ZNF521 Enhances MLL-AF9-Dependent Hematopoietic Stem Cell Transformation in Acute Myeloid Leukemias by Altering the Gene Expression Landscape

Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34(+) cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34(+) cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones

Predominant VH1-69 IgBCR Clones Show Higher Expression of CD5 in Heterogeneous Chronic Lymphocytic Leukemia Populations

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones

Regulatory Role of microRNAs Targeting the Transcription Co-Factor ZNF521 in Normal Tissues and Cancers

Powerful bioinformatics tools have provided a wealth of novel miRNA–transcription factor networks crucial in controlling gene regulation. In this review, we focus on the biological functions of miRNAs targeting ZNF521, explaining the molecular mechanisms by which the dysregulation of this axis contributes to malignancy. ZNF521 is a stem cell-associated co-transcription factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells. The aberrant expression of ZNF521 transcripts, frequently associated with miRNA deregulation, has been detected in several tumors including pancreatic, hepatocellular, gastric, bladder transitional cell carcinomas as well as in breast and ovarian cancers. miRNA expression profiling tools are currently identifying a multitude of miRNAs, involved together with oncogenes and TFs in the regulation of oncogenesis, including ZNF521, which may be candidates for diagnostic and prognostic biomarkers of cancer

Dual chain extension effect and antibacterial properties of biomolecules interleaved within LDH dispersed into PBS by in situ polymerization

International audienceNanocomposites based on poly(butylene succinate) (PBS) and hydrotalcite-type anionic clays (HTs) organo-modified with biomolecules characterized by antibacterial and/or antioxidant activities, such as L-ascorbic acid (ASA), phloretic acid (HPP), L-tyrosine (TYR) and L-tryptophan (TRP), have been prepared by in situ polymerization. From XRD analysis and rheology experiments in a molten polymer state, intercalated HT hybrid platelets acting here as a hybrid filler are found to be well dispersed into polymers while providing a chain extension effect on PBS. Moreover, the molecules, when hosted within a HT interlayer gap, do preserve their pristine antibacterial activity, both in HT and in the resulting PBS composites. In particular, under the experimental conditions tested, HT/ASA and HT/TYR present the best combination of both properties (chain extension effect and antibacterial), especially versus E. coli as high as 90 and 97% of inhibition, respectively, using 2.5 wt% hybrid filler only. These findings open future applications for PBS associated with the hybrid HT filler as multifunctional materials in active packaging applications

ZNF423: A New Player in Estrogen Receptor-Positive Breast Cancer

Preventive therapy can target hormone-responsive breast cancer (BC) by treatment with selective estrogen receptor modulators (SERMs) and reduce the incidence of BC. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) with relevant predictive values, SNPs in the ZNF423 gene were associated with decreased risk of BC during SERM therapy, and SNPs in the Cathepsin O gene with an increased risk. ZNF423, which was not previously associated with BC is a multifunctional transcription factor known to have a role in development, neurogenesis, and adipogenesis and is implicated in other types of cancer. ZNF423 is transcriptionally controlled by the homolog ZNF521, early B cell factor transcription factor, epigenetic silencing of the promoter by CpG island hyper-methylation, and also by ZNF423 itself in an auto-regulatory loop. In BC cells, ZNF423 expression is found to be induced by estrogen, dependent on the binding of the estrogen receptor and calmodulin-like 3 to SNPs in ZNP423 intronic sites in proximity to consensus estrogen response elements. ZNF423 has also been shown to play a mechanistic role by trans-activating the tumor suppressor BRCA1 and thus modulating the DNA damage response. Even though recent extensive trial studies did not classify these SNPs with the highest predictive values, for inclusion in polygenic SNP analysis, the mechanism unveiled in these studies has introduced ZNF423 as a factor important in the control of the estrogen response. Here, we aim at providing an overview of ZNF423 expression and functional role in human malignancies, with a specific focus on its implication in hormone-responsive BC

Turning Stem Cells Bad: Generation of Clinically Relevant Models of Human Acute Myeloid Leukemia through Gene Delivery- or Genome Editing-Based Approaches

Acute myeloid leukemia (AML), the most common acute leukemia in the adult, is believed to arise as a consequence of multiple molecular events that confer on primitive hematopoietic progenitors unlimited self-renewal potential and cause defective differentiation. A number of genetic aberrations, among which a variety of gene fusions, have been implicated in the development of a transformed phenotype through the generation of dysfunctional molecules that disrupt key regulatory mechanisms controlling survival, proliferation, and differentiation in normal stem and progenitor cells. Such genetic aberrations can be recreated experimentally to a large extent, to render normal hematopoietic stem cells “bad”, analogous to the leukemic stem cells. Here, we wish to provide a brief outline of the complementary experimental approaches, largely based on gene delivery and more recently on gene editing, employed over the last two decades to gain insights into the molecular mechanisms underlying AML development and progression and on the prospects that their applications offer for the discovery and validation of innovative therapies

ZNF521 Represses Osteoblastic Differentiation in Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (hADSCs) are multipotent mesenchymal cells that can differentiate into adipocytes, chondrocytes, and osteocytes. During osteoblastogenesis, the osteoprogenitor cells differentiate into mature osteoblasts and synthesize bone matrix components. Zinc finger protein 521 (ZNF521/Zfp521) is a transcription co-factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells, where it has been shown to inhibit adipogenic differentiation. The present study is aimed at determining the effects of ZNF521 on the osteoblastic differentiation of hADSCs to clarify whether it can influence their osteogenic commitment. The enforced expression or silencing of ZNF521 in hADSCs was achieved by lentiviral vector transduction. Cells were cultured in a commercial osteogenic medium for up to 20 days. The ZNF521 enforced expression significantly reduced osteoblast development as assessed by the morphological and molecular criteria, resulting in reduced levels of collagen I, alkaline phosphatase, osterix, osteopontin, and calcium deposits. Conversely, ZNF521 silencing, in response to osteoblastic stimuli, induced a significant increase in early molecular markers of osteogenesis and, at later stages, a remarkable enhancement of matrix mineralization. Together with our previous findings, these results show that ZNF521 inhibits both adipocytic and osteoblastic maturation in hADSCs and suggest that its expression may contribute to maintaining the immature properties of hADSCs

IBtkα Activates the β-Catenin-Dependent Transcription of MYC through Ubiquitylation and Proteasomal Degradation of GSK3β in Cancerous B Cells

The IBTK gene encodes the IBtkα protein that is a substrate receptor of E3 ubiquitin ligase, Cullin 3. We have previously reported the pro-tumorigenic activity of Ibtk in MYC-dependent B-lymphomagenesis observed in Eμ-myc transgenic mice. Here, we provide mechanistic evidence of the functional interplay between IBtkα and MYC. We show that IBtkα, albeit indirectly, activates the β-catenin-dependent transcription of the MYC gene. Of course, IBtkα associates with GSK3β and promotes its ubiquitylation, which is associated with proteasomal degradation. This event increases the protein level of β-catenin, a substrate of GSK3β, and results in the transcriptional activation of the MYC and CCND1 target genes of β-catenin, which are involved in the control of cell division and apoptosis. In particular, we found that in Burkitt’s lymphoma cells, IBtkα silencing triggered the downregulation of both MYC mRNA and protein expression, as well as a strong decrease of cell survival, mainly through the induction of apoptotic events, as assessed by using flow cytometry-based cell cycle and apoptosis analysis. Collectively, our results shed further light on the complex puzzle of IBtkα interactome and highlight IBtkα as a potential novel therapeutic target to be employed in the strategy for personalized therapy of B cell lymphoma

Development of Cyclic Peptides Targeting the Epidermal Growth Factor Receptor in Mesenchymal Triple-Negative Breast Cancer Subtype

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections

Objectives: Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2).Methods: We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR.Results: We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2.Conclusions: The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2