cytokine production, which contribute to regulating and coordinating the immune response. Th17 and Treg cells are among the most intriguing CD4+ T-cell subpopulations in terms of genetics and metabolism. Gene expression modulation processes rely on and are linked to metabolic changes in cells. Lactylation is a new model that combines metabolism and gene modulation to drive Th17/Treg differentiation and functional processes. The focus of this review is on the metabolic pathways that impact lymphocyte gene modulation in a functionally relevant manner.

New Insights for Polyphenolic Compounds as Naturally Inspired Proteasome Inhibitors.

Polyphenols, an important class of natural products, are widely distributed in plant-based foods. These compounds are endowed with several biological activities and exert protective effects in various physiopathological contexts, including cancer. We herein investigated novel potential mechanisms of action of polyphenols, focusing on the proteasome, which has emerged as an attractive therapeutic target in cancers such as multiple myeloma. We carried out a structure-based virtual screening study using the DrugBank database as a repository of FDA-approved polyphenolic molecules. Starting from 86 polyphenolic compounds, based on the theoretical binding affinity and the interactions established with key residues of the chymotrypsin binding site, we selected 2 promising candidates, namely Hesperidin and Diosmin. The further assessment of the biologic activity highlighted, for the first time, the capability of these two molecules to inhibit the ß5-proteasome activity and to exert anti-tumor activity against proteasome inhibitor-sensitive or resistant multiple myeloma cell lines.

RANBP1, a member of the nuclear-cytoplasmic trafficking-regulator complex, is the terminal-striking point of the SGK1-dependent Th17+ pathological differentiation.

The Th17+ arrangement is critical for orchestrating both innate and acquired immune responses. In this context, the serum and glucocorticoid regulated kinase 1 (SGK1) exerts a key role in the governance of IL-23R-dependent Th17+ maturation, through the phosphorylation-dependent control of FOXO1 localization. Our previous work has shown that some of the SGK1-key functions are dependent on RAN-binding protein 1 (RANBP1), a terminal gene in the nuclear transport regulation. Here, we show that RANBP1, similarly to SGK1, is modulated during Th17+ differentiation and that RANBP1 fluctuations mediate the SGK1-dependent effects on Th17+ maturation. RANBP1, as the final effector of the SGK1 pathway, affects FOXO1 transport from the nucleus to the cytoplasm, thus enabling RORγt activation. In this light, RANBP1 represents the missing piece, in an essential and rate-limiting manner, underlying the Th17+ immune asset.

Uncovering Novel Capsaicin Inhibitory Activity towards Human Carbonic Anhydrase Isoforms IX and XII by Combining In Silico and In Vitro Studies.

Hot pepper (Capsicum annuum) represents one of the most widespread functional foods of the Mediterranean diet, and is associated with a reduced risk of developing cardiovascular disease, cancer, and mental disorders. In particular, its bioactive spicy molecules, named Capsaicinoids, exhibit polypharmacological properties. Among them, Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the most studied and reported in variegated scientific contributions for its beneficial effects, often linked to mechanisms of action unrelated to the activation of Transient Receptor Potential Vanilloid 1 (TRPV1). In this study, we present the application of in silico methods to Capsaicin for evaluating its inhibitory activity against the tumor-associated human (h) expressed CA IX and XII. In vitro assays confirmed Capsaicin inhibitory activity towards the most relevant tumor-related hCA isoforms. In particular, the hCAs IX and XII showed an experimental KI value of 0.28 µM and 0.064 µM, respectively. Then, an A549 model of non-small cell lung cancer, typically characterized by an elevated expression of hCA IX and XII, was employed to test the inhibitory effects of Capsaicin in vitro under both normoxic and hypoxic conditions. Finally, the migration assay revealed that Capsaicin [10 µM] inhibits cells from moving in the A549 cells model.

Natural Agents as Novel Potential Source of Proteasome Inhibitors with Anti-Tumor Activity: Focus on Multiple Myeloma.

Multiple myeloma (MM) is an aggressive and incurable disease for most patients, characterized by periods of treatment, remission and relapse. The introduction of new classes of drugs, such as proteasome inhibitors (PIs), has improved survival outcomes in these patient populations. The proteasome is the core of the ubiquitin-proteasome system (UPS), a complex and conserved pathway involved in the control of multiple cellular processes, including cell cycle control, transcription, DNA damage repair, protein quality control and antigen presentation. To date, PIs represent the gold standard for the treatment of MM. Bortezomib was the first PI approved by the FDA, followed by next generation of PIs, namely carfilzomib and ixazomib. Natural agents play an important role in anti-tumor drug discovery, and many of them have recently been reported to inhibit the proteasome, thus representing a new potential source of anti-MM drugs. Based on the pivotal biological role of the proteasome and on PIs' significance in the management of MM, in this review we aim to briefly summarize recent evidence on natural compounds capable of inhibiting the proteasome, thus triggering anti-MM activity.

RANBP1 (RAN Binding Protein 1): The Missing Genetic Piece in Cancer Pathophysiology and Other Complex Diseases.

RANBP1 encoded by RANBP1 or HTF9A (Hpall Tiny Fragments Locus 9A), plays regulatory functions of the RAN-network, belonging to the RAS superfamily of small GTPases. Through this function, RANBP1 regulates the RANGAP1 activity and, thus, the fluctuations between GTP-RAN and GDP-RAN. In the light of this, RANBP1 take actions in maintaining the nucleus-cytoplasmic gradient, thus making nuclear import-export functional. RANBP1 has been implicated in the inter-nuclear transport of proteins, nucleic acids and microRNAs, fully contributing to cellular epigenomic signature. Recently, a RANBP1 diriment role in spindle checkpoint formation and nucleation has emerged, thus constituting an essential element in the control of mitotic stability. Over time, RANBP1 has been demonstrated to be variously involved in human cancers both for the role in controlling nuclear transport and RAN activity and for its ability to determine the efficiency of the mitotic process. RANBP1 also appears to be implicated in chemo-hormone and radio-resistance. A key role of this small-GTPases related protein has also been demonstrated in alterations of axonal flow and neuronal plasticity, as well as in viral and bacterial metabolism and in embryological maturation. In conclusion, RANBP1 appears not only to be an interesting factor in several pathological conditions but also a putative target of clinical interest.

The Tumour Suppressor Fhit Protein Activates C-Raf Ubiquitination and Degradation in Human Melanoma Cells by Interacting with Hsp90.

Fhit protein expression is reduced in the majority of human tumors; moreover, its restoration both triggers apoptosis of cancer cells and suppresses tumor formation in a large number of preclinical models of cancers. In the following study, we observed that Fhit expression is significantly reduced in human melanoma cells, and their in vivo growth is blocked by a recombinant adenovirus carrying the FHIT gene. Importantly, we found here that Fhit physically interacts with Hsp90. Since Hsp90 is a chaperone with a crucial function in the conformational maturation and stabilization of C-Raf, we also investigated whether Fhit could interfere with the Hsp90/C-Raf protein complex in melanoma. Interestingly, the administration of the Hsp90 inhibitor 17-AAG, in combination with Fhit protein overexpression in melanoma cells, reacts synergistically to increase C-Raf ubiquitination and degradation. These data reveal Hsp90 as a novel interactor of Fhit and suggest that FHIT activity restoration could represent a helpful strategy for suppressing the oncogenic C-Raf pathway in the therapy of human melanoma.

Germline Testing in a Cohort of Patients at High Risk of Hereditary Cancer Predisposition Syndromes: First Two-Year Results from South Italy.

Germline pathogenic variants (PVs) in oncogenes and tumour suppressor genes are responsible for 5 to 10% of all diagnosed cancers, which are commonly known as hereditary cancer predisposition syndromes (HCPS). A total of 104 individuals at high risk of HCPS were selected by genetic counselling for genetic testing in the past 2 years. Most of them were subjects having a personal and family history of breast cancer (BC) selected according to current established criteria. Genes analysis involved in HCPS was assessed by next-generation sequencing (NGS) using a custom cancer panel with high- and moderate-risk susceptibility genes. Germline PVs were identified in 17 of 104 individuals (16.3%) analysed, while variants of uncertain significance (VUS) were identified in 21/104 (20.2%) cases. Concerning the germline PVs distribution among the 13 BC individuals with positive findings, 8/13 (61.5%) were in the BRCA1/2 genes, whereas 5/13 (38.4%) were in other high- or moderate-risk genes including PALB2, TP53, ATM and CHEK2. NGS genetic testing showed that 6/13 (46.1%) of the PVs observed in BC patients were detected in triple-negative BC. Interestingly, the likelihood of carrying the PVs in the moderate-to-high-risk genes calculated by the cancer risk model BOADICEA was significantly higher in pathogenic variant carriers than in negative subjects. Collectively, this study shows that multigene panel testing can offer an effective diagnostic approach for patients at high risk of hereditary cancers.

Th17-Gene Expression Profile in Patients with Chronic Venous Disease and Venous Ulcers: Genetic Modulations and Preliminary Clinical Evidence.

Chronic venous disease is a condition globally widespread, resulting in a disabling pathological disorder. The CD4 + Th17+ (Cluster Differentiation 4) lymphocytes represent a regulative factor for innate immunity related to the development of complex diseases. Recently, these mechanisms have been associated with vascular disease. The aim of this work is to validate whether the Th17 response correlates with the development of CVI (Chronic venous insufficiency)and CVLUs (chronic venous limbs ulcers) and whether Th17 markers can be used, both as intrinsic risk factors and diagnostic markers, for disease development. PBL derived from peripheral blood samples of patients and controls were subjected to gene expression analysis for IL23R, IL17, SGK1, TGFß, RORγ, FOXO1, and RANBP1 by qRT-PCR and immunoblot. A post hoc correlation, the diagnostic performance of the target genes, and multivariable analyses were properly conducted. The main expression markers of the CD4 + Th17+ switch were strongly activated in chronic venous insufficiency and in advanced ulceration. The correlation analysis demonstrated the inter-dependence on Th17's signature modulation. ROC (Receiver Operating Characteristic) analysis defined, for the examined genes, a clinical value as the potential diagnostic markers. Multi-logistic regression studies showed that Th17 markers behave as empirical risk factors for CVD (chronic venous disease) development. Taken together, the present data provide a new hypothesis for the TH17-dependent pathogenesis of CVD, favoring the possibility for the development of new diagnostic, preventive, and therapeutic approaches.

Corrigendum: Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides.

[This corrects the article DOI: 10.3389/fmolb.2021.715263.].

Overcome Chemoresistance: Biophysical and Structural Analysis of Synthetic FHIT-Derived Peptides.

The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. On the basis of genetic evidence, it has been postulated that the FHIT protein may function as tumor suppressor, implying a role for the FHIT protein in carcinogenesis. Recently, Gaudio et al. reported that FHIT binds and delocalizes annexin A4 (ANXA4) from plasma membrane to cytosol in paclitaxel-resistant lung cancer cells, thus restoring their chemosensitivity to the drug. They also identified the smallest protein sequence of the FHIT still interacting with ANXA4, ranging from position 7 to 13: QHLIKPS. This short sequence of FHIT protein was not only able to bind ANXA4 but also to hold its target in the cytosol during paclitaxel treatment, thus avoiding ANXA4 translocation to the inner side of the cell membrane. Starting from these results, to obtain much information about structure requirements involved in the interaction of the peptide mentioned above, we synthetized a panel of seven peptides through an Ala-scan approach. In detail, to study the binding of FHIT derived peptides with ANXA4, we applied a combination of different biophysical techniques such as differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and microscale thermophoresis (MST). Circular dichroism (CD) and nuclear magnetic resonance (NMR) were used to determine the conformational structure of the lead peptide (7-13) and peptides generated from ala-scan technique. The application of different biophysical and structural techniques, integrated by a preliminary biological evaluation, allowed us to build a solid structure activity relationship on the synthesized peptides.

A Familial Form of Epidermolysis Bullosa Simplex Associated with a Pathogenic Variant in KRT5.

Epidermolysis bullosa simplex is a disease that belongs to a group of genodermatoses characterised by the formation of superficial bullous lesions caused by minor mechanical trauma to the skin. The skin fragility observed in the EBS is mainly caused by pathogenic variants in the KRT5 and KRT14 genes that compromise the mechanical stability of epithelial cells. By performing DNA sequencing in a female patient with EBS, we found the pathogenic variant c.967G>A (p.Val323Met) in the KRT5 gene. This variant co-segregated with EBS in the family pedigree and was transmitted in an autosomal dominant inheritance manner. This is the first report showing a familial form of EBS due to this pathogenic variant.

Case Report: Identification of a Novel Pathogenic Germline TP53 Variant in a Family With Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is an inherited autosomal dominant disease characterized by a predisposition to many cancers. Germline pathogenic variants in TP53 are primarily responsible for LFS. By performing a targeted sequencing panel in a proband with liver carcinoma having a deceased son affected by osteosarcoma, we found the novel heterozygous frameshift variant c.645del (p.Ser215Argfs*32) in the TP53 gene. This variant co-segregated with typical LFS cancers in the family pedigree, consistent with the pathogenicity of this novel and previously undescribed TP53 variant.

Natural Products Extracted from Fungal Species as New Potential Anti-Cancer Drugs: A Structure-Based Drug Repurposing Approach Targeting HDAC7.

Mushrooms can be considered a valuable source of natural bioactive compounds with potential polypharmacological effects due to their proven antimicrobial, antiviral, antitumor, and antioxidant activities. In order to identify new potential anticancer compounds, an in-house chemical database of molecules extracted from both edible and non-edible fungal species was employed in a virtual screening against the isoform 7 of the Histone deacetylase (HDAC). This target is known to be implicated in different cancer processes, and in particular in both breast and ovarian tumors. In this work, we proposed the ibotenic acid as lead compound for the development of novel HDAC7 inhibitors, due to its antiproliferative activity in human breast cancer cells (MCF-7). These promising results represent the starting point for the discovery and the optimization of new HDAC7 inhibitors and highlight the interesting opportunity to apply the "drug repositioning" paradigm also to natural compounds deriving from mushrooms.

In Silico Identification and Biological Evaluation of Antioxidant Food Components Endowed with IX and XII hCA Inhibition.

The tumor-associated isoenzymes hCA IX and hCA XII catalyze the hydration of carbon dioxide to bicarbonate and protons. These isoforms are highly overexpressed in many types of cancer, where they contribute to the acidification of the tumor environment, promoting tumor cell invasion and metastasis. In this work, in order to identify novel dual hCA IX and XII inhibitors, virtual screening techniques and biological assays were combined. A structure-based virtual screening towards hCA IX and XII was performed using a database of approximately 26,000 natural compounds. The best shared hits were submitted to a thermodynamic analysis and three promising best hits were identified and evaluated in terms of their hCA IX and XII inhibitor activity. In vitro biological assays were in line with the theoretical studies and revealed that syringin, lithospermic acid, and (-)-dehydrodiconiferyl alcohol behave as good hCA IX and hCA XII dual inhibitors.

Multi-Targeting Bioactive Compounds Extracted from Essential Oils as Kinase Inhibitors.

Essential oils (EOs) are popular in aromatherapy, a branch of alternative medicine that claims their curative effects. Moreover, several studies reported EOs as potential anti-cancer agents by inducing apoptosis in different cancer cell models. In this study, we have considered EOs as a potential resource of new kinase inhibitors with a polypharmacological profile. On the other hand, computational methods offer the possibility to predict the theoretical activity profile of ligands, discovering dangerous off-targets and/or synergistic effects due to the potential multi-target action. With this aim, we performed a Structure-Based Virtual Screening (SBVS) against X-ray models of several protein kinases selected from the Protein Data Bank (PDB) by using a chemoinformatics database of EOs. By evaluating theoretical binding affinity, 13 molecules were detected among EOs as new potential kinase inhibitors with a multi-target profile. The two compounds with higher percentages in the EOs were studied more in depth by means Induced Fit Docking (IFD) protocol, in order to better predict their binding modes taking into account also structural changes in the receptor. Finally, given its good binding affinity towards five different kinases, cinnamyl cinnamate was biologically tested on different cell lines with the aim to verify the antiproliferative activity. Thus, this work represents a starting point for the optimization of the most promising EOs structure as kinase inhibitors with multi-target features.

7q35 Microdeletion and 15q13.3 and Xp22.33 Microduplications in a Patient with Severe Myoclonic Epilepsy, Microcephaly, Dysmorphisms, Severe Psychomotor Delay and Intellectual Disability.

Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ∼240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 (CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.

A familial t(4;8) translocation segregates with epilepsy and migraine with aura.

Three relatives carrying a t(4;8)(p15.2;p23.2) translocation had juvenile myoclonic epilepsy, self-limited photosensitive occipital epilepsy and migraine with aura. The t(4;8) translocation interrupted the coding sequence of CSMD1 gene and occurred immediately to the 3'UTR of STIM2 gene. STIM2 was overexpressed in the patient carrying the unbalanced translocation, and all three individuals had a single functional copy of CSMD1. Array CGH study disclosed that these three individuals also carried a deletion at 5q12.3 that involves the RGS7BP gene. The overall results favor the view that CSMD1, STIM2, and RGS7BP genes could contribute to epilepsy and migraine phenotypes in our family.

IBTK Haploinsufficiency Affects the Tumor Microenvironment of Myc-Driven Lymphoma in E-myc Mice.

The tumor microenvironment is a dynamic and interactive supporting network of various components, including blood vessels, cytokines, chemokines, and immune cells, which sustain the tumor cell's survival and growth. Murine models of lymphoma are useful to study tumor biology, the microenvironment, and mechanisms of response to therapy. Lymphomas are heterogeneous hematologic malignancies, and the complex microenvironment from which they arise and their multifaceted genetic basis represents a challenge for the generation and use of an appropriate murine model. So, it is important to choose the correct methodology. Recently, we supported the first evidence on the pro-oncogenic action of IBTK in Myc-driven B cell lymphomagenesis in mice, inhibiting apoptosis in the pre-cancerous stage. We used the transgenic Eµ-myc mouse model of non-Hodgkin's lymphoma and Ibtk hemizygous mice to evaluate the tumor development of Myc-driven lymphoma. Here, we report that the allelic loss of Ibtk alters the immunophenotype of Myc-driven B cell lymphomas, increasing the rate of pre-B cells and affecting the tumor microenvironment in Eµ-myc mice. In particular, we observed enhanced tumor angiogenesis, increasing pro-angiogenic and lymphangiogenic factors, such as VEGF, MMP-9, CCL2, and VEGFD, and a significant recruitment of tumor-associated macrophages in lymphomas of Ibtk+/- Eµ-myc compared to Ibtk+/+ Eµ-myc mice. In summary, these results indicate that IBTK haploinsufficiency promotes Myc tumor development by modifying the tumor microenvironment.