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.

Targeting Ovarian Cancer Cell Cytotoxic Drug Resistance Phenotype with Xanthium strumarium L. Extract.

Emerging drugs aim at targeting the genomic integrity and replication machinery in ovarian cancer. While the antiproliferative activity of Xanthium strumarium L. extract (XFC), a traditional herbal medicine, is believed to alter the mitotic apparatus of Chinese hamster ovary epithelial cells, its capacity to target and overcome the chemoresistance phenotype in ovarian cancer is unknown. Among the cancer cell lines tested, we found that the best proliferation inhibitory effect for XFC was against ovarian cancer cells and ranged from 30 to 35 µg/mL. XFC efficiently targeted both the cytotoxic drug chemoresistance phenotype of SKOV-3 cells and of the chemosensitive ES-2 cells. Early apoptosis and late apoptosis were effectively induced by XFC extract in ES-2 cells, whereas late apoptosis and necrosis events were triggered in SKOV-3 cells. Cell cycling regulation was trapped by XFC extract in the G2/M phase in both the ES-2 and SKOV-3 cell models. This effect was, in part, attributable to increased dose-dependent tubulin polymerization, which was increased in SKOV-3 cells. Whereas XFC extract triggered poly (ADP-Ribose) polymerase (PARP) cleavage in both ES-2 and SKOV-3 cells, it only lowered Nrf2 in ES-2 cells and phosphorylated Akt levels in SKOV-3 cells. Interestingly, cell cycling regulators Cdk4, Cyclin D3, and p27 were all decreased in SKOV-3 cells. XFC extracts were effective in inhibiting in vitro migration in both ovarian cancer cell models. Our data support the potential anticancer targeting of chemoresistant human ovarian cancer cells phenotype by XFC extract.

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.

A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype.

Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells.

Identification of GPM6A and GPM6B as potential new human lymphoid leukemia-associated oncogenes.

BACKGROUND: Previously, we found that the Graffi murine leukemia virus (MuLV) is able to induce a wide spectrum of hematologic malignancies in vivo. Using high-density oligonucleotide microarrays, we established the gene expression profiles of several of these malignancies, thereby specifically focusing on genes deregulated in the lymphoid sub-types. We observed over-expression of a variety of genes, including Arntl2, Bfsp2, Gfra2, Gpm6a, Gpm6b, Nln, Fbln1, Bmp7, Etv5 and Celsr1 and, in addition, provided evidence that Fmn2 and Parm-1 may act as novel oncogenes. In the present study, we assessed the expression patterns of eight selected human homologs of these genes in primary human B-cell malignancies, and explored the putative oncogenic potential of GPM6A and GPM6B. METHODS: The gene expression levels of the selected human homologs were tested in human B-cell malignancies by semi-quantitative RT-PCR. The protein expression profiles of human GPM6A and GPM6B were analyzed by Western blotting. The localization and the effect of GPM6A and GPM6B on the cytoskeleton were determined using confocal and indirect immunofluorescence microscopy. To confirm the oncogenic potential of GPM6A and GPM6B, classical colony formation assays in soft agar and focus forming assays were used. The effects of these proteins on the cell cycle were assessed by flow cytometry analysis. RESULTS: Using semi-quantitative RT-PCR, we found that most of the primary B-cell malignancies assessed showed altered expression patterns of the genes tested, including GPM6A and GPM6B. Using confocal microscopy, we found that the GPM6A protein (isoform 3) exhibits a punctate cytoplasmic localization and that the GPM6B protein (isoform 4) exhibits a peri-nuclear and punctate cytoplasmic localization. Interestingly, we found that exogenous over-expression of both proteins in NIH/3T3 cells alters the actin and microtubule networks and induces the formation of long filopodia-like protrusions. Additionally, we found that these over-expressing NIH/3T3 cells exhibit anchorage-independent growth and enhanced proliferation rates. Cellular transformation (i.e., loss of contact inhibition) was, however, only observed after exogenous over-expression of GPM6B. CONCLUSIONS: Our results indicate that several human homologs of the genes found to be deregulated in Graffi MuLV experimental mouse models may serve as candidate biomarkers for human B-cell malignancies. In addition, we found that GPM6A and GPM6B may act as novel oncogenes in the development of these malignancies.

Characterization and identification of PARM-1 as a new potential oncogene.

BACKGROUND: The Graffi murine retrovirus is a powerful tool to find leukemia associated oncogenes. Using DNA microarrays, we recently identified several genes specifically deregulated in T- and B-leukemias induced by this virus. RESULTS: In the present study, probsets associated with T-CD8+ leukemias were analyzed and we validated the expression profile of the Parm-1 gene. PARM-1 is a member of the mucin family. We showed that human PARM-1 is an intact secreted protein accumulating predominantly, such as murine PARM-1, at the Golgi and in the early and late endosomes. PARM-1 colocalization with α-tubulin suggests that its trafficking within the cell involves the microtubule cytoskeleton. Also, the protein co-localizes with caveolin-1 which probably mediates its internalization. Transient transfection of both mouse and human Parm-1 cDNAs conferred anchorage- and serum-independent growth and enhanced cell proliferation. Moreover, deletion mutants of human PARM-1 without either extracellular or cytoplasmic portions seem to retain the ability to induce anchorage-independent growth of NIH/3T3 cells. In addition, PARM-1 increases ERK1/2, but more importantly AKT and STAT3 phosphorylation. CONCLUSIONS: Our results strongly suggest the oncogenic potential of PARM-1.

Binding and repressive activities of apolipoprotein E3 and E4 isoforms on the human ApoD promoter.

Apolipoprotein D (ApoD) gene expression is increased in several neurological disorders such as Alzheimer's disease (AD) and multiple sclerosis. We previously showed that transgenic mice that overexpress human ApoD show a better resistance against paraquat or OC43 coronavirus-induced neurodegeneration. Here, we identified several nuclear factors from the cortex of control and OC43-infected mice which bind a fragment of the proximal ApoD promoter in vitro. Of interest, we detected apolipoprotein E (ApoE). Human ApoE consists of three isoforms (E2, E3, and E4) with the E4 and E2 alleles representing a greater and a lower risk for developping AD, respectively. Our results show that ApoE is located in the nucleus and on the ApoD promoter in human hepatic and glioblastoma cells lines. Furthermore, overexpression of ApoE3 and ApoE4 isoforms but not ApoE2 significantly inhibited the ApoD promoter activity in U87 cells (E3/E3 genotype) cultured under normal or different stress conditions while ApoE knock-down by siRNA had a converse effect. Consistent with these results, we also demonstrated by ChIP assay that E3 and E4 isoforms, but not E2, bind the ApoD promoter. Moreover, using the Allen Brain Atlas in situ hybridization database, we observed an inverse correlation between ApoD and ApoE mRNA expression during development and in several regions of the mouse brain, notably in the cortex, hippocampus, plexus choroid, and cerebellum. This negative correlation was also observed for cortex layers IV-VI based on a new Transcriptomic Atlas of the Mouse Neocortical Layers. These findings reveal a new function for ApoE by regulating ApoD gene expression.

Decreasing stearoyl-CoA desaturase-1 expression inhibits ß-catenin signaling in breast cancer cells.

Stearoyl-CoA desaturase-1 (SCD1) is an endoplasmic reticulum anchored enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly palmytoleyl-CoA and oleyl-CoA. Recent studies have revealed a function for SCD1 in the modulation of signaling processes related to cell proliferation, survival and transformation to cancer. We used MCF7 and MDA-MB-231 cells to analyze the role of SCD1 in the metastatic acquisition of breast cancer cells. Silencing SCD1 expression in breast cancer cells has no effect on cell viability but the levels of cell proliferation, cell cycle genes' expressions and the phosphorylation state of ERK1/2 MAPK are significantly reduced. Decreasing SCD1 expression also reduces the level of GSK3 phosphorylation, indicating higher activity of the kinase. Using cells fractionation, immunofluorescence and a ß-catenin/TCF-responsive reporter construct, we demonstrate that lowering SCD1 expression leads to a decrease of ß-catenin amounts within the nucleus and to inhibition of its transactivation capacity. Moreover, MDA-MB-231 cells transfected with the SCD1 siRNA show a lower invasive potential than the control cells. Taken together, our data demonstrate that low SCD1 expression is associated with a decrease in the proliferation rate of breast cancer cells associated with a decrease in ERK1/2 activation. SCD1 silencing also inhibits GSK3 phosphorylation, lowering ß-catenin translocation to the nucleus, and, subsequently, its transactivation capacity and the expression of its target genes. Finally, we show that silencing SCD1 impairs the epithelial to mesenchymal transition-like behavior of the cells, a characteristic of metastatic breast cancer.

Gene profiling of Graffi murine leukemia virus-induced lymphoid leukemias: identification of leukemia markers and Fmn2 as a potential oncogene.

The Graffi murine leukemia virus induces a large spectrum of leukemias in mice and thus provides a good model to compare the transcriptome of all types of leukemias. We analyzed the gene expression profiles of both T and B leukemias induced by the virus with DNA microarrays. Given that we considered that a 4-fold change in expression level was significant, 388 probe sets were associated to B, to T, or common to both leukemias. Several of them were not yet associated with lymphoid leukemia. We confirmed specific deregulation of Fmn2, Arntl2, Bfsp2, Gfra2, Gpm6a, and Gpm6b in B leukemia, of Nln, Fbln1, and Bmp7 in T leukemias, and of Etv5 in both leukemias. More importantly, we show that the mouse Fmn2 induced an anchorage-independent growth, a drastic modification in cell shape with a concomitant disruption of the actin cytoskeleton. Interestingly, we found that human FMN2 is overexpressed in approximately 95% of pre-B acute lymphoblastic leukemia with the highest expression levels in patients with a TEL/AML1 rearrangement. These results, surely related to the role of FMN2 in meiotic spindle maintenance, suggest its important role in leukemogenesis. Finally, we propose a new panel of genes potentially involved in T and/or B leukemias.

Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: implication of PARP-1, APEX-1 and ERK1/2 catalytic activities.

Human Apolipoprotein D (apoD) is upregulated under several stress conditions and pathological situations such as neurodegenerative diseases and cancers. We previously showed that apoD mRNA expression is induced in growth-arrested cells and demonstrated the specific binding of nuclear proteins to the region -514 to -475 of the promoter. Such region contains a pair of Serum Responsive Elements (SRE), an Ets-Binding Site (EBS) and a Glucocorticoid Responsive Element (GRE). In this study, we show that Parp-1, HnRNP-U, CBF-A, BUB-3, Kif4, APEX-1 and Ifi204 bind these regulatory elements of the apoD promoter. Specific binding of HnRNP-U and Parp-1 was confirmed by Electrophoretic Mobility Shift Assay (EMSA). In a biotin pull-down assay, Kif4 and BUB-3 bind preferentially the SRE1 and the EBS-GRE sites, respectively, while APEX-1 seems recruited indirectly to these elements. We found that the mRNA expression of some of these binding factors is upregulated in growth-arrested cells and that these proteins also transactivate the apoD promoter. In agreement with these results, mutants of APEX-1 and of Parp-1 defective for their DNA-binding and catalytic activities could not transactivate the promoter. The knockdown of Parp-1 and HnRNP-U and the use of specific inhibitors of MEK1/2 and of Parp-1 also inhibited the induction of apoD gene expression. Moreover, ERK1/2 was found activated in a biphasic manner post serum-starvation and the inhibition of Parp-1 causes a sustained activation of ERK2 but not ERK1 for up to 2h. Altogether, these findings demonstrate the importance of Parp-1, APEX-1 and ERK1/2 catalytic activities in the growth arrest-induced apoD gene expression.