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Pyriplatin, cis-diammine(pyridine)chloroplatinum(II), a platinum-based antitumor drug candidate, is a cationic compound with anticancer properties in mice and is a substrate for organic cation transporters that facilitate oxaliplatin uptake. Unlike cisplatin and oxaliplatin, which form DNA cross-links, pyriplatin binds DNA in a monofunctional manner. The antiproliferative effects of pyriplatin, alone and in combination with known anticancer drugs (paclitaxel, gemcitabine, SN38, cisplatin, and 5-fluorouracil), were evaluated in a panel of epithelial cancer cell lines, with direct comparison to cisplatin and oxaliplatin. The effects of pyriplatin on gene expression and platinum–DNA adduct formation were also investigated. Pyriplatin exhibited cytotoxic effects against human cell lines after 24 hours (IC50 = 171–443 μmol/L), with maximum cytotoxicity in HOP-62 non–small cell lung cancer cells after 72 hours (IC50 = 24 μmol/L). Pyriplatin caused a G2-M cell cycle block similar to that induced by cisplatin and oxaliplatin. Induction of apoptotsis and DNA damage response was supported by Annexin-V analysis and detection of phosphorylated Chk2 and H2AX. Treatment with pyriplatin increased CDKN1/p21 and decreased ERCC1 mRNA expression. On a platinum-per-nucleotide basis, pyriplatin–DNA adducts are less cytotoxic than those of cisplatin and oxaliplatin. The mRNA levels of genes implicated in drug transport and DNA damage repair, including GSTP1 and MSH2, correlate with pyriplatin cellular activity in the panel of cell lines. Synergy occurred for combinations of pyriplatin with paclitaxel. Because its spectrum of activity differs significantly from those of cisplatin or oxaliplatin, pyriplatin is a lead compound for developing novel drug candidates with cytotoxicity profiles unlike those of drugs currently in use. Mol Cancer Ther; 10(9); 1709–19. ©2011 AACR.

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

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.

All H3K27Ac peaks called in non-promoter regions in IMR5

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.

Supplemental Figures S7. Supplemental Figures S7. Effects of JQ1 and OTX015 on BRD4 binding to MYD88 promoter region in DLBCL cell lines.

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

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.

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

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).

Purpose: In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-containing proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015.Experimental Design: We assessed the preclinical activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo experiments to identify the mechanism of action and the genetic features associated with sensitivity to the compound.Results: OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo experiments showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, especially with mTOR and BTK inhibitors. Gene expression signatures associated with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compound induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell–like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11.Conclusions: Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clinical investigation of OTX015 as single agent and in combination therapies. Clin Cancer Res; 21(7); 1628–38. ©2015 AACR.

Supplemental Figures S1-2. Supplemental Figures S1: effects of OTX015 on cell cycle and cell growth in DLBCL cell lines. Supplemental Figures S2: effects of OTX015 on apoptosis in DLBCL cell lines.

Purpose: Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma.Experimental Design: The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control.Results: OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes.Conclusions: We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470–81. ©2015 AACR.

List of all Super-enhancers identified in IMR5 cells

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).

Supplemental Table S4: List of gene-sets associated with the sensitivity to OTX015.

Supplemental Figures S6. Supplemental Figures S6: OTX015 effects on the production of IL-4 and IL-10 in DLBCL cell lines.

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.

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

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

Supplemental Figures S3-5. Supplemental Figures S3: the gene expression changes induced by OTX015 in DLBCL cell lines. Supplemental Figures S4: network of genes affected by OTX015. Supplemental Figures S5: similar biologic effects of OTX015 and JQ1.

Supplemental Table S1: Cell lines and growth medium.

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

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

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

Acquired resistance to protein kinase C (PKC) modulators may explain the failure of clinical trials in patients with cancer. Herein, we established a human colon cancer cell line resistant to PEP005, a drug that inhibits PKCα and activates PKCδ. Colo205-R cells, selected by stepwise exposure to PEP005, were >300-fold more resistant to PEP005 than parental Colo205-S cells and were cross-resistant to phorbol 12-myristate 13-acetate, bryostatin, bistratene A, and staurosporine. No PKCα or PKCδ mutation was detected in Colo205-S and Colo205-R cells. Changes in Colo205-R cells were reminiscent of the epithelial-to-mesenchymal transition (EMT) phenotype. Accordingly, Colo205-R cells were more invasive than Colo205-S in Matrigel assays and in mouse xenografts. We also found an increased mRNA expression of several EMT genes, such as those encoding for transforming growth factor-β and vimentin, along with a decreased mRNA expression of genes involved in epithelial differentiation, such as CDH1 (E-cadherin), CLDN4 (claudin 4), S100A4, and MUC1, in Colo205-R compared with Colo205-S cells in vitro and in vivo. Interestingly, high expression of ET-1 was shown in Colo205-R cells and correlated with low sensitivity to PEP005 and staurosporine in a panel of 10 human cancer cell lines. Inhibition of the ET-1 receptor ETR-A with bosentan restored the antiproliferative effects of PEP005 in Colo205-R cells and decreased the invasive properties of this cell line. Exogenous exposure to ET-1 and silencing ET-1 expression using small interfering RNA modulated cell signaling in Colo205-S and Colo205-R. In summary, acquired resistance to PEP005 was associated with expression of EMT markers and activates the ET-1/ETR-A cell signaling. [Cancer Res 2009;69(10):4260–69]