Regulation of Extracellular HMGB-1 Alarmin Levels by CIGB-300 Anticancer Peptide In Vitro and In Vivo
Daylen Aguilar-NoriegaYing YiJamilet MirandaYanelda GarcíaDania M. VázquezYaqin LanRicardo BringasWen LiYasser PereraSilvio E. Perea
Aug 2026· Kinases and Phosphatases· 0 citations· 32 references
TL;DR
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.
Abstract
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.
The extracellular cyclic GMP-AMP (cGAMP)-ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) axis is an emerging pharmacological target that links tumor-intrinsic DNA stress to antitumor immunity. Tumor cells can generate cGAMP in response to chromosomal instability, micronuclear rupture, replication stress, and therapy-induced DNA damage. After export into the tumor microenvironment, extracellular cGAMP may be transferred to antigen-presenting cells and activate stimulator of interferon genes (STING)-dependent type I interferon and C-X-C motif chemokine ligand 10 (CXCL10) programs, thereby supporting dendritic-cell activation, immune priming, and cytotoxic T-cell recruitment. ENPP1 restricts this process by degrading extracellular cGAMP and by contributing to nucleotide catabolism associated with AMP- and adenosine-dependent immunosuppression. Accordingly, pharmacological ENPP1 inhibition differs from direct STING agonism by preserving endogenous tumor-derived cGAMP rather than imposing exogenous receptor activation. This Review summarizes the mechanistic basis, pharmacological rationale, and translational challenges of targeting the extracellular cGAMP-ENPP1 axis in cancer. We discuss ENPP1 inhibitors and cGAMP-stabilizing approaches, focusing on mechanism of action, pharmacokinetic/pharmacodynamic (PK/PD) relationships, target engagement, therapeutic window, and potential immunotoxicological constraints. We also propose a biomarker-guided framework incorporating cGAMP-generating capacity, ENPP1 expression and enzymatic activity, STING-response competence, and on-treatment pharmacodynamic conversion. Finally, we evaluate rational combinations with radiotherapy, chemotherapy, DNA damage response-targeted agents, immune checkpoint blockade, and immune-metabolic modulators. Clinical translation will require patient stratification, schedule-aware combination design, and robust pharmacodynamic validation in early-phase studies.
Kailang Mu, Rui-qi Liao, Jun Xie et al.· European Journal of Pharmaco...· 0 citations
BACKGROUND
Evidences indicate that tumor-derived exosomes released by cancer cells are key mediators of intercellular communication and disease progression in various cancers. This study aimed to investigate the expression of key molecules endothelin-1 (ET-1) and high-mobility group box 1 (HMGB1), as well as the enzymatic activity of matrix metalloproteinase-9 (MMP-9) in serum-derived exosomes from patients with chronic lymphocytic leukemia (CLL).
METHODS
Serum samples were obtained from twenty-five patients with CLL and twenty-five controls. Exosomes were purified from serum and further characterized through dynamic light scattering (DLS), atomic force microscopy (AFM), and flow cytometry. Real-time PCR was used to determine the mRNA expression of ET-1 and HMGB1 in isolated exosomes. Protein patterns of isolated exosomes were visualized by SDS-PAGE, and then MMP-9 enzymatic activity was assessed using gelatin zymography.
RESULTS
Isolated exosomes from serum exhibited the expected morphology and marker expression based on characterization data obtained from DLS, AFM, and flow cytometry assays. Real-time PCR results indicated a significant overexpression of ET-1 and HMGB1 in CLL-derived exosomes compared to healthy controls (p < 0.05). In gelatin zymography, the activity of the MMP-9 dimer was significantly higher in patient-derived exosomes than that of controls (p < 0.001).
CONCLUSION
These findings showed that serum-derived exosomes from CLL patients have higher expression of ET-1 and HMGB1 and higher MMP-9 enzymatic activity. These alterations suggest that exosomal ET-1, HMGB1, and MMP-9 may serve as potential non-invasive biomarkers for disease monitoring and warrant further investigation regarding their biological and clinical significance in CLL.
Maryam Ghotbbahaei, Amirhossein Amoei, Maryam Lotfi et al.· Cancer Treatment and Researc...· 0 citations
BACKGROUND
MALAT1 has been validated to favor the progression of acute lymphoblastic leukemia (ALL), but its detailed mechanism remains obscure. This study explored the functional roles of ALL cells-derived exosomal MALAT1 in chemoresistance and malignant growth of ALL cells, as well as its underlying mechanisms.
METHODS
The expression of target molecules was evaluated by qRT-PCR, Western blotting, immunofluorescence, and immunohistochemical staining. CCK-8, EdU staining and flow cytometry were conducted to determine cell proliferation and apoptosis. RIP, RNA-pull down, Co-IP, and MeRIP were used to investigate molecular mechanisms. ALL cells were injected into nude mice to evaluate in vivo tumor formation.
RESULTS
MALAT1 and methyltransferase-like 14 (METTL14) were up-regulated in ALL, which exhibited a positive correlation. METTL14-mediated m6A modification raised MALAT1 stability and expression, and consequently facilitated ALL cell growth and apoptosis inhibition. Furthermore, MALAT1 was packaged into ALL cells-derived exosomes by hnRNPA2B1, and then transferred to NK92-MI cells. Exosomal MALAT1 suppressed tripartite motif-containing 27 (TRIM27)-mediated ubiquitination of High-mobility group box 1 (HMGB1) in NK92-MI cells, thereby leading to adriamycin resistance and malignant development of ALL cells.
CONCLUSION
ALL cells-derived exosomal MALAT1 was up-regulated by METTL14-mediated m6A modification, and subsequently restrained HMGB1 ubiquitination and degradation in NK92-MI cells, which resulted in adriamycin resistance and malignant growth of ALL cells. Therefore, inhibition of METTL14/MALAT1/HMGB1 axis might be a therapeutic strategy for ALL patients.
HIGHLIGHTS
(1)METTL14 and MALAT1 were positively correlated in ALL samples.(2)METTL14 increased MALAT1 stability and expression in an IGF2BP1-mediated m6A manner.(3)ALL cells transferring exosomal MALAT1 suppressed TRIM27-mediated HMGB1 ubiquitination in NK92-MI cells.(4)HMGB1 silencing in NK92-MI cells enhanced adriamycin sensitivity of ALL cells.(5)MALAT1/HMGB1 axis inhibition delayed in vivo ALL growth via NK cell activation.
Xiaofang Hong, Daiyan Yang, Jianhao Xing et al.· Cancer Immunology and Immuno...· 0 citations
Objective: Hepatocellular carcinoma remains a major contributor to cancer mortality worldwide, with low five-year survival rates despite treatments, underscoring the need for more effective and targeted therapies. Metformin, an oral antihyperglycemic agent, has gained attention for its potential anticancer properties. The present study aims to investigate the effects of metformin on key inflammatory cytokines involved in hepatocellular carcinoma (HCC) progression, as well as on the expression of c-Jun, a key subunit of the AP-1.Method: HepG2 cells were treated with metformin at a range of concentrations from 500 μM to 10 mM for 24 hours. To evaluate the anticancer effects of metformin, cell viability was assessed using the trypan blue exclusion method, while IL-6 and IL-8 concentrations were quantified by ELISA. Furthermore, c-Jun protein expression was examined by immunocytochemistry.Results: Metformin treatment led to a significant reduction in the levels of IL-6 and IL-8 in HepG2 cells compared to control group (p
Tuğba Soydaş, Merve Eskici, M. Tunçdemir· Interdisciplinary Medical Jo...· 0 citations
Metastasis and immunosuppression remain major barriers to effective treatment of lung adenocarcinoma (LUAD), yet the metabolic mechanisms governing the pro-tumor functions of tumor-associated macrophages are incompletely understood. In this study, we identified Uridine Phosphorylase 1 (UPP1) as a macrophage-enriched metabolic regulator associated with LUAD progression. By integrating single-cell RNA sequencing with clinical cohort analyses, we found that UPP1 was preferentially expressed in tumor-associated macrophages and was associated with adverse clinical outcomes. Functional and mechanistic studies demonstrated that dysregulated UPP1 disrupted nucleotide homeostasis, leading to mitochondrial reactive oxygen species accumulation and mitochondrial DNA leakage. These mitochondrial stress signals activated the cGAS-STING pathway, which preferentially engaged NLRP3 inflammasome signaling rather than canonical antiviral responses. Consequently, macrophages underwent pyroptosis and released elevated levels of interleukin-1β (IL-1β). Through paracrine signaling, macrophage-derived IL-1β promoted epithelial-mesenchymal transition in LUAD cells and enhanced their invasive capacity in vitro. Consistent with these findings, co-injection of UPP1-overexpressing macrophages significantly increased spontaneous lung metastasis in vivo. Clinically, elevated UPP1 expression served as an independent predictor of poor survival. Furthermore, pharmacological blockade of this signaling cascade or neutralization of IL-1β attenuated macrophage-induced malignant phenotypes in tumor cells, highlighting the therapeutic relevance of this pathway. Collectively, our findings identify a macrophage-specific immunometabolic circuit in which UPP1-driven mitochondrial stress activates the mtROS-cGAS-NLRP3 axis, promoting IL-1β-dependent macrophage-tumor crosstalk and metastatic progression. These results suggest that UPP1 may serve as both a prognostic biomarker and a potential therapeutic target in LUAD.
Mingtao Feng, Chao Gao, Yue-fei Yang et al.· Cell Death Discovery· 0 citations
Recent studies have revealed that nucleic acid sensing by the endolysosomal TLRs (TLR7, TLR8, and TLR9) requires coordinated activity of the lysosomal peptide transporter SLC15A4. This peptide transporter interacts with the adaptor protein TASL and is necessary for the recruitment and activation of the transcription factor IRF5. The importance of this signaling pathway in the pathogenesis of autoimmune disease has been highlighted by human genetic variants in SLC15A4, TASL, and IRF5 that are associated with elevated risk of developing systemic lupus erythematosus (SLE), while pre-clinical studies in mouse have shown that genetic knock-out of these genes is protective in multiple autoimmune or inflammatory disease models.
Using structure-based drug design, we have developed a series of novel small molecule SLC15A4 inhibitors. Further optimization of the potency and drug-like properties of these molecules has led to discovery of potential first-in-class lead compounds that exhibit nanomolar cellular potency, favorable in vitro ADME and off-target profiles, and suitable PK properties in mouse that allow for once-daily oral dosing.
In vitro studies show that SLC15A4 inhibition results in the intracellular degradation of the TASL adapter protein and blockade of inflammatory cytokine production in response to TLR7/8/9 agonists. Using in vivo mouse models of TLR-driven acute cytokine production, treatment with an advanced tool compound NTX-348 results in complete suppression of IRF5 phosphorylation and type I interferon production in response to TLR7 or TLR9 agonist treatment.
These results highlight our early progress developing novel, drug-like inhibitors of SLC15A4. SLC15A4 inhibition represent a novel therapeutic approach that may provide clinical benefit as an oral therapy for SLE and other immune-mediated diseases.
All authors are employees or contractors of Nimbus Therapeutics
Therapeutic Approaches to Autoimmunity (THER)
J. McElwee, Ana Antić, A. Basavapathruni et al.· Journal of Immunology· 0 citations