Colibrimycins, Novel Halogenated Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase (PKS-NRPS) Compounds Produced by Streptomyces sp. Strain CS147.

The improvement of genome sequencing techniques has brought to light the biosynthetic potential of actinomycetes due to the large number of gene clusters they present compared to the number of known compounds. Genome mining is a recent strategy in the search for novel bioactive compounds, which involves the analysis of sequenced genomes to identify uncharacterized natural product biosynthetic gene clusters, many of which are cryptic or silent under laboratory conditions, and to develop experimental approaches to identify their products. Owing to the importance of halogenation in terms of structural diversity, bioavailability, and bioactivity, searching for new halogenated bioactive compounds has become an interesting issue in the field of natural product discovery. Following this purpose, a screening for halogenase coding genes was performed on 12 Streptomyces strains isolated from fungus-growing ants of the Attini tribe. Using the bioinformatics tools antiSMASH and BLAST, six halogenase coding genes were identified. Some of these genes were located within biosynthetic gene clusters (BGCs), which were studied by construction of several mutants for the identification of the putative halogenated compounds produced. The comparison of the metabolite production profile of wild-type strains and their corresponding mutants by ultrahigh-performance liquid chromatography-UV and high-performance liquid chromatography-mass spectrometry allowed us the identification of a novel family of halogenated compounds in Streptomyces sp. strain CS147, designated colibrimycins. IMPORTANCE Genome mining has proven its usefulness in the search for novel bioactive compounds produced by microorganisms, and halogenases comprise an interesting starting point. In this work, we have identified a new halogenase coding gene that led to the discovery of novel lipopetide nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS)-derived natural products, the colibrimycins, produced by Streptomyces sp. strain CS147, isolated from the Attini ant niche. Some colibrimycins display an unusual α-ketoamide moiety in the peptide structure. Although its biosynthetic origin remains unknown, its presence might be related to a hypothetical inhibition of virus proteases, and, together with the presence of the halogenase, it represents a feature to be incorporated in the arsenal of structural modifications available for combinatorial biosynthesis.

CXCR4-targeted nanotoxins induce GSDME-dependent pyroptosis in head and neck squamous cell carcinoma.

BACKGROUND: Therapy resistance, which leads to the development of loco-regional relapses and distant metastases after treatment, constitutes one of the major problems that head and neck squamous cell carcinoma (HNSCC) patients currently face. Thus, novel therapeutic strategies are urgently needed. Targeted drug delivery to the chemokine receptor 4 (CXCR4) represents a promising approach for HNSCC management. In this context, we have developed the self-assembling protein nanotoxins T22-PE24-H6 and T22-DITOX-H6, which incorporate the de-immunized catalytic domain of Pseudomonas aeruginosa (PE24) exotoxin A and the diphtheria exotoxin (DITOX) domain, respectively. Both nanotoxins contain the T22 peptide ligand to specifically target CXCR4-overexpressing HNSCC cells. In this study, we evaluate the potential use of T22-PE24-H6 and T22-DITOX-H6 nanotoxins for the treatment of HNSCC. METHODS: T22-PE24-H6 and T22-DITOX-H6 CXCR4-dependent cytotoxic effect was evaluated in vitro in two different HNSCC cell lines. Both nanotoxins cell death mechanisms were assessed in HNSCC cell lines by phase-contrast microscopy, AnnexinV/ propidium iodide (PI) staining, lactate dehydrogenase (LDH) release assays, and western blotting. Nanotoxins antitumor effect in vivo was studied in a CXCR4+ HNSCC subcutaneous mouse model. Immunohistochemistry, histopathology, and toxicity analyses were used to evaluate both nanotoxins antitumor effect and possible treatment toxicity. GSMDE and CXCR4 expression in HNSCC patient tumor samples was also assessed by immunohistochemical staining. RESULTS: First, we found that both nanotoxins exhibit a potent CXCR4-dependent cytotoxic effect in vitro. Importantly, nanotoxin treatment triggered caspase-3/Gasdermin E (GSDME)-mediated pyroptosis. The activation of this alternative cell death pathway that differs from traditional apoptosis, becomes a promising strategy to bypass therapy resistance. In addition, T22-PE24-H6 and T22-DITOX-H6 displayed a potent antitumor effect in the absence of systemic toxicity in a CXCR4+ subcutaneous HNSCC mouse model. Lastly, GSDME was found to be overexpressed in tumor tissue from HNSCC patients, highlighting the relevance of this strategy. CONCLUSIONS: Altogether, our results show that T22-PE24-H6 and T22-DITOX-H6 represent a promising therapy for HNSCC patients. Remarkably, this is the first study showing that both nanotoxins are capable of activating caspase-3/GSDME-dependent pyroptosis, opening a novel avenue for HNSCC treatment.

A Novel CXCR4-Targeted Diphtheria Toxin Nanoparticle Inhibits Invasion and Metastatic Dissemination in a Head and Neck Squamous Cell Carcinoma Mouse Model.

Loco-regional recurrences and metastasis represent the leading causes of death in head and neck squamous cell carcinoma (HNSCC) patients, highlighting the need for novel therapies. Chemokine receptor 4 (CXCR4) has been related to loco-regional and distant recurrence and worse patient prognosis. In this regard, we developed a novel protein nanoparticle, T22-DITOX-H6, aiming to selectively deliver the diphtheria toxin cytotoxic domain to CXCR4+ HNSCC cells. The antimetastatic effect of T22-DITOX-H6 was evaluated in vivo in an orthotopic mouse model. IVIS imaging system was utilized to assess the metastatic dissemination in the mouse model. Immunohistochemistry and histopathological analyses were used to study the CXCR4 expression in the cancer cells, to evaluate the effect of the nanotoxin treatment, and its potential off-target toxicity. In this study, we report that CXCR4+ cancer cells were present in the invasive tumor front in an orthotopic mouse model. Upon repeated T22-DITOX-H6 administration, the number of CXCR4+ cancer cells was significantly reduced. Similarly, nanotoxin treatment effectively blocked regional and distant metastatic dissemination in the absence of systemic toxicity in the metastatic HNSCC mouse model. The repeated administration of T22-DITOX-H6 clearly abrogates tumor invasiveness and metastatic dissemination without inducing any off-target toxicity. Thus, T22-DITOX-H6 holds great promise for the treatment of CXCR4+ HNSCC patients presenting worse prognosis.

Novel Endometrial Cancer Models Using Sensitive Metastasis Tracing for CXCR4-Targeted Therapy in Advanced Disease.

Advanced endometrial cancer (EC) lacks therapy, thus, there is a need for novel treatment targets. CXCR4 overexpression is associated with a poor prognosis in several cancers, whereas its inhibition prevents metastases. We assessed CXCR4 expression in EC in women by using IHC. Orthotopic models were generated with transendometrial implantation of CXCR4-transduced EC cells. After in vitro evaluation of the CXCR4-targeted T22-GFP-H6 nanocarrier, subcutaneous EC models were used to study its uptake in tumor and normal organs. Of the women, 91% overexpressed CXCR4, making them candidates for CXCR4-targeted therapies. Thus, we developed CXCR4+ EC mouse models to improve metastagenesis compared to current models and to use them to develop novel CXCR4-targeted therapies for unresponsive EC. It showed enhanced dissemination, especially in the lungs and liver, and displayed 100% metastasis penetrance at all clinically relevant sites with anti-hVimentin IHC, improving detection sensitivity. Regarding the CXCR4-targeted nanocarrier, 60% accumulated in the SC tumor; therefore, selectively targeting CXCR4+ cancer cells, without toxicity in non-tumor organs. Our CXCR4+ EC models will allow testing of novel CXCR4-targeted drugs and development of nanomedicines derived from T22-GFP-H6 to deliver drugs to CXCR4+ cells in advanced EC. This novel approach provides a therapeutic option for women with metastatic, high risk or recurrent EC that have a dismal prognosis and lack effective therapies.

GSDMD-dependent pyroptotic induction by a multivalent CXCR4-targeted nanotoxin blocks colorectal cancer metastases.

Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro. Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4+ CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4+ SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4+ cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.

Self-assembling protein nanocarrier for selective delivery of cytotoxic polypeptides to CXCR4+ head and neck squamous cell carcinoma tumors.

Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma (HNSCC) mortality. The overexpression of chemokine receptor 4 (CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4+ tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4+ HNSCC cells, achieving a high accumulation in CXCR4+ tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4+ cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted protein-only nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.