Silvio E. Perea

HMGB-1 is an alarmin representative of DAMP playing a central role in immunogenic cell death (ICD), a necessary condition in the dialog established between dying tumor cells and the immune system during some anticancer therapies. Therefore, early screening for ICD inducers represents a major priority in drug development today. In this work, we investigated the effect elicited by the clinical-grade CIGB-300 peptide, which impairs Protein Kinase CK2-mediated phosphorylation and other CK2 signaling connected kinases. Here, HMGB-1 extracellular release was investigated in an 18-cell line panel from blood malignancies, uterine-cervical cancer and NSCLC treated with CIGB-300 at equipotent doses (IC₅₀) over 24 h. Interestingly, CIGB-300 treatment upregulated the HMGB-1 protein levels at the culture supernatant in most of the cell lines (p = 0.01) and fold-change increases ≥ 2 were associated with intrinsic cell line sensitivity towards CIGB-300’s cytotoxic effect. However, the HMGB-1 release by CIGB-300 was context-specific with clear induction on blood and uterine-cervical cancer cells and a diffused response pattern in NSCLC. Importantly, CIGB-300 treatment of blood cancer patients enrolled in a Phase I study induced plasma HMGB-1 alarmin in 4 out of 7 subject who received the entire treatment plan. Altogether, our data reveal for the first time that CIGB-300 treatment is able to induce extracellular HMGB-1 release in vitro and in vivo which could be indicative of ICD induction in some kinds of tumors; furthermore, the induction of extracellular HMGB-1 alarmin as a putative CIGB-300 response biomarker merits further investigation.

HMGB-1 is an alarmin representative of DAMP playing a central role in the immuno-genic cell death (ICD), a necessary condition in the dialogue stablished between dying tumor cells and immune system during some anticancer therapies. Therefore, early screening for ICD inducers represents a major priority in drug development today. In this work, we investigated the effect elicited by the clinical-grade CIGB-300 peptide, which impairs the Protein Kinase CK2-mediated phosphorylation and other CK2 signaling connected kinases. Here, the HMGB-1 extracellular release was investigated in an 18-cell line panel from blood malignancies, uterine-cervical cancer and NSCLC treated with CIGB-300 at equipotent doses (IC50) during 24 hours. Interestingly, CIGB-300 treatment upregulated the HMGB-1 protein levels at the culture supernatant in most of the cell lines (p=0.01) and fold-change increase ≥2 trended to be associated with intrinsic cell line sen-sitivity towards CIGB-300 cytotoxic effect. However, the HMGB-1 release by CIGB-300 was context-specific with clear induction on blood and uterine-cervical cancer cells and a diffused response pattern in NSCLC. Importantly, CIGB-300 treatment of blood cancer patients enrolled in a Phase I study induced plasma HMGB-1 alarmin in 4 out of 7 subject who received the entire treatment plan. Altogether, our data reveal for the first time that CIGB-300 treatment is able to induce extracellular HMGB-1 release in vitro and in vivo which could be indicative of ICD induction in some kind of tumors, also the induction of extracellular HMGB-1 alarmin as putative CIGB-300 response biomarker merits further investigation.

Alarmins are endogenous molecules that alert the immune system to circumvent the invading antigens in the host. Here, we investigated the serum levels of the high mobility group box 1 (HMGB-1) and nucleophosmin alarmins in a cohort of 162 newly diagnosed patients with non-small cell lung cancer and 60 age-matched healthy individuals. Interestingly, patients exhibited lower median values of HMGB-1 than healthy individuals, but higher levels of HMGB-1 were associated with a tumor size of > 2.0 cm. In addition, nucleophosmin levels were significantly increased in patients compared with healthy individuals, and those with tumors of > 2 cm in diameter also displayed higher circulating nucleophosmin levels. Furthermore, a strong association was reported between HMGB-1 and nucleophosmin levels in patients (r = 0.679; P = 0.0001) compared with healthy individuals. Interestingly, the strongest association between both alarmins was observed in patients with a Spread Through Airspaces pattern and tumors of less than 2 cm in size (r = 0.900; P = 0.0001). Co-expression of both alarmins was also observed in tumor tissues, although to a lesser extent. Altogether, our data indicate that high levels of each alarmin were individually associated with larger tumor size, however the most significant associations between both alarmins in NSCLC seemed to be primarily linked to early disease progression parameters.

3D cell systems for in vitro experimental studies are able to mimic the in vivo efficacy of drugs, before testing on animals, however many studies are still needed for mimicking the physiological environment with 3D cell growth systems. The mechano-physical properties of the microenvironment are relevant for the invasiveness of cancer cells and also for their drug resistance. 3D tumoroids of human oral squamous cell carcinoma (OSCC) CAL27 cells of different stiffness are here produced using a tunable PEG-silk fibroin hydrogel (PSF) and the anti-tumor activity of the CIGB300 peptide, an anticancer therapeutic peptide, on these 3D tumoroids models is assessed. Moreover, spectroscopical studies on CIGB300 peptide are here also reported for analyzing its structure, stability, aggregation and diffusion properties. For the first time, the CIGB300 peptide diffusion in tunable silk fibroin hydrogels of different stiffness is investigated over-time by fluorescence spectroscopy as potential tool in drug-screening using hydrogel-based 3D tumoroids.

Proteome-wide scale in a dose- and time-depending setting is crucial to fully understand the pharmacological mechanism of anticancer drugs as well as identification of candidates for drug response biomarkers. Here, we investigated the effect of the CIGB-300 anticancer peptide at IC₅₀ and IC₈₀ doses during 1 and 4 h of treatment on the squamous lung cancer cell (NCI-H226) proteome. An overwhelming dose-dependent inhibitory effect with minor up-regulated proteins was observed by increasing CIGB-300 dose level. Functional enrichment was also CIGB-300 dose-dependent with common or exclusively regulated proteins in each dose and time settings. A protein core involving small molecule biosynthesis, aldehyde metabolism and metabolism of nucleobases was regulated irrespectively to the dose or the treatment time. Importantly, a group of proteins linked to NSCLC tumor biology, poor clinical outcome and some Protein Kinase CK2 substrates, were significantly regulated by treating with both CIGB-300 doses. Likewise, we observed a consistent downregulation of different proteins that had been already reported to be inhibited by CIGB-300 in lung adenocarcinoma and acute myeloid leukemia. Overall, our proteomics-guided strategy based on time and drug dose served to uncover novel clues supporting the CIGB-300 cytotoxic effect and also to identify putative pharmacodynamic biomarkers in NSCLC.

Proteome-wide scale in a dose- and time-depending setting is crucial in drug-regulated proteomics to fully understand the pharmacological mechanism of anticancer drugs as well as the identification of proteins suitable for drug response biomarkers. Here, we investigated the effect of the CIGB-300 anticancer peptide at IC ₅₀ and IC ₈₀ dose levels during 1 and 4 hours of treatment on squamous lung cancer cell (NCI-H226) proteome. A dose-dependent inhibitory effect of the global proteomics while slight change of the upregulated proteins was observed, by increasing CIGB-300 dose level. The biological processes that were more represented among the modulated proteins were those related to small molecule biosynthetic process, pyridine-containing compound metabolic process and nucleobase-containing small molecule metabolism. Importantly, NSCLC previously described biomarkers and Protein Kinase CK2 substrates were significantly modulated by treating both doses of CIGB-300. These proteins, which are associated with both metabolism and cell survival, reinforce the hypothesis of the therapeutic potential of CIGB-300 as anticancer treatment for squamous non-small cell lung cancer and merits be validated as CIGB-300 response biomarkers in NSCLC patients. Overall, our proteomics-guided strategy based on time and drug dose served not only to deep into the mechanism of action of CIGB-300 but also to identify candidate proteins for pharmacodynamic and drug response biomarkers.

Alarmins are endogenous molecules that alert the immune system to circumvent the invading antigens in the host. They are basically sensed by specific cellular receptors, leading to recruitment and activation of dendritic cells to mount innate and adaptive immune responses. However, a dual role of alarmins as mediators in either antitumor immunity or tumor progression has been also reported. Here, we investigated the status of the HMGB-1 alarmin and the alarmin-like NPM/B23 in 162 debut NSCLC patients and 60 age-matched healthy individuals as control. NSCLC patients exhibited lower median values of circulating HMGB-1 compared to control (30.04 versus 37.30 ng/ml, p=0.02), but higher levels of HMGB-1 were positively correlated with tumor size > 2.0 cm (p=0.004) irrespectively of tumor histology. Otherwise, circulating NPM/B23 levels were significantly increased in NSCLC patients respect to control (812 versus 551 pg/ml, p=0.00027). Likewise, patients with tumors > 2 cm exhibited higher levels NPM/B23. Strong association was observed between HMGB-1 and NPM/B23 in serum of NSCLC patients (r=0.679 p=0.0001) compared to healthy individuals who exhibited a weaker correlation (r=0.278 p=0.031). However, the strongest correlation between both alarmins was observed in patients with STAS and tumors ≤ 2 cm (r=0.900 p=0.0001). Co-expression of HMGB-1 and NPM/B23 was also observed in tumor tissues although to a lesser extent. Our data unveil that concomitant circulating HMGB-1 and NPM/B23 could serve as a putative alarmin-based biomarker of NSCLC disease progression.

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 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 of HPV-16 E7. However, the actual role of this targeting in CIGB-300’s 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 blot 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 from HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduces the CK2-mediated phosphorylation of E7, as well as its binding to the tumour 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 the CIGB-300 triggers cell death in cervical cancer cells.