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-Regulated Proteome Reveals Common and Tailored Response Patterns of AML Cells to CK2 Inhibition.

Protein kinase CK2 is a highly pleiotropic and ubiquitously expressed Ser/Thr kinase with instrumental roles in normal and pathological states, including neoplastic phenotype in solid tumor and hematological malignancies. In line with previous reports, CK2 has been suggested as an attractive prognostic marker and molecular target in acute myeloid leukemia (AML), a blood malignant disorder that remains as an unmet medical need. Accordingly, this work investigates the complex landscape of molecular and cellular perturbations supporting the antileukemic effect exerted by CK2 inhibition in AML cells. To identify and functionally characterize the proteomic profile differentially modulated by the CK2 peptide-based inhibitor CIGB-300, we carried out LC-MS/MS and bioinformatic analysis in human cell lines representing two differentiation stages and major AML subtypes. Using this approach, 109 and 129 proteins were identified as significantly modulated in HL-60 and OCI-AML3 cells, respectively. In both proteomic profiles, proteins related to apoptotic cell death, cell cycle progression, and transcriptional/translational processes appeared represented, in agreement with previous results showing the impact of CIGB-300 in AML cell proliferation and viability. Of note, a group of proteins involved in intracellular redox homeostasis was specifically identified in HL-60 cell-regulated proteome, and flow cytometric analysis also confirmed a differential effect of CIGB-300 over reactive oxygen species (ROS) production in AML cells. Thus, oxidative stress might play a relevant role on CIGB-300-induced apoptosis in HL-60 but not in OCI-AML3 cells. Importantly, these findings provide first-hand insights concerning the CIGB-300 antileukemic effect and draw attention to the existence of both common and tailored response patterns triggered by CK2 inhibition in different AML backgrounds, a phenomenon of particular relevance with regard to the pharmacologic blockade of CK2 and personalized medicine.

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.

Entomological factors affecting the low endemicity of Chagas disease in Nazca, Southwestern Peru.

Chagas disease is prevalent in Peru. The province of Nazca, in the southwestern region of the country, shows a high intradomiciliary infestation rate of Triatoma infestans bugs. Although the vector is present, the number of Chagas disease cases appears to be much lower than those reported in the neighboring region of Arequipa. We examined 624 T. infestans from Nazca to determine the current Trypanosoma cruzi infection rates, and found that no bugs were infected with this parasite. These results contrast with those found in Arequipa, where 19-30% triatomines have been reported infected. To compare their vectorial capacity, we infected 30 T. infestans specimens, selected both from Nazca and Arequipa, by feeding bugs on T. cruzi-infected mice. The parasites developed all stages expected in the vector; furthermore, the infective stage, metacyclic trypomastigote, was found in both insect populations from the second week after infection. In addition, those insects that accepted to be fed with mice blood defecated immediately after finishing blood meal, indicating that they might be efficient vectors. We maintain that differences observed in infection rates between vectors from Nazca and Arequipa may be explained by differences in host availability. In Arequipa hosts are mainly small animals, whereas in Nazca the main blood source comes from birds, which are refractory to the infection.