onc201 and Colorectal-Neoplasms

The purpose of the present research was to explore the therapeutic impact of raw lacquer extract from Toxicodendron vernicifluum on colorectal cancer cells and to investigate the outcome of raw lacquer extract and ONC201 co-treatment on the activity of colorectal cancer cells.. The cells of HCT116 were treated with raw lacquer extract, ONC201, or co-treatment. Subsequently, MTT, trypan blue staining, colony formation, annexin V/propidium iodide staining, wound healing, and transwell assays were performed to assess the effects of raw lacquer extract, ONC201 and the synthesis effect of co-treatment on cell activity, survival, proliferation, apoptosis, migration, and invasion in HCT116 cells. Western blotting and immunostaining assay were also performed to detect the expressions of tumor necrosis factor-related apoptosis-inducing ligand, death receptor-5, cleaved caspase-8, p-mTOR/mTOR, and p-S6K/S6K in cells.. The results showed that ONC201 and raw lacquer extract had effective anti-cancer effects on HCT116 cells. ONC201 and raw lacquer extract treatment on colorectal cancer cells inhibited cell viability and growth, as well as induced cell apoptosis and cell death of HCT116. The migration and invasion of HCT116 cells were also inhibited. Significantly, raw lacquer extract and ONC201 cotreatment further enhanced the anti-colorectal cancer cell activity in HCT116 cells. Western blotting and immunostaining assay showed that raw lacquer extract in combination with ONC201 induced tumor necrosis factor-related apoptosis-inducing ligand/death receptor-5 expression activation, inhibited the expression of cleaved caspase-8/procaspase-8, and reduced the expression of p-mTOR/mTOR and p-S6K/S6K.. These results indicated that raw lacquer extract in combination with ONC201 enhanced the inhibitory effects on colorectal cancer cell activity.

Topics: Apoptosis; Caspase 8; Cell Proliferation; Colorectal Neoplasms; Humans; Lacquer; Ligands; Receptors, TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases; Toxicodendron; Tumor Necrosis Factors

Activation of the TRAIL proapoptotic pathway can promote cancer cell apoptosis. Histone deacetylases (HDACs) also are promising drug targets for cancers, and their synergistic effect with TRAIL can improve the inhibitory effect on cancer cells. Therefore, the development of highly TRAIL-sensitive HDAC inhibitors might be a promising strategy for the treatment of cancers. We synthesized a series of HDAC inhibitors by introducing effective fragments sensitive to TRAIL. Compound IIc showed good inhibitory activity against HDAC1 and HCT116 cells and showed higher sensitivity to activating the expression of the TRAIL protein and promoting the apoptosis of HCT-116 cells compared with ONC201. The inhibitory activity of compound IIc (25 mg/kg) in the HCT-116 xenograft model was significantly greater than those of the positive control drugs (ONC201, chidamide). These findings suggested that development of highly TRAIL-sensitive HDAC inhibitors as colorectal tumor cancer drugs.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Histone Deacetylase Inhibitors; Humans; Imidazoles; Pyridines; Pyrimidines; TNF-Related Apoptosis-Inducing Ligand

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

Topics: Alternative Splicing; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Computational Biology; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Pyridines; Pyrimidines; RNA, Messenger; Signal Transduction; Tetrazolium Salts; Thiazoles; Transcription Factor CHOP; Tumor Microenvironment; Up-Regulation

Small molecule ONC201 is an investigational anti-tumor agent that upregulates intra-tumoral TRAIL expression and the integrated stress response pathway. A Phase I clinical trial using ONC201 therapy in advanced cancer patients has been completed and the drug has progressed into Phase II trials in several cancer types. Colorectal cancer (CRC) remains one of the leading causes of cancer worldwide and metastatic disease has a poor prognosis. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents.. We investigated the potential combination of VEGF inhibitors such as bevacizumab and its murine-counterpart; along with other anti-angiogenic agents and ONC201 in both CRC xenograft and patient-derived xenograft (PDX) models. We utilized non-invasive imaging and immunohistochemistry to determine potential mechanisms of action.. Our results demonstrate significant tumor regression or complete tumor ablation in human xenografts with the combination of ONC201 with bevacizumab, and in syngeneic MC38 colorectal cancer xenografts using a murine VEGF-A inhibitor. Imaging demonstrated the impact of this combination on decreasing tumor growth and tumor metastasis. Our results indicate that ONC201 and anti-angiogenic agents act through distinct mechanisms while increasing tumor cell death and inhibiting proliferation.. With the use of both a murine VEGF inhibitor in syngeneic models, and bevacizumab in human cell line-derived xenografts, we demonstrate that ONC201 in combination with anti-angiogenic therapies such as bevacizumab represents a promising approach for further testing in the clinic for the treatment of CRC.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Bevacizumab; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Disease Models, Animal; Drug Synergism; Heterocyclic Compounds, 4 or More Rings; Human Umbilical Vein Endothelial Cells; Humans; Imidazoles; Mice; Models, Biological; Neovascularization, Pathologic; Pyridines; Pyrimidines; Tumor Burden; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

Topics: Animals; Cell Death; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Dose-Response Relationship, Drug; Female; HCT116 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Imidazoles; Killer Cells, Natural; Mice; Mice, Nude; Neoplasm Metastasis; Pyridines; Pyrimidines; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Xenograft Model Antitumor Assays

Cancer stem cells (CSCs) correlate with recurrence, metastasis and poor survival in clinical studies. Encouraging results from clinical trials of CSC inhibitors have further validated CSCs as therapeutic targets. ONC201 is a first-in-class small molecule imipridone in Phase I/II clinical trials for advanced cancer. We have previously shown that ONC201 targets self-renewing, chemotherapy-resistant colorectal CSCs via Akt/ERK inhibition and DR5/TRAIL induction. In this study, we demonstrate that the anti-CSC effects of ONC201 involve early changes in stem cell-related gene expression prior to tumor cell death induction. A targeted network analysis of gene expression profiles in colorectal cancer cells revealed that ONC201 downregulates stem cell pathways such as Wnt signaling and modulates genes (ID1, ID2, ID3 and ALDH7A1) known to regulate self-renewal in colorectal, prostate cancer and glioblastoma. ONC201-mediated changes in CSC-related gene expression were validated at the RNA and protein level for each tumor type. Accordingly, we observed inhibition of self-renewal and CSC markers in prostate cancer cell lines and patient-derived glioblastoma cells upon ONC201 treatment. Interestingly, ONC201-mediated CSC depletion does not occur in colorectal cancer cells with acquired resistance to ONC201. Finally, we observed that basal expression of CSC-related genes (ID1, CD44, HES7 and TCF3) significantly correlate with ONC201 efficacy in >1000 cancer cell lines and combining the expression of multiple genes leads to a stronger overall prediction. These proof-of-concept studies provide a rationale for testing CSC expression at the RNA and protein level as a predictive and pharmacodynamic biomarker of ONC201 response in ongoing clinical studies.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Cell Line, Tumor; Cell Survival; Central Nervous System Neoplasms; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Glioblastoma; HCT116 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Hyaluronan Receptors; Imidazoles; Inhibitor of Differentiation Protein 1; Neoplastic Stem Cells; Pyridines; Pyrimidines; Transcriptome; Wnt Signaling Pathway

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Enzyme Activation; Gene Knockdown Techniques; Gene Silencing; Heterocyclic Compounds, 4 or More Rings; Humans; Imidazoles; Morpholines; Mutation; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; TOR Serine-Threonine Kinases

Self-renewing colorectal cancer stem/progenitor cells (CSC) contribute to tumor maintenance and resistance to therapy. Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. ONC201/TIC10 is a first-in-class antitumor agent that induces TRAIL pathway-mediated cell death in cancer cells without observed toxicity. We have previously described that ONC201/TIC10 exposure leads to transcriptional induction of the TRAIL gene via transcription factor Foxo3a, which is activated by dual inactivation of Akt and ERK. The Akt and ERK pathways serve as important targets in CSCs. Foxo3a is a key mediator of Akt and ERK-mediated CSC regulation. We hypothesized that the potent antitumor effect of ONC201/TIC10 in colorectal cancer involves targeting CSCs and bulk tumor cells. ONC201/TIC10 depletes CD133(+), CD44(+), and Aldefluor(+) cells in vitro and in vivo. TIC10 significantly inhibits colonosphere formation of unsorted and sorted 5-fluorouracil-resistant CSCs. ONC201/TIC10 significantly reduces CSC-initiated xenograft tumor growth in mice and prevents the passage of these tumors. ONC201/TIC10 treatment also decreased xenograft tumor initiation and was superior to 5-fluorouracil treatment. Thus, ONC201/TIC10 inhibits CSC self-renewal in vitro and in vivo. ONC201/TIC10 inhibits Akt and ERK, consequently activating Foxo3a and significantly induces cell surface TRAIL and DR5 expression in both CSCs and non-CSCs. ONC201/TIC10-mediated anti-CSC effect is significantly blocked by the TRAIL sequestering antibody RIK-2. Overexpression of Akt, DR5 knockdown, and Foxo3a knockdown rescues ONC201/TIC10-mediated depletion of CD44(+) cells and colonosphere inhibition. In conclusion, ONC201/TIC10 is a promising agent for colorectal cancer therapy that targets both non-CSCs and CSCs in an Akt-Foxo3a-TRAIL-dependent manner.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; HCT116 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Imidazoles; Mice, Nude; Mice, SCID; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Xenograft Model Antitumor Assays