Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing.

The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10 melanoma syngeneic tumor model demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes.

Corrigendum: Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing.

[This corrects the article DOI: 10.3389/fimmu.2024.1355945.].

The TH1902 Docetaxel Peptide-Drug Conjugate Inhibits Xenografts Growth of Human SORT1-Positive Ovarian and Triple-Negative Breast Cancer Stem-like Cells.

Background: Breast and ovarian cancer stem cells (CSC) can contribute to the invasive and chemoresistance phenotype of tumors. TH1902, a newly developed sortilin (SORT1)-targeted peptide-docetaxel conjugate is currently in phase-1 clinical trial. Whether TH1902 impacts the chemoresistance phenotype of human triple-negative breast CSC (hTNBCSC) and ovarian CSC (hOvCSC) is unknown. Methods and Results: Immunophenotyping of hTNBCSC and hOvCSC was performed by flow cytometry and confirmed the expression of SORT1, and of CSC markers CD133, NANOG, and SOX2. Western blotting demonstrated the expression of the drug efflux pumps from the P-gp family members, ABCB1 and ABCB5. The cellular uptake of the fluorescent Alexa488-peptide from TH1902 was inhibited upon siRNA-mediated repression of SORT1 or upon competition with SORT1 ligands. In contrast to docetaxel, TH1902 inhibited in vitro migration, induced cell apoptosis and lead to G2/M cell cycle arrest of the hTNBCSC. These events were unaffected by the presence of the P-gp inhibitors cyclosporine A or PSC-833. In vivo, using immunosuppressed nude mice xenografts, TH1902 significantly inhibited the growth of hTNBCSC and hOvCSC xenografts (~80% vs. ~35% for docetaxel) when administered weekly as intravenous bolus for three cycles at 15 mg/kg, a dose equivalent to the maximal tolerated dose of docetaxel. Therapeutic efficacy was further observed when carboplatin was combined to TH1902. Conclusions: Overall, TH1902 exerts a superior anticancer activity than the unconjugated docetaxel, in part, by circumventing the CSC drug resistance phenotype that could potentially reduce cancer recurrence attributable to CSC.

The Peptide-Drug Conjugate TH1902: A New Sortilin Receptor-Mediated Cancer Therapeutic against Ovarian and Endometrial Cancers.

Sortilin (SORT1) receptor-mediated endocytosis functions were exploited for this new approach for effective and safe treatments of gynecological cancers. Here, high expression of SORT1 was found in >75% of the clinically annotated ovarian and endometrial tumors analyzed by immunohistochemistry. Therefore, the anticancer properties of the peptide-drug conjugate TH1902, a peptide that targets SORT1 and which is linked to docetaxel molecules, were investigated both in vitro using ovarian and endometrial cancer cell cultures and in vivo using xenograft models. In vitro, TH1902 inhibited cell proliferation and triggered higher SORT1-dependent cell apoptosis than unconjugated docetaxel did in ES-2 and SKOV3 ovarian cancer cell lines. The uptake of the Alexa488-TH19P01 peptide from TH1902 was reduced upon siRNA-mediated silencing of SORT1. In vivo, weekly administration of TH1902 showed better tolerability compared to equivalent docetaxel doses and inhibited tumor growth in ovarian and endometrial xenograft mice models. TH1902 as a single agent inhibited ovarian tumor growth more than either of the unconjugated taxanes or carboplatin. Furthermore, TH1902 combination with carboplatin also demonstrated better efficacy when compared to both taxanes-carboplatin combinations. Overall, TH1902 shows better in vivo efficacy, compared to that of docetaxel and even paclitaxel, against SORT1-positive ovarian and endometrial cancers and could be safely combined with carboplatin.

New Peptide-Drug Conjugates for Precise Targeting of SORT1-Mediated Vasculogenic Mimicry in the Tumor Microenvironment of TNBC-Derived MDA-MB-231 Breast and Ovarian ES-2 Clear Cell Carcinoma Cells.

Vasculogenic mimicry (VM) is defined as the formation of microvascular channels by genetically deregulated cancer cells and is often associated with high tumor grade and cancer therapy resistance. This microcirculation system, independent of endothelial cells, provides oxygen and nutrients to tumors, and contributes also in part to metastasis. VM has been observed in ovarian cancer and in triple negative breast cancer (TNBC) and shown to correlate with decreased overall cancer patient survival. Thus, strategies designed to inhibit VM may improve cancer patient treatments. In this study, sortilin (SORT1) receptor was detected in in vitro 3D capillary-like structures formed by ES-2 ovarian cancer and MDA-MB-231 TNBC-derived cells when grown on Matrigel. SORT1 gene silencing or antibodies directed against its extracellular domain inhibited capillary-like structure formation. In vitro, VM also correlated with increased gene expression of matrix metalloproteinase-9 (MMP-9) and of the cancer stem cell marker CD133. In vivo ES-2 xenograft model showed PAS+/CD31- VM structures (staining positive for both SORT1 and CD133). TH1904, a Doxorubicin-peptide conjugate that is internalized by SORT1, significantly decreased in vitro VM at low nM concentrations. In contrast, VM was unaffected by unconjugated Doxorubicin or Doxil (liposomal Doxorubicin) up to µM concentrations. TH1902, a Docetaxel-peptide conjugate, altered even more efficiently in vitro VM at pM concentrations. Overall, current data evidence for the first time that 1) SORT1 itself exerts a crucial role in both ES-2 and MDA-MB-231 VM, and that 2) VM in these cancer cell models can be efficiently inhibited by the peptide-drug conjugates TH1902/TH1904. These new findings also indicate that both peptide-drug conjugates, in addition to their reported cytotoxicity, could possibly inhibit VM in SORT1-positive TNBC and ovarian cancer patients.

TH1902, a new docetaxel-peptide conjugate for the treatment of sortilin-positive triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of cancers which lacks the expression and/or amplification of targetable biomarkers (ie, estrogen receptor, progestrogen receptor, and human epidermal growth factor receptor 2), and is often associated with the worse disease-specific outcomes than other breast cancer subtypes. Here, we report that high expression of the sortilin (SORT1) receptor correlates with the decreased survival in TNBC patients, and more importantly in those bearing lymph node metastases. By exploiting SORT1 function in ligand internalization, a new anticancer treatment strategy was designed to target SORT1-positive TNBC-derived cells both in vitro and in two in vivo tumor xenografts models. A peptide (TH19P01), which requires SORT1 for internalization and to which many anticancer drugs could be conjugated, was developed. In vitro, while the TH19P01 peptide itself did not exert any antiproliferative or apoptotic effects, the docetaxel-TH19P01 conjugate (TH1902) exerted potent antiproliferative and antimigratory activities when tested on TNBC-derived MDA-MB-231 cells. TH1902 triggered faster and more potent apoptotic cell death than did unconjugated docetaxel. The apoptotic and antimigratory effects of TH1902 were both reversed by two SORT1 ligands, neurotensin and progranulin, and on siRNA-mediated silencing of SORT1. TH1902 also altered microtubule polymerization and triggered the downregulation of the anti-apoptotic Bcl-xL biomarker. In vivo, both i.p. and i.v. administrations of TH1902 led to greater tumor regression in two MDA-MB-231 and HCC-70 murine xenograft models than did docetaxel, without inducing neutropenia. Altogether, the data demonstrates the high in vivo efficacy and safety of TH1902 against TNBC through a SORT1 receptor-mediated mechanism. This property allows for selective treatment of SORT1-positive TNBC and makes TH1902 a promising avenue for personalized therapy with the potential of improving the therapeutic window of cytotoxic anticancer drugs such as docetaxel.

Cerebrovascular Angiogenic Reprogramming upon LRP1 Repression: Impact on Sphingosine-1-Phosphate-Mediated Signaling in Brain Endothelial Cell Chemotactism.

Switches in sphingolipid metabolism have recently been associated with oncogenic transformation, and a role for the low-density lipoprotein receptor-related protein 1 (LRP1) in sphingosine-1-phosphate (S1P) proangiogenic signaling inferred. S1P signaling crosstalk with LRP1 in brain microvascular endothelial cells (HBMEC) is however unclear. Transient in vitro siLRP1 gene silencing was compared to stable shLRP1 knockdown. We observed decreased expression of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor for which multiple binding sites are found within the promoter sequences of all five S1P receptor members, upon stable but not transient LRP1 repression. Chemotactic migration of brain EC isolated from Lrp1(EC)-/- mice and of stable shLRP1 HBMEC became unresponsive to S1P, partly due to altered ERK and p38 MAPK pathways, whereas chemotactism remained unaltered following transient in vitro siLRP1 repression. Diminished S1P1, S1P3, and S1P5 expression were observed in stable shLRP1 HBMEC and in brain EC isolated from Lrp1(EC)-/- mice. Overexpression of LRP1 cluster IV rescued S1P-mediated cell migration through increased S1P3 transcription in shLRP1 HBMEC. Our study highlights an adaptive signaling crosstalk between LRP1 and specific S1P receptors which may regulate the angiogenic response of brain EC and be targeted at the blood-brain barrier in future therapeutic strategies.

Cavin-2 Functions as a Suppressive Regulator in TNF-induced Mesenchymal Stromal Cell Inflammation and Angiogenic Phenotypes.

Tumour necrosis factor (TNF)-α activation of mesenchymal stromal cells (MSC) enhances their tumour-suppressive properties and tumour-homing ability. The molecular actors involved are unknown. We found that TNF induced MSC migration and tubulogenesis which correlated with a dose-dependent increase in Cavin-1 and Cavin-3 transcript levels. TNF triggered cyclooxygenase (COX)-2 expression, whereas specific siRNA-mediated gene silencing of Cavin-2 resulted in an amplified COX-2 expression, tubulogenesis, and migratory response partially due to a rapid and sustained increase in NF-κB phosphorylation status. Our results highlight a suppressive role for the caveolar component Cavin-2 in the angiogenic and inflammatory regulation of TNF-activated MSC.

Olive oil compounds inhibit the paracrine regulation of TNF-α-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression.

The established causal relationship between the chronic inflammatory microenvironment, tumor development and cancer recurrence has provided leads for developing novel preventive strategies. Accumulating experimental, clinical and epidemiological data has provided support for the chemopreventive properties of olive oil compounds traditionally found within the Mediterranean diet. In this study, we investigated whether tyrosol (Tyr), hydroxytyrosol, oleuropein and oleic acid (OA), four compounds contained in extra virgin olive oil, can prevent tumor necrosis factor (TNF)-α-induced expression of cyclooxygenase (COX)-2 (an inflammation biomarker) in a human glioblastoma cell (U-87 MG) model. We found that Tyr and OA significantly inhibited TNF-α-induced COX-2 gene and protein expression, as well as PGE2 secretion. Both compounds also inhibited TNF-α-induced JNK and ERK phosphorylation, whereas only Tyr inhibited TNF-α-induced NF-κB phosphorylation. Paracrine-regulated migration of human brain microvascular endothelial cells (HBMECs) was assessed using growth factor-enriched conditioned media (CM) isolated from U-87 MG cells. We found that while PGE2 triggered HBMEC migration, the CM isolated from U-87 MG cells, where either COX-2 or NF-κB had been silenced or had been treated with Tyr or OA, exhibited decreased chemotactic properties. These observations demonstrate that olive oil compounds inhibit the effect of the chronic inflammatory microenvironment on glioblastoma progression through TNF-α actions and may be useful in cancer chemoprevention.

Selective JAK/STAT3 signalling regulates transcription of colony stimulating factor-2 and -3 in Concanavalin-A-activated mesenchymal stromal cells.

Human bone marrow-derived mesenchymal stromal cells (MSCs) express Toll-like receptors (TLRs) and produce cytokines and chemokines, all of which contribute to these cells' immunomodulatory and proangiogenic properties. Among the secreted cytokines, colony-stimulating factors (CSFs) regulate angiogenesis through activation of endothelial cell proliferation and migration. Since MSC are recruited within hypoxic tumors where they signal paracrine-regulated angiogenesis, the aim of this study was to evaluate which CSF members are expressed and are inducible in activated MSC. Furthermore, we investigated the JAK/STAT signal transducing pathway that may impact on CSF transcription. MSC were activated with Concanavalin-A (ConA), a TLR-2/6 agonist as well as a membrane type-1 matrix metalloproteinase (MT1-MMP) inducer, and we found increased transcription of granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP. Gene silencing of either STAT3 or MT1-MMP prevented ConA-induced phosphorylation of STAT3, and reversed ConA effects on CSF-2 and CSF-3. Treatment with the Janus Kinase (JAK)2 inhibitor AG490 antagonized the ConA induction of MT1-MMP and CSF-2, while the pan-JAK inhibitor Tofacitinib reversed ConA-induced CSF-2 and -3 gene expression. Silencing of JAK2 prevented the ConA-mediated increase of CSF-2, while silencing of JAK1, JAK3 and TYK2 prevented the increase in CSF-3. Given that combined TLR-activation and locally-produced CSF-2 and CSF-3 could regulate immunomodulation and neovascularization, pharmacological targeting of TLR-2/6-induced MT1-MMP/JAK/STAT3 signalling pathway may prevent MSC contribution to tumor development.

Epigallocatechin gallate targeting of membrane type 1 matrix metalloproteinase-mediated Src and Janus kinase/signal transducers and activators of transcription 3 signaling inhibits transcription of colony-stimulating factors 2 and 3 in mesenchymal stromal cells.

BACKGROUND: CSF-2 and CSF-3 confer proangiogenic and immunomodulatory properties to mesenchymal stromal cells (MSCs). RESULTS: Transcriptional regulation of CSF-2 and CSF-3 in concanavalin A-activated MSCs requires MT1-MMP signaling and is inhibited by EGCG. CONCLUSION: The chemopreventive properties of diet-derived EGCG alter MT1-MMP-mediated intracellular signaling. SIGNIFICANCE: Pharmacological targeting of MSCs proangiogenic functions may prevent their contribution to tumor development. Epigallocatechin gallate (EGCG), a major form of tea catechins, possesses immunomodulatory and antiangiogenic effects, both of which contribute to its chemopreventive properties. In this study, we evaluated the impact of EGCG treatment on the expression of colony-stimulating factors (CSF) secreted from human bone marrow-derived mesenchymal stromal cells (MSCs), all of which also contribute to the immunomodulatory and angiogenic properties of these cells. MSCs were activated with concanavalin A (ConA), a Toll-like receptor (TLR)-2 and TLR-6 agonist as well as a membrane type 1 matrix metalloproteinase (MT1-MMP) inducer, which increased granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP gene expression. EGCG antagonized the ConA-induced CSF-2 and CSF-3 gene expression, and this process required an MT1-MMP-mediated sequential activation of the Src and JAK/STAT pathways. Gene silencing of MT1-MMP expression further demonstrated its requirement in the phosphorylation of Src and STAT3, whereas overexpression of a nonphosphorylatable MT1-MMP mutant (Y573F) abrogated CSF-2 and CSF-3 transcriptional increases. Given that MSCs are recruited within vascularizing tumors and are believed to contribute to tumor angiogenesis, possibly through secretion of CSF-2 and CSF-3, our study suggests that diet-derived polyphenols such as EGCG may exert chemopreventive action through pharmacological targeting of the MT1-MMP intracellular signaling.