OTX015 has no significant effect on non-malignant human fibroblasts. (a) Representative pictures of human fibroblasts treated for 72 h with 500 nM OTX015 or DMSO control. Scale bar=100 µm. (b) Growth of fibroblasts treated with 500 nM OTX015 (black) or DMSO control (grey) monitored over 96 h using the xCELLigence system. The slope (1/h) of both curves was calculated, and did not show a significant difference (right) using the Student's t test.

Supplementary Figure Legends 1-2 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

OTX015 reduces cell proliferation and induces apoptosis in MYCN amplified human xenografts. (a) Representative pictures of IMR5 xenograft tumor sections stained with hematoxylin/eosin, and immunohistochemical staining for apoptotic cells (cleaved caspase 3) and proliferating cells (Mib-1/Ki 67) are shown. Scale bar=250 µm. The relative fraction of positively stained cells for cleaved caspase 3 (b) and Mib-1 (c) were calculated from three representative images from each xenograft tumor and are shown as box plots. Statistical difference between groups was assessed by Student's t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Differentially expressed genes in IMR5 cells after OTX015 and JQ1 treatment respectively

Supplementary Figure 2 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Westermann et al. as well as Valentijn et al. MYCN target gene list and expression changes after OTX015 treatment as well as BRD4 binding

GSEA of cancer gene sets after treatment with OTX015 and/or JQ1(GSEA, Broad institute)

OTX015 effect on cell viability positively correlates with MYCN status. (a) IC50 of the IMR 5, Chp 134, Chp 212, SK N-BE(2), IMR-32, SK-N-BE, NB69, SK N AS and GI-M-EN neuroblastoma cell lines measured using MTT assays. (b) Maximum reduction of viability in IMR 5, Chp 134, Chp 212, SK N-BE(2), IMR-32, SK-N-BE, NB69, SK N AS and GI-M-EN cells treated for 72 h with 6 µM OTX015 and measured using MTT assays. (c) Relative MYCN mRNA (top) and protein (bottom) expression in all analyzed neuroblastoma cell lines. (d/e) The IC50 values of the cell lines shown in (a) were plotted against MYCN mRNA expression (d) or MYCN protein expression (e), which is shown in (c). Both graphs show weak anti-correlative tendencies in both cell lines (MYCN mRNA p=0.22, MYCN protein p=0.16).

GSEA of gene sets describing general cellular processes after treatment with OTX015 and/or JQ1(GSEA, Broad institute)

Supplementary Figure 1 from Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

List of all genes from hg19 with changes in BRD4 binding using BRD4- ChIP-seq in IMR5 cells with changes after treatment with OTX015

BET inhibition preferentially disrupts expression of genes associated with super-enhancers. (a) ChIP-seq profiles for H3K27Ac binding at representative super-enhancer-associated gene loci, including MYCN, NCOR2, BCOR and GLI2, in MYCN-amplified IMR-5 cells. The x-axis shows genomic position and the y-axis the histone mark signal in units of reads per million per base pair (rpm/bp). The gene model is depicted below and scale bars above the binding profiles. (b) The Fisher exact test shows enrichment of super-enhancer associated genes among the downregulated genes (logFC < 0 and q < 0.05) by OTX015 or JQ1 treatment. (c) Cumulative distribution plots of the top 200 super-enhancer-associated genes and their fold-change measured using genes which are differentially regulated in IMR-5 cells by OTX015 treatment, JQ1 treatment or the overlap between these datasets (left to right). Plots show a shift towards negative logFC for super-enhancer associated genes (blue curves) compared to genes not associated with super-enhancers (black curves). (d) Box plots of log2-fold changes in the top 200 genes associated with super-enhancers (SE) and regular enhancers (non-SE) in genes differentially regulated (compared to DMSO-treated controls) in IMR-5 cells treated with OTX015, JQ1 or the overlap from both datasets (left to right). Statistical significance was determined using a two-sided Mann-Whitney test.

All H3K27Ac peaks called in non-promoter regions in IMR5

Mechanisms of reduced cell viability induced by OTX015 are cell line dependent. OTX015 reduces cell proliferation over time and induces cell cycle changes as well as apoptosis. (a) Representative photomicrographs of neuroblastoma cell lines treated with OTX015 for 72 h. Scale bar=200 µm. (b) The slope (1/h) of cell proliferation over time measured using xCELLigence of cell lines treated with OTX015. Significance was measured with the Student's t test. (c) Cell proliferation monitored using the xCELLigence system over time in neuroblastoma cell lines treated with 500 nM OTX015 (black) or DMSO control (grey). (d) Cell lines were treated 72 h with 500 nM OTX015 or DMSO, then fixed and stained with propidium iodide for flow cytometry. The distribution of OTX015-treated cells in different stages of the cell cycle is shown. (e) Fraction of apoptotic cells measured in the cell death ELISA assay after 72 h treatment with 250 nM or 500 nM OTX015 or DMSO control. Significant differences between treated cells versus controls were calculated by Student's t test. * p < 0.05, ** p < 0.01. (f) Cell proliferation measured in the BrdU ELISA after 72 h of treatment with 250 nM or 500 nM OTX015 or DMSO. Significant differences between treatment groups and the controls were calculated by Student's t test.

BET bromodomain inhibition leads to disruption of MYCN driven gene-expression programs even at high levels of MYCN. (a) Enrichment of the published gene expression signature for JQ1-treated human MYCN-amplified neuroblastoma cell lines (11) in differentially expressed genes in IMR-5 cells after treatment with OTX015, JQ1 or DMSO control. (b) Enrichment of the published gene expression signature for MYC target genes (16) in differentially expressed genes in IMR-5 cells after treatment with OTX015, JQ1 or DMSO control. (c) Enrichment of the published gene expression signature for JQ1-treated human MYCN-amplified neuroblastoma cell lines (11) in differentially expressed genes in mNB-A1 cells after treatment of with OTX015, JQ1 or DMSO control. (d) Enrichment of the published gene expression signature for MYC target genes (16) in differentially expressed genes in mNB-A1 cells after treatment with OTX015, JQ1 or DMSO control (* p < 0.05, ** p < 0.01, *** p < 0.001). (e) GSEA (version 2.0, Broad Institute) of differentially expressed genes in IMR5 cells after treatment with OTX015 or JQ1 using gene sets representing signatures of JQ1 as well as MYC target genes (11, 16).

List of all Super-enhancers identified in IMR5 cells

Cancer‐associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in‐house and public, including single‐cell analyses), genetically engineered mouse PDAC tissues, and patient‐derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin‐positive CAFs were significantly associated with shorter overall survival of patients. Periostin‐positive CAFs were characterized by high proliferation and protein synthesis rates and low α‐smooth muscle actin expression and were found in peri‐/pre‐tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin‐positive CAFs were associated with immune‐related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin‐positive CAFs and podoplanin‐positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin‐positive CAFs identified an inflammatory CAF (iCAF)‐like subset, whereas periostin‐positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin‐positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin‐positive CAF is associated with an immune‐related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Pancreatic ductal adenocarcinoma (PDAC) incidence and related-deaths are increasing worldwide. PDAC is characterized by poor prognosis due to late diagnosis, high metastatic capacity and resistance to therapy. This is partially due to its specific microenvironment, where the stroma is prominent over tumor cells. Besides the oral and gut microbiota, the intratumor microbiome, i.e. the bacterial and fungal microorganisms present within the tumor, was recently introduced as a new partner of the tumor microenvironment of PDAC modulating pancreatic carcinogenesis, intratumor immune infiltrates, and response to chemotherapy. In this review, we propose an overview of current knowledge about the roles of bacteria and fungi in PDAC development and biology, and discuss potential therapeutic implications.

Abstract Background. Notch pathway is involved in tumor biology including cell proliferation, migration, drug resistance, and epithelial-to-mesenchymal transition (EMT). Notch activation leads to a proteolytic cleavage, followed by the release of the Notch intracellular domain (NICD), which translocates into the nucleus and activates target genes. Notch1 and Notch4 expressions were described as mutually exclusive in some tumors, with an increased aggressiveness linked to low Notch1/high Notch4 expression phenotype. What about the role of their basal activation in cancers? The aim of this work is to characterize the role of basal Notch1 and Notch4 pathway on cell proliferation, motility and sensitivity to Notch inhibition in human pancreatic (PDAC), head&neck (H&N), colorectal (CRC), cholangio (CK), and hepato (HCC) carcinoma cell lines. Materials and Methods. Cell lines were selected regarding their basal expression of NICD4 using western blot (one low/one high Notch4 basal activation) for each tumor type. Chosen cell lines were then characterized, by western blot and/or RT-qPCR, for Notch signaling (NICD1, HES1, NUMB), EMT status (vimentin, E-cadherin) and survival pathways (pAKT, pERK). Basal cell proliferation and motility were studied using MTT and wound-healing assay. The effects of a Notch inhibitor, PF-03084014, were assessed on cell proliferation and Notch signaling pathway by MTT and western blot. Results. In PDAC, H&N, and CRC cell lines, high NICD4 expression was associated with low NICD1, HES1, and NUMB expressions, whereas the opposite was observed in low NICD4 expressing cells. Interestingly, all high NICD4/low NICD1 cells displayed a mesenchymal phenotype with high vimentin and low E-cadherin expressions. This phenotype was also associated with an increased proliferation rate and basal cell motility in all tumor types, except for HCC. In addition, basal ERK phosphorylation was increased in the aggressive cells (high NICD4/low NICD1). In PDAC, H&N, and CRC cell lines, PF-03084014 showed higher antiproliferative effects in high NICD4/low NICD1 cells compared to low NICD4/high NICD1 cells. In all cell lines, PF-03084014 displayed no effect on Notch4 activation, but abrogated Notch1 activation in all low NICD4/high NICD1 cells. Interestingly, PF-03084014 decreased HES1 expression in all cells, with increased effects in the most sensitive cells (high NICD4/low NICD1). Further analysis of PF-04084014 effects on cell signaling pathways will be displayed at the conference. Conclusions. In this study, high Notch4 activation is shown to be correlated with low NICD1, HES1 and NUMB expressions. This phenotype is associated with an increased aggressiveness of human cancer cell lines, and an increased sensitivity to Notch inhibition. As expected, the strongest HES1 inhibition was observed in cell lines with an increased sensitivity to Notch inhibition. Since the inhibition of Notch pathway is an interesting topic for anticancer therapy, this study could help to select tumor types that may be good candidate for Notch inhibitors in the clinics. Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Annemilaï Tijeras-Raballand. Basal Notch1 and Notch4 activation as potential markers of aggressiveness and sensitivity to Notch inhibition in human cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3684.

Abstract Background. Head and neck (H&N) and liver carcinomas are considered as the most deadly cancers in the world. Despite advances in diagnosis and treatment, survival rates remain low mainly due to locoregional relapse, possibly triggered by the activation of epithelial to mesenchymal transition (EMT). EMT is a critical step for tumor progression, increasing motility and invasiveness of tumor cells. M1 and M2 macrophages, major components of the tumor microenvironment, were recently described as involved in EMT. Moreover, proteins expressed by cancer cells, such as CCL2 and CSF1, and promoting M2 differentiation, have been correlated with poor prognosis, and tumor aggressiveness. The aim of this study is to understand the interactions between M1/M2 macrophages and H&N or liver cancer cells considering their EMT status. Materials and Methods. M1 and M2 macrophages were obtained from THP-1 cell after exposure to PMA followed by LPS/IFNg for M1 or IL4/IL13 for M2. Differentiation was validated by immunofluorescence (IF) using CD14 for monocyte, CD68 for macrophage, CD80 for M1, and CD163 for M2. H&N, cholangiocarcinoma (CK), and hepatocellular carcinoma (HCC) cells were characterized for their EMT status (E-cadherin/vimentin expressions) by western blot. In each tumor type, 1 epithelial and 1 mesenchymal cells were selected to analyze the effects of conditioned medium (CM) from cancer cells on macrophages differentiation, using IF. Furthermore, CCL2 and CSF1 mRNA levels were evaluated in the selected cancer cell lines. In addition, the effect of M1 and M2 CM on cancer cell proliferation, migration, and invasion were also analyzed by MTT, wound-healing, and Boyden chamber assays. Results. Using our two steps methods, monocytes were first differentiated into macrophages (decrease of CD14 and increase of CD68 expression), and then into M1 and M2 (increase of CD80 or CD163 expression, respectively). Based on cell lines EMT status, SQ20B, SNU1196, and C3A (epithelial) and Hep2, SNU1079, and SKHep1 (mesenchymal) cells were selected. Macrophages exposed to CM from epithelial cells displayed a M1 phenotype, while macrophages exposed to CM from mesenchymal cells exhibited a M2 phenotype. Interestingly, higher mRNA expression of CCL2 and CSF1 mRNA were detected in mesenchymal cells compared to epithelial cells. In H&N and CK cell lines, M1 CM displayed increased antiproliferative effects on epithelial cells, whereas M1 CM displayed antiproliferative effects only on mesenchymal HCC cells. M1 and M2 CM increased migration and invasion in all selected cell lines, except for migration in the epithelial HCC cell line. Further characterization of M1 and M2 macrophages will be displayed at the conference. Conclusions. This study showed that EMT status of cancer cells modulates macrophages differentiation. In fact, mesenchymal cells showed elevated levels of CCL2 and CSF1 expression, promoting M2 differentiation. Conversely, M1 and M2 displayed differential effects on H&N and liver carcinomas cell lines. These results open up new perspectives on the role of M1/M2 macrophages in EMT-dependent tumors, with the aim of developing new therapeutic approaches for patients with EMT-dependent tumors. Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Sandrine Faivre, Annemilaï Tijeras-Raballand. Bidirectional EMT-dependent modulation of cancer cell and M1/M2 macrophage differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5103.