A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer
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eLife assessment
This useful manuscript presents a new therapeutic formulation and these solid findings have potential clinical significance as the efficacy of CK21 is relevant in various pancreatic cancer models.Further validation studies would help to strengthen the findings.
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Abstract
Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, ( E )–19-[(1’-benzoyloxy-1’-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.
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Author Response
Reviewer #2 (Public Review):
The authors describe the synthesis and testing of the anti-cancer activity of a new molecule CK21 against pancreatic cancer mouse models. This part of the study is very strong showing regression of pancreatic tumors at non-toxic concentrations, which is very hard to achieve for practically uncurable pancreatic cancer. Authors synthesized CK21 as an analog of a known inhibitor of RNA synthesis which is very toxic. The authors did very little attempt to understand whether the mechanism of anti-cancer efficacy of CK2 is similar to this known inhibitor of transcription or not. One cannot compare gene expression profiles between untreated and CK21-treated cells, taking into account that CK2 may inhibit the expression of all genes. The effect of CK2 on general transcription needs to be tested …
Author Response
Reviewer #2 (Public Review):
The authors describe the synthesis and testing of the anti-cancer activity of a new molecule CK21 against pancreatic cancer mouse models. This part of the study is very strong showing regression of pancreatic tumors at non-toxic concentrations, which is very hard to achieve for practically uncurable pancreatic cancer. Authors synthesized CK21 as an analog of a known inhibitor of RNA synthesis which is very toxic. The authors did very little attempt to understand whether the mechanism of anti-cancer efficacy of CK2 is similar to this known inhibitor of transcription or not. One cannot compare gene expression profiles between untreated and CK21-treated cells, taking into account that CK2 may inhibit the expression of all genes. The effect of CK2 on general transcription needs to be tested first, and then based on this data absolute changes in the expression of genes may be considered for the revealing of the mechanism of activity of CK21.
We also appreciated the toxicity concerns; thus, we designed the transcriptomic analysis on the human organoid cultured cells for early time points of 3, 6, 9 and 12 h, and with a CK21 concentration of 50nM, to ensure that at the time of harvest, the cells were ~100% viable. At these time points, many genes were upregulated but defined by IPA as enriched for cell death (apoptosis and necrosis), senescence and cell cycle arrest (Fig 5). This led us to hypothesize that the direct effect of CK21 on the tumor cells is the induction of apoptosis, but via multiple pathways.
Reviewer #3 (Public Review):
This manuscript describes CK21, a modified version of Triptolide, a natural compound with antcancer activities, to improve its bioavailability. The authors tested the compound in two human pancreatic cancer cell lines, in vitro and in vivo. The authors also use two human organoid lines derived from pancreatic cancer, and mouse KC and KPC cell lines. In all models, CK21 treatment induces dose-dependent cytotoxicity. In vivo, CK21 causes tumor regression. The authors perform gene expression analysis and show that treated organoids have generally lower transcription, consistent with cytotoxicity, and a reduction in the KFkB pathway activation.
Key experiments that would strengthen the current manuscript are: the inclusion of normal cell lines and organoids, too, presumably, show no cytotoxic effect. If that is the case, the authors would have the opportunity to compare responses and determine whether a tumor-specific mechanism can be defined.
Our in vivo studies suggest that CK21 is more specific to tumors, as CK21 ≤3 mg/kg treated mice were 100% viable and gained weight comparably to no treatment group (Fig.2d). Furthermore, in vitro studies with primary fibroblast cells indicate that comparable significant toxicity to CK21 after 72h culture was observed at 500 nM (Fig.s2). In contrast, CK21 induced significant toxicity in AsPC1 and Panc-1 cells at 50 nM (Fig. 1f.)
The authors observe that few gene changes - besides from overall lowering in transcription, occur upon treatment with CK21. They suggest that the drug acts through inhibition of the NFkB pathway and an increase in reactive oxygen species (ROS). However, no experiments to test whether either/both of these findings explain the cytotoxic effect (rescue experiments would be particularly valuable).
We performed a rescue study using an ROS inhibitor (acetylcysteine) but observed no significant effect (data not shown). We speculate that ROS and/or NF-B might function synergistically; additionally, it is possible that other mechanisms might be involved in the anti-tumor effects of CK21.
In the last figure, the authors text whether CK21 is immunosuppressive by testing immunity against a mis-matched tumor cell line (using KPC tumors, mixed strain, in mixed strain mice). The immunity against HLA mis-matched cells is a very strong immune reaction, and mild immune suppression might be missed, which diminishes the value of these findings.
KPC-960 tumor cells were derived from KPC (C57BL/6 background); therefore, KPC-960 tumors were HLA matched with host C57BL/6 mice. We were surprised to observe spontaneous rejection of the KPC-960 tumor line, since this contrasts with Torres et al. 2013. We speculate that this could be due to the increased number of passages resulting in antigenic drift, which may result in the accumulation of mutations that induce spontaneous rejection.
We agree that there might be mild immunosuppression that we did not detect; we have included this caveat in the discussion. KC-6141 tumor cells used as CTL targets were from KC mice (mixed background – B6.129).
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eLife assessment
This useful manuscript presents a new therapeutic formulation and these solid findings have potential clinical significance as the efficacy of CK21 is relevant in various pancreatic cancer models.Further validation studies would help to strengthen the findings.
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Reviewer #1 (Public Review):
In this manuscript, Tian et al. describe a novel modified version of the pro-drug triptolide, CK21, and provide evidence for its improved pharmacokinetics and its safety and efficacy in multiple xenograft models of pancreatic cancer. The authors performed transcriptomic analysis upon CK21 treatment which revealed that downregulation of NF-kB and mitochondrial dysfunction induce apoptosis and therefore lead to tumor regression. Downregulation of NF-kB and induction of apoptosis was then validated in vitro and in vivo. These findings have potential clinical significance as the efficacy of CK21 in preclinical PDAC models is compelling. However, there are also some limitations to their experiments and more validation studies are necessary to strengthen their findings regarding the mechanism of action of the …
Reviewer #1 (Public Review):
In this manuscript, Tian et al. describe a novel modified version of the pro-drug triptolide, CK21, and provide evidence for its improved pharmacokinetics and its safety and efficacy in multiple xenograft models of pancreatic cancer. The authors performed transcriptomic analysis upon CK21 treatment which revealed that downregulation of NF-kB and mitochondrial dysfunction induce apoptosis and therefore lead to tumor regression. Downregulation of NF-kB and induction of apoptosis was then validated in vitro and in vivo. These findings have potential clinical significance as the efficacy of CK21 in preclinical PDAC models is compelling. However, there are also some limitations to their experiments and more validation studies are necessary to strengthen their findings regarding the mechanism of action of the drug. Specifically, the authors suggest that mitochondrial dysfunction is responsible for the observed apoptosis; however, this is not demonstrated. Additionally, side-by-side comparisons to other clinical triptolide analogs to show CK21 is at least as efficacious as other analogs in vivo would be valuable, especially since other analogs have been shown to synergize with conventional chemotherapy in PDAC mouse models, whereas CK21 does not appear to. Moreover, assessing whether CK21 is efficacious in syngeneic orthotopic PDAC models is critical, especially since CK21 was shown to have an impact on NF-KB which plays a major role in the immune compartment and triptolide has been shown to be immunosuppressive.
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Reviewer #2 (Public Review):
The authors describe the synthesis and testing of the anti-cancer activity of a new molecule CK21 against pancreatic cancer mouse models. This part of the study is very strong showing regression of pancreatic tumors at non-toxic concentrations, which is very hard to achieve for practically uncurable pancreatic cancer. Authors synthesized CK21 as an analog of a known inhibitor of RNA synthesis which is very toxic. The authors did very little attempt to understand whether the mechanism of anti-cancer efficacy of CK2 is similar to this known inhibitor of transcription or not. One cannot compare gene expression profiles between untreated and CK21-treated cells, taking into account that CK2 may inhibit the expression of all genes. The effect of CK2 on general transcription needs to be tested first, and then based …
Reviewer #2 (Public Review):
The authors describe the synthesis and testing of the anti-cancer activity of a new molecule CK21 against pancreatic cancer mouse models. This part of the study is very strong showing regression of pancreatic tumors at non-toxic concentrations, which is very hard to achieve for practically uncurable pancreatic cancer. Authors synthesized CK21 as an analog of a known inhibitor of RNA synthesis which is very toxic. The authors did very little attempt to understand whether the mechanism of anti-cancer efficacy of CK2 is similar to this known inhibitor of transcription or not. One cannot compare gene expression profiles between untreated and CK21-treated cells, taking into account that CK2 may inhibit the expression of all genes. The effect of CK2 on general transcription needs to be tested first, and then based on this data absolute changes in the expression of genes may be considered for the revealing of the mechanism of activity of CK21.
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Reviewer #3 (Public Review):
This manuscript describes CK21, a modified version of Triptolide, a natural compound with ant-cancer activities, to improve its bioavailability. The authors tested the compound in two human pancreatic cancer cell lines, in vitro and in vivo. The authors also use two human organoid lines derived from pancreatic cancer, and mouse KC and KPC cell lines. In all models, CK21 treatment induces dose-dependent cytotoxicity. In vivo, CK21 causes tumor regression. The authors perform gene expression analysis and show that treated organoids have generally lower transcription, consistent with cytotoxicity, and a reduction in the KFkB pathway activation.
Key experiments that would strengthen the current manuscript are: the inclusion of normal cell lines and organoids, too, presumably, show no cytotoxic effect. If that is …
Reviewer #3 (Public Review):
This manuscript describes CK21, a modified version of Triptolide, a natural compound with ant-cancer activities, to improve its bioavailability. The authors tested the compound in two human pancreatic cancer cell lines, in vitro and in vivo. The authors also use two human organoid lines derived from pancreatic cancer, and mouse KC and KPC cell lines. In all models, CK21 treatment induces dose-dependent cytotoxicity. In vivo, CK21 causes tumor regression. The authors perform gene expression analysis and show that treated organoids have generally lower transcription, consistent with cytotoxicity, and a reduction in the KFkB pathway activation.
Key experiments that would strengthen the current manuscript are: the inclusion of normal cell lines and organoids, too, presumably, show no cytotoxic effect. If that is the case, the authors would have the opportunity to compare responses and determine whether a tumor-specific mechanism can be defined.
The authors observe that few gene changes - besides from overall lowering in transcription, occur upon treatment with CK21. They suggest that the drug acts through inhibition of the NFkB pathway and an increase in reactive oxygen species (ROS). However, no experiments to test whether either/both of these findings explain the cytotoxic effect (rescue experiments would be particularly valuable).
In the last figure, the authors text whether CK21 is immunosuppressive by testing immunity against a mis-matched tumor cell line (using KPC tumors, mixed strain, in mixed strain mice). The immunity against HLA mis-matched cells is a very strong immune reaction, and mild immune suppression might be missed, which diminishes the value of these findings.
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