MNDA, a PYHIN factor involved in transcriptional regulation and apoptosis control in leukocytes.

Inflammation control is critical during the innate immune response. Such response is triggered by the detection of molecules originating from pathogens or damaged host cells by pattern-recognition receptors (PRRs). PRRs subsequently initiate intra-cellular signalling through different pathways, resulting in i) the production of inflammatory cytokines, including type I interferon (IFN), and ii) the initiation of a cascade of events that promote both immediate host responses as well as adaptive immune responses. All human PYRIN and HIN-200 domains (PYHIN) protein family members were initially proposed to be PRRs, although this view has been challenged by reports that revealed their impact on other cellular mechanisms. Of relevance here, the human PYHIN factor myeloid nuclear differentiation antigen (MNDA) has recently been shown to directly control the transcription of genes encoding factors that regulate programmed cell death and inflammation. While MNDA is mainly found in the nucleus of leukocytes of both myeloid (neutrophils and monocytes) and lymphoid (B-cell) origin, its subcellular localization has been shown to be modulated in response to genotoxic agents that induce apoptosis and by bacterial constituents, mediators of inflammation. Prior studies have noted the importance of MNDA as a marker for certain forms of lymphoma, and as a clinical prognostic factor for hematopoietic diseases characterized by defective regulation of apoptosis. Abnormal expression of MNDA has also been associated with altered levels of cytokines and other inflammatory mediators. Refining our comprehension of the regulatory mechanisms governing the expression of MNDA and other PYHIN proteins, as well as enhancing our definition of their molecular functions, could significantly influence the management and treatment strategies of numerous human diseases. Here, we review the current state of knowledge regarding PYHIN proteins and their role in innate and adaptive immune responses. Emphasis will be placed on the regulation, function, and relevance of MNDA expression in the control of gene transcription and RNA stability during cell death and inflammation.

IKAROS: from chromatin organization to transcriptional elongation control.

IKAROS is a master regulator of cell fate determination in lymphoid and other hematopoietic cells. This transcription factor orchestrates the association of epigenetic regulators with chromatin, ensuring the expression pattern of target genes in a developmental and lineage-specific manner. Disruption of IKAROS function has been associated with the development of acute lymphocytic leukemia, lymphoma, chronic myeloid leukemia and immune disorders. Paradoxically, while IKAROS has been shown to be a tumor suppressor, it has also been identified as a key therapeutic target in the treatment of various forms of hematological malignancies, including multiple myeloma. Indeed, targeted proteolysis of IKAROS is associated with decreased proliferation and increased death of malignant cells. Although the molecular mechanisms have not been elucidated, the expression levels of IKAROS are variable during hematopoiesis and could therefore be a key determinant in explaining how its absence can have seemingly opposite effects. Mechanistically, IKAROS collaborates with a variety of proteins and complexes controlling chromatin organization at gene regulatory regions, including the Nucleosome Remodeling and Deacetylase complex, and may facilitate transcriptional repression or activation of specific genes. Several transcriptional regulatory functions of IKAROS have been proposed. An emerging mechanism of action involves the ability of IKAROS to promote gene repression or activation through its interaction with the RNA polymerase II machinery, which influences pausing and productive transcription at specific genes. This control appears to be influenced by IKAROS expression levels and isoform production. In here, we summarize the current state of knowledge about the biological roles and mechanisms by which IKAROS regulates gene expression. We highlight the dynamic regulation of this factor by post-translational modifications. Finally, potential avenues to explain how IKAROS destruction may be favorable in the treatment of certain hematological malignancies are also explored.

Gene expression profiling unveils the temporal dynamics of CIGB-300-regulated transcriptome in AML cell lines.

BACKGROUND: Protein kinase CK2 activity is implicated in the pathogenesis of various hematological malignancies like Acute Myeloid Leukemia (AML) that remains challenging concerning treatment. This kinase has emerged as an attractive molecular target in therapeutic. Antitumoral peptide CIGB-300 blocks CK2 phospho-acceptor sites on their substrates but it also binds to CK2α catalytic subunit. Previous proteomic and phosphoproteomic experiments showed molecular and cellular processes with relevance for the peptide action in diverse AML backgrounds but earlier transcriptional level events might also support the CIGB-300 anti-leukemic effect. Here we used a Clariom S HT assay for gene expression profiling to study the molecular events supporting the anti-leukemic effect of CIGB-300 peptide on HL-60 and OCI-AML3 cell lines. RESULTS: We found 183 and 802 genes appeared significantly modulated in HL-60 cells at 30 min and 3 h of incubation with CIGB-300 for p < 0.01 and FC > = │1.5│, respectively; while 221 and 332 genes appeared modulated in OCI-AML3 cells. Importantly, functional enrichment analysis evidenced that genes and transcription factors related to apoptosis, cell cycle, leukocyte differentiation, signaling by cytokines/interleukins, and NF-kB, TNF signaling pathways were significantly represented in AML cells transcriptomic profiles. The influence of CIGB-300 on these biological processes and pathways is dependent on the cellular background, in the first place, and treatment duration. Of note, the impact of the peptide on NF-kB signaling was corroborated by the quantification of selected NF-kB target genes, as well as the measurement of p50 binding activity and soluble TNF-α induction. Quantification of CSF1/M-CSF and CDKN1A/P21 by qPCR supports peptide effects on differentiation and cell cycle. CONCLUSIONS: We explored for the first time the temporal dynamics of the gene expression profile regulated by CIGB-300 which, along with the antiproliferative mechanism, can stimulate immune responses by increasing immunomodulatory cytokines. We provided fresh molecular clues concerning the antiproliferative effect of CIGB-300 in two relevant AML backgrounds.

CIGB-300 Peptide Targets the CK2 Phospho-Acceptor Domain on Human Papillomavirus E7 and Disrupts the Retinoblastoma (RB) Complex in Cervical Cancer Cells.

CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate's phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7-pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells.

Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection.

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

CIGB-300 Anticancer Peptide Differentially Interacts with CK2 Subunits and Regulates Specific Signaling Mediators in a Highly Sensitive Large Cell Lung Carcinoma Cell Model.

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α' catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide.

Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300.

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), an advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2α catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML-targeted therapy.

Treatment with an Anti-CK2 Synthetic Peptide Improves Clinical Response in COVID-19 Patients with Pneumonia. A Randomized and Controlled Clinical Trial.

The instrumental role of CK2 in the SARS-CoV-2 infection has pointed out this protein kinase as promising therapeutic target in COVID-19. Anti-SARS-CoV-2 activity has been reported by CK2 inhibitors in vitro; however, no anti-CK2 clinical approach has been investigated in COVID-19. This trial aimed to explore the safety and putative clinical benefit of CIGB-325, an anti-CK2 peptide previously assessed in cancer patients. A monocentric, controlled, and therapeutic exploratory trial of intravenous CIGB-325 in adults hospitalized with COVID-19 was performed. Twenty patients were randomly assigned to receive CIGB-325 (2.5 mg/kg/day during 5-consecutive days) plus standard-of-care (10 patients) or standard-of-care alone (10 patients). Adverse events were classified by the WHO Adverse Reaction Terminology. Parametric and nonparametric statistical analyses were performed according to the type of variable. Considering the small sample size, differences between groups were estimated by Bayesian analysis. CIGB-325 induced transient mild and/or moderate adverse events such as pruritus, flushing, and rash in some patients. Both therapeutic regimens were similar with respect to SARS-CoV-2 clearance in nasopharynx swabs over time. However, CIGB-325 significantly reduced the median number of pulmonary lesions (9.5 to 5.5, p = 0.042) at day 7 and the proportion of patients with such an effect was also higher according to Bayesian analysis (pDif > 0; 0.951). Also, CIGB-325 significantly reduced the CPK (p = 0.007) and LDH (p = 0.028) plasma levels at day 7. Our preliminary findings suggest that this anti-CK2 clinical approach could be combined with standard-of-care in COVID-19 in larger studies.

Phosphoproteomic Landscape of AML Cells Treated with the ATP-Competitive CK2 Inhibitor CX-4945.

Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.

Clinical-Grade Peptide-Based Inhibition of CK2 Blocks Viability and Proliferation of T-ALL Cells and Counteracts IL-7 Stimulation and Stromal Support.

Despite remarkable advances in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), relapsed cases are still a major challenge. Moreover, even successful cases often face long-term treatment-associated toxicities. Targeted therapeutics may overcome these limitations. We have previously demonstrated that casein kinase 2 (CK2)-mediated phosphatase and tensin homologue (PTEN) posttranslational inactivation, and consequent phosphatidylinositol 3-kinase (PI3K)/Akt signaling hyperactivation, leads to increased T-ALL cell survival and proliferation. We also revealed the existence of a crosstalk between CK2 activity and the signaling mediated by interleukin 7 (IL-7), a critical leukemia-supportive cytokine. Here, we evaluated the impact of CIGB-300, a the clinical-grade peptide-based CK2 inhibitor CIGB-300 on T-ALL biology. We demonstrate that CIGB-300 decreases the viability and proliferation of T-ALL cell lines and diagnostic patient samples. Moreover, CIGB-300 overcomes IL-7-mediated T-ALL cell growth and viability, while preventing the positive effects of OP9-delta-like 1 (DL1) stromal support on leukemia cells. Signaling and pull-down experiments indicate that the CK2 substrate nucleophosmin 1 (B23/NPM1) and CK2 itself are the molecular targets for CIGB-300 in T-ALL cells. However, B23/NPM1 silencing only partially recapitulates the anti-leukemia effects of the peptide, suggesting that CIGB-300-mediated direct binding to CK2, and consequent CK2 inactivation, is the mechanism by which CIGB-300 downregulates PTEN S380 phosphorylation and inhibits PI3K/Akt signaling pathway. In the context of IL-7 stimulation, CIGB-300 blocks janus kinase / signal transducer and activator of transcription (JAK/STAT) signaling pathway in T-ALL cells. Altogether, our results strengthen the case for anti-CK2 therapeutic intervention in T-ALL, demonstrating that CIGB-300 (given its ability to circumvent the effects of pro-leukemic microenvironmental cues) may be a valid tool for clinical intervention in this aggressive malignancy.