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 anticancer peptide regulates the protein kinase CK2-dependent phosphoproteome.

Casein-kinase CK2 is a Ser/Thr protein kinase that fosters cell survival and proliferation of malignant cells. The CK2 holoenzyme, formed by the association of two catalytic alpha/alpha' (CK2α/CK2α') and two regulatory beta subunits (CK2ß), phosphorylates diverse intracellular proteins partaking in key cellular processes. A handful of such CK2 substrates have been identified as targets for the substrate-binding anticancer peptide CIGB-300. However, since CK2ß also contains a CK2 phosphorylation consensus motif, this peptide may also directly impinge on CK2 enzymatic activity, thus globally modifying the CK2-dependent phosphoproteome. To address such a possibility, firstly, we evaluated the potential interaction of CIGB-300 with CK2 subunits, both in cell-free assays and cellular lysates, as well as its effect on CK2 enzymatic activity. Then, we performed a phosphoproteomic survey focusing on early inhibitory events triggered by CIGB-300 and identified those CK2 substrates significantly inhibited along with disturbed cellular processes. Altogether, we provided here the first evidence for a direct impairment of CK2 enzymatic activity by CIGB-300. Of note, both CK2-mediated inhibitory mechanisms of this anticancer peptide (i.e., substrate- and enzyme-binding mechanism) may run in parallel in tumor cells and help to explain the different anti-neoplastic effects exerted by CIGB-300 in preclinical cancer models.

Characterization of low-abundance species in the active pharmaceutical ingredient of CIGB-300: A clinical-grade anticancer synthetic peptide.

CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation.

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines.

BACKGROUND: Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment. METHODS: The human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity. RESULTS: We demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic ß-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment. CONCLUSIONS: Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

Pharmacologic inhibition of the CK2-mediated phosphorylation of B23/NPM in cancer cells selectively modulates genes related to protein synthesis, energetic metabolism, and ribosomal biogenesis.

B23/NPM is a multifunctional nucleolar protein frequently overexpressed, mutated, or rearranged in neoplastic tissues. B23/NPM is involved in diverse biological processes and is mainly regulated by heteroligomer association and posttranslational modification, phosphorylation being a major posttranslational event. While the role of B23/NPM in supporting and/or driving malignant transformation is widely recognized, the particular relevance of its CK2-mediated phosphorylation remains unsolved. Interestingly, the pharmacologic inhibition of such phosphorylation event by CIGB-300, a clinical-grade peptide drug, was previously associated to apoptosis induction in tumor cell lines. In this work, we sought to identify the biological processes modulated by CIGB-300 in a lung cancer cell line using subtractive suppression hybridization and subsequent functional annotation clustering. Our results indicate that CIGB-300 modulates a subset of genes involved in protein synthesis (ES = 8.4, p < 0.001), mitochondrial ATP metabolism (ES = 2.5, p < 0.001), and ribosomal biogenesis (ES = 1.5, p < 0.05). The impairment of these cellular processes by CIGB-300 was corroborated at the molecular and cellular levels by Western blot (P-S6/P-4EBP1, translation), confocal microscopy (JC-1, mitochondrial potential), qPCR (45SrRNA/p21, ribosome biogenesis), and electron microscopy (nucleolar structure, ribosome biogenesis). Altogether, our findings provide new insights on the potential relevance of the CK2-mediated phosphorylation of B23/NPM in cancer cells, revealing at the same time the potentialities of its pharmacological manipulation for cancer therapy. Finally, this work also suggests several candidate gene biomarkers to be evaluated during the clinical development of the anti-CK2 peptide CIGB-300.

Mechanisms of cellular uptake, intracellular transportation, and degradation of CIGB-300, a Tat-conjugated peptide, in tumor cell lines.

CIGB-300 is a cyclic synthetic peptide that induces apoptosis in malignant cells, elicits antitumor activity in cancer animal models, and shows tumor reduction signs when assayed in first-in-human phase I trial in patients with cervical tumors. CIGB-300 impairs phosphorylation by casein kinase 2 through targeting the substrate's phosphoacceptor domain. CIGB-300 was linked to the cell penetrating peptide Tat to facilitate the delivery into cells. Previously, we showed that CIGB-300 had a differential antiproliferative behavior in different tumor cell lines. In this work, we studied differential antiproliferative behavior in terms of cellular uptake, intracellular transportation, and degradation in tumor cell lines with dissimilar sensitivity to CIGB-300. The internalization of CIGB-300 was studied in different malignant cell lines. We found that the cell membrane heparan sulfate proteoglycans act as main receptors for extracellular CIGB-300 uptake. The most sensitive tumor cell lines showed higher intracellular incorporation of CIGB-300 in comparison to less sensitive cell lines. Furthermore, CIGB-300 uptake is time- and concentration-dependent in all studied cell lines. It was shown that CIGB-300 has the ability to penetrate cells mainly by direct membrane translocation. However, a minor proportion of the peptide uses an energy-dependent endocytic pathway mechanism to gain access into cells. CIGB-300 is internalized and transported into cells preferentially by caveolae-mediated endocytosis. Lysosomes are involved in CIGB-300 degradation; highly sensitive cell lines showed degradation at earlier times compared to low sensitive cells. Altogether, our data suggests a mechanism of internalization, vesicular transportation, and degradation for CIGB-300 in tumor cells.

Sensitivity of tumor cells towards CIGB-300 anticancer peptide relies on its nucleolar localization.

CIGB-300 is a novel anticancer peptide that impairs the casein kinase 2-mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB-300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB-300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB-300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB-300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB-300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull-down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB-300-treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB-300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide-based drug should entail its more efficient intracellular delivery at such subcellular localization.

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo.

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.

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.

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.

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research.

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.

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.

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.

Proteomic profile regulated by the anticancer peptide CIGB-300 in non-small cell lung cancer (NSCLC) cells.

CIGB-300 is a proapoptotic peptide-based drug that abrogates the CK2-mediated phosphorylation. This peptide has antineoplastic effect on lung cancer cells in vitro and in vivo. To understand the mechanisms involved on such anticancer activity, the NCI-H125 cell line proteomic profile after short-term incubation (45 min) with CIGB-300 was investigated. As determined by 2-DE or 2D-LC-MS/MS, 137 proteins changed their abundances more than 2-fold in response to the CIGB-300 treatment. The expression levels of proteins related to ribosome biogenesis, metastasis, cell survival and proliferation, apoptosis, and drug resistance were significantly modulated by the presence of CIGB-300. The protein translation process was the most affected (23% of the identified proteins). From the proteome analysis of the NCI-H125 cell line, novel potentialities for CIGB-300 as anticancer agent were evidenced.

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.

Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization.

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.

Safety and preliminary efficacy data of a novel casein kinase 2 (CK2) peptide inhibitor administered intralesionally at four dose levels in patients with cervical malignancies.

BACKGROUND: Cervical cancer is now considered the second leading cause of death among women worldwide, and its incidence has reached alarming levels, especially in developing countries. Similarly, high grade squamous intraepithelial lesion (HSIL), the precursor stage for cervical cancer, represents a growing health problem among younger women as the HSIL management regimes that have been developed are not fully effective. From the etiological point of view, the presence of Human Papillomavirus (HPV) has been demonstrated to play a crucial role for developing cervical malignancies, and viral DNA has been detected in 99.7% of cervical tumors at the later stages. CIGB-300 is a novel cyclic synthetic peptide that induces apoptosis in malignant cells and elicits antitumor activity in cancer animal models. CIGB-300 impairs the Casein Kinase (CK2) phosphorylation, by targeting the substrate's phosphoaceptor domain. Based on the perspectives of CIGB-300 to treat cancer, this "first-in-human" study investigated its safety and tolerability in patients with cervical malignancies. METHODS: Thirty-one women with colposcopically and histologically diagnosed microinvasive or pre-invasive cervical cancer were enrolled in a dose escalating study. CIGB-300 was administered sequentially at 14, 70, 245 and 490 mg by intralesional injections during 5 consecutive days to groups of 7 - 10 patients. Toxicity was monitored daily until fifteen days after the end of treatment, when patients underwent conization. Digital colposcopy, histology, and HPV status were also evaluated. RESULTS: No maximum-tolerated dose or dose-limiting toxicity was achieved. The most frequent local events were pain, bleeding, hematoma and erythema at the injection site. The systemic adverse events were rash, facial edema, itching, hot flashes, and localized cramps. 75% of the patients experienced a significant lesion reduction at colposcopy and 19% exhibited full histological regression. HPV DNA was negative in 48% of the previously positive patients. Long term follow-up did not reveal recurrences or adverse events. CONCLUSION: CIGB 300 was safe and well tolerated. This is the first clinical trial where a drug has been used to target the CK2 phosphoaceptor domain providing an early proof-of-principle of a possible clinical benefit.