p53 (IC50 = 0.37 μM); NF-κB (IC50 = 0.47 μM); FACT
FACT (facilitates chromatin transcription) complex [1]
FACT (facilitates chromatin transcription) complex [2]

CBL0137 is a potent inducer of apoptosis in pancreatic cancer cell lines and toxic to both pancreatic cancer stem cells and bulk tumor cells that are actively proliferating. Inhibiting cellular stress pathways controlled by NF-B and HSF-1 while simultaneously activating p53 is possible with CBL0137 and related molecules[2]. CBL0137 binds DNA without introducing any kind of chemical alterations, making it non-genotoxic. The Facilitates Chromatin Transcription (FACT) complex, a chromatin remodeling complex involved in transcription, replication, and DNA repair, is functionally inactivated as a result of CBL0137 binding to DNA. FACT is lost from the nucleoplasm and becomes trapped in chromatin in CBL0137-treated cells, which inhibits FACT-dependent transcription, including NF-kB-mediated transcription. Additionally, chromatin trapping of FACT results in the phosphorylation and activation of p53 in a casein kinase 2 (CK2)-dependent manner[3].

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CBL0137 HCl (CBLC137) showed cytotoxicity to human fibrosarcoma HT1080 cells with an IC₅₀ value, and was less toxic to normal diploid fibroblasts (Wi38, MEF) (P < 0.001). Treatment with 2 μM CBLC137 for 24 hours induced cell cycle arrest in tumor cells (HT1080, RCC45, MiaPaca) but had little effect on normal cells (Wi38, NKE-hTERT). It activated p53 by promoting phosphorylation of p53 Ser³⁹² via casein kinase 2 (CK2) and inhibited NF-κB-dependent transcription by trapping FACT in chromatin, without inducing detectable DNA damage (comet assay and H2AX phosphorylation assay confirmed no DNA breaks). Additionally, it reduced the soluble fraction of FACT subunits (SSRP1, SPT16) and blocked NF-κB nuclear-cytoplasmic shuttling and DNA binding [1]
CBL0137 HCl (CBLC137) was a potent inducer of apoptosis in pancreatic cancer cell lines (MiaPaCa-2, PANC-1, BxPC-3), as evidenced by cleavage of caspases 3, 7, 8, 9 and PARP1 after 4-24 hours of treatment with 2 μM. It inhibited the viability of pancreatic cancer cells and drug-resistant cancer stem cells (CSCs), reducing the proportion of CSC surface markers (CD24ᵂⁱ CD44ᵂⁱ) and preventing gemcitabine-induced "side population" enrichment. It showed synergistic effects with gemcitabine in colony formation assays (MiaPaCa-2, PANC-1) and inhibited gemcitabine-induced transcriptional responses, including NF-κB target gene expression and RRM1/RRM2 subunits of ribonucleotide reductase (RNR) [2]

In mice, CBL0137 is effective against several Pancreatic ductal adenocarcinoma (PDA) models, including orthotopic gemcitabine resistant PANC-1 model and patient derived xenografts, in which CBL0137 anti-tumor effect correlated with overexpression of FACT[1]. According to its proposed mechanism of action, CBL0137 targets glioblastoma (GBM), penetrates the blood-brain barrier, and is effective in both TMZ-responsive and -resistant orthotopic models. The ability of this medication to cross the blood-brain barrier, especially when given intravenously, is encouraging for its potential to treat CNS tumors. In orthotopic models, intravenously administered drugs have higher bioavailability than those administered orally because they accumulate more in tumor tissue. The normal buildup of CBL0137 in brain tissue does not result in detectable neurotoxicity[3].

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CBL0137 HCl (CBLC137) exhibited antitumor activity in various human tumor xenograft mouse models. For renal cell carcinoma Caki-1, colon carcinoma DLD-1, melanoma Mel-7, and pancreatic ductal adenocarcinoma (PDA), treatment with CBLC137 significantly reduced tumor volume compared to vehicle (P < 0.005, ANOVA), with 5-10 mice per group. In MMTV-neu transgenic mice with spontaneous mammary tumors, oral gavage of 100 mg/kg CBLC137 reduced the soluble fraction of SSRP1 and SPT16 in tumors 24 hours post-treatment (P < 0.05) [1]
CBL0137 HCl (CBLC137) showed efficacy in orthotopic and patient-derived xenograft (PDX) models of pancreatic cancer. In orthotopic gemcitabine-resistant PANC-1 models (n=6-7/group), intravenous administration of 90 mg/kg CBLC137 once weekly for 4 weeks reduced tumor volume, and combination with gemcitabine (40 mg/kg i.p. Q4d) enhanced antitumor effects. In PDX models (PDX#13756, #13590; n=5-10/group), 50-90 mg/kg CBLC137 i.v. once weekly (alone or combined with 20 mg/kg gemcitabine i.p. Q4d) for 4 weeks inhibited tumor growth, with antitumor effects correlating with FACT overexpression [2]

CBL0137 hydrochloride is applied to MiaPaca2 and BxPC-3 cells for 4 or 24 hours. Protease and phosphatase inhibitors are present in 1× Cell Culture Lysis Reagent, which is used to harvest cells. Lysates 5 to 20 μg are transferred to PVDF membranes after being separated on SDS-PAGE gels. Antibodies that target the proteins SSRP1, SPT16, RRM1 and RRM2 are used to probe blots. The loading control is GAPDH. ECL kit is used to visualize proteins[1].

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CK2 kinase activity assay was performed using CK2β immunoprecipitated from HT1080 cell lysates treated with CBL0137 HCl (CBLC137), quinacrine, or UV. A peptide substrate corresponding to the C-terminus of p53 (amino acids 311-393) was used to measure CK2-mediated phosphorylation induced by the drug [1]

Cells are resuspended in serum-free Dulbecco's Modified Eagle Medium (DMEM) and exposed to varying concentrations of CBL0137 hydrochloride for 1h. Then, 105 cells from each treatment condition are plated in 3 wells of a 6-well plate in 2 mL of serum-free DMEM/F12 medium supplemented with 0.4% BSA, 0.2×B27, 10 ng/mL recombinant EGF, and containing 0.25% agarose. In three wells of a 6-well plate with regular FBS-containing medium, 103 cells from each treatment condition are plated. Seven to fifteen days after plating, colonies are counted under an inverted microscope.

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Cytotoxicity assay: Tumor cells (HT1080, RCC45, MiaPaca) and normal cells (Wi38, NKE-hTERT) were treated with different concentrations of CBL0137 HCl (CBLC137) for 24-72 hours, and cell viability was detected by Cell Titer Blue assay or methylene blue staining. Cell cycle analysis: Cells were stained with propidium iodide after 24-hour treatment with 2 μM CBLC137, and analyzed by flow cytometry (FACS). Western blot: HT1080 cells were treated with CBLC137 (0.8-2 μM) for 8-16 hours, and p53 Ser³⁹² phosphorylation, FACT subunit expression, and caspase/PARP1 cleavage were detected. RT-PCR/qPCR: HT1080 or MiaPaCa-2 cells were treated with 1-2 μM CBLC137 for 2-24 hours, and mRNA expression of NF-κB target genes (IL-8, TNF, IκBα) and RNR subunits (RRM1, RRM2) was quantified. Chromatin immunoprecipitation (ChIP): HT1080 cells were treated with 1-2 μM CBLC137 for 2 hours (with/without TNF stimulation), and binding of SSRP1, p65, and RNA Pol II to NF-κB-dependent gene promoters (IL-8, TNF) was analyzed. CSC-related assays: PANC-1 cells were treated with 3 μM CBLC137 for 1 hour, stained with Hoechst 33342 or CSC surface markers (CD24, CD44), and analyzed by flow cytometry; colony formation in 2D/3D media was counted after drug treatment [1][2]

Ketamine/xylazine is used to create a deep anesthesia in 10-week-old female athymic nude mice (n = 8 per treatment group). Each mouse has its pancreas tail inoculated with 2 106 PANC-1 cells via laparotomy. When the tumor was detected by ultrasound two weeks after the vaccination, treatment started. There are the following routines: 1) Vehicles, gavage of sterile water and 100 mg/kg captisol 2) 100 mg/mL captisol diluted with 50 to 90 mg/kg CBL0137 hydrochloride and given intravenously once a week via the tail vein; 3) 10 to 20 mg/kg CBL0137 hydrochloride given orally five days on and two days off. To measure tumors, digital calipers are used. The equation LW2/2 is used to determine the tumor volume, where L is the longest dimension and W is the dimension that is perpendicular to W. Mice are monitored for at least 90 days after the start of treatment or until at least one tumor per mouse reached 1000 mm3, whichever occurs first.

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Xenograft tumor models: Human tumor cells (Caki-1, DLD-1, Mel-7, PDA, PANC-1) or PDX tissues were inoculated into nude mice or SCID mice. When tumors reached the specified volume, CBL0137 HCl (CBLC137) was administered intravenously at 50-90 mg/kg once weekly, alone or combined with gemcitabine (20-40 mg/kg i.p. every 4 days) for 4 weeks. Tumor volume was measured periodically, and mice were euthanized 1 week post-treatment for tumor collection and analysis. Spontaneous tumor model: MMTV-neu transgenic mice with palpable mammary tumors were given 100 mg/kg CBLC137 via oral gavage, and tumor lysates were collected 24 hours later to detect soluble FACT subunits by Western blot [1][2]

CBL0137 HCl (CBLC137) exhibits selective toxicity to tumor cells, with an IC₅₀ value for tumor cells (HT1080, C8) lower than that for normal diploid fibroblasts (Wi38, MEF). Comet assay (no tail moment increase) and loss of histone H2AX phosphorylation confirmed that CBLC137 does not induce detectable DNA damage in HeLa or HT1080 cells [1]

[1]. Sci Transl Med . 2011 Aug 10;3(95):95ra74.

[2]. Oncotarget . 2014 Nov 30;5(22):11038-53.

[3]. Neuro Oncol . 2017 Feb 1;19(2):186-196.

Effective cancer eradication requires treatment targeting multiple pathways. The p53 and nuclear factor κB (NF-κB) pathways are aberrantly regulated in almost all tumors, making them ideal targets for therapeutic activation and inhibition, respectively. We isolated and optimized the structure of the small molecule compound curaxins, which simultaneously activates p53 and inhibits NF-κB without causing detectable genotoxicity. Curaxins exhibited anticancer activity in all tested mouse xenograft models of human tumors. This paper reports that the effects of curaxins on p53 and NF-κB, and their toxicity to cancer cells, stem from “chromatin trapping” of the FACT (promoting chromatin transcription) complex. This inaccessibility of the FACT complex leads to phosphorylation of p53 Ser(392) by casein kinase 2 and inhibition of NF-κB-dependent transcription, which requires FACT complex activity during the elongation phase. These results suggest that FACT is a potential anticancer target that can simultaneously regulate multiple signaling pathways that are frequently dysregulated in cancer without inducing DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs. [1]

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Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest cancers due to the lack of effective treatments. Curaxins are a class of small molecule compounds with anticancer activity that have been demonstrated in various mouse cancer models. The lead compound CBL0137 has recently entered a phase I clinical trial. Curaxins regulate multiple important signaling pathways involved in the pathogenesis of PDA by inhibiting the chromatin remodeling complex FACT. FACT is overexpressed in a variety of tumors, with the highest overexpression rate (59%) in PDA. In this study, the efficacy of CBL0137 alone or in combination with the current standard therapy gemcitabine in different PDA models was tested in vitro and in vivo in mouse models. Studies have found that CBL0137, alone, effectively induces apoptosis in pancreatic cancer cell lines and is toxic not only to proliferating tumor cells but also to pancreatic cancer stem cells. In mouse models, CBL0137 is effective against various pancreatic ductal adenocarcinoma (PDA) models, including the orthotopic gemcitabine-resistant PANC-1 model and patient-derived xenograft models, where the antitumor effect of CBL0137 is associated with FACT overexpression. Furthermore, we observed a synergistic effect between CBL0137 and gemcitabine, possibly due to CBL0137's ability to inhibit multiple gemcitabine-induced transcriptional programs, including the NF-κB response and ribonucleotide reductase expression, which is one of the cellular targets of gemcitabine. These data suggest that the efficacy of CBL0137 monotherapy and combination therapy with gemcitabine should be tested in a phase II clinical trial in patients with pancreatic ductal adenocarcinoma (PDA). [2]

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Pancreatic ductal adenocarcinoma (PDA) remains one of the deadliest cancers due to the lack of effective treatments. Curaxins are small molecule compounds with anticancer activity that have been demonstrated in various mouse cancer models. The lead compound of Curaxin, CBL0137, has recently entered a phase I clinical trial. Curaxins regulate multiple important signaling pathways involved in the pathogenesis of PDA by inhibiting the chromatin remodeling complex FACT. FACT is overexpressed in a variety of tumors, with the highest overexpression rate (59%) in PDA. In this study, we tested the efficacy of CBL0137 monotherapy and combination therapy with the current standard therapy gemcitabine in different PDA models in vitro and in mouse models. The study found that CBL0137 alone can effectively induce apoptosis in pancreatic cancer cell lines and is toxic not only to proliferating tumor cells but also to pancreatic cancer stem cells. In mouse models, CBL0137 was effective against a variety of pancreatic ductal adenocarcinoma (PDA) models, including the orthotopic gemcitabine-resistant PANC-1 model and patient-derived xenograft models, where the antitumor effect of CBL0137 was associated with overexpression of FACT. In addition, we observed a synergistic effect between CBL0137 and gemcitabine, which may be due to CBL0137’s ability to inhibit gemcitabine-induced transcriptional programs, including the NF-κB response and the expression of ribonucleotide reductase, which is one of the targets of gemcitabine in cells. These data suggest that the efficacy of CBL0137 monotherapy and combination therapy with gemcitabine should be tested in a phase II clinical trial in patients with pancreatic ductal adenocarcinoma (PDA). [3]

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CBL0137 HCl (CBLC137) is a lead compound curaxin, a class of small molecule anticancer drugs that target the FACT complex. [1][2]
Its mechanism of action involves “chromatin capture” of FACT, thereby activating p53 (through CK2-mediated Ser³⁹² phosphorylation) and inhibiting NF-κB (blocking transcriptional elongation) without causing genotoxicity. [1]
FACT is overexpressed in a variety of tumors (overexpression rate of 59% in pancreatic ductal adenocarcinoma), and the antitumor effect of CBL0137 is related to the expression level of FACT. [2]
It can eliminate drug-resistant cancer stem cells and enhance the efficacy of gemcitabine by inhibiting gemcitabine-induced FACT expression. NF-κB response and RNR expression [2]
CBL0137 has entered phase I clinical trials and is planned to conduct phase II clinical trials (monotherapy or combination with gemcitabine) in pancreatic cancer patients [2]

C21H24N2O2-HCL

372.89

372.16

C, 67.64; H, 6.76; Cl, 9.51; N, 7.51; O, 8.58

1197397-89-9

CBL0137;1197996-80-7

44519123

Off-white to yellow solid powder

5.39

2

3

6

26

466

0

CC(C1C=C2C(N(CCNC(C)C)C3=CC=C(C=C32)C(C)=O)=CC=1)=O.Cl

IXRKBBVMDMKAEB-UHFFFAOYSA-N

InChI=1S/C21H24N2O2.ClH/c1-13(2)22-9-10-23-20-7-5-16(14(3)24)11-18(20)19-12-17(15(4)25)6-8-21(19)23;/h5-8,11-13,22H,9-10H2,1-4H3;1H

1-[6-acetyl-9-[2-(propan-2-ylamino)ethyl]carbazol-3-yl]ethanone;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 0.75mg/ml

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 (Please use freshly prepared in vivo formulations for optimal results.)