Kinase experiment:

MiaPaca2 and BxPC-3 cells are treated with CBL0137 hydrochloride for 4 or 24 h. Cells are harvested in 1× Cell Culture Lysis Reagent containing protease and phosphatase inhibitors. Lysates 5 to 20 μg are separated on SDS-PAGE gels and transferred to PVDF membranes. Blots are probed with antibodies specific for SSRP1, SPT16, RRM1, and RRM2. GAPDH is used as a loading control. Proteins are visualized using ECL kit[1].

Cell experiment:

Cells are resuspended in serum free Dulbecco's Modified Eagle Medium (DMEM) and treated with different concentrations of CBL0137 hydrochloride for 1h. After that 105 cells from each treatment condition are plated in 3 wells of 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. 103 cells from each treatment condition are plated in 3 wells of 6-well plate in regular FBS containing medium. Colonies are counted using inverted microscope 7 to 15 days after plating[1].

Animal experiment:

10-week old female athymic nude mice (n=8 per treatment group) are deeply anesthetized with ketamine/xylazine. Using laparotomy, 2×106 PANC-1 cells are inoculated into the tail of the pancreas of each mouse. Two weeks following inoculation (tumor presence confirmed by ultrasound), treatment commenced. The following regimens are used: 1) vehicles, 100 mg/kg captisol i.v. and sterile water via gavage, 2) 50 to 90 mg/kg CBL0137 hydrochloride in 100 mg/mL captisol i.v. delivered via tail vein once per week, 3) 10 to 20 mg/kg CBL0137 hydrochloride p.o. via oral gavage, 5 days on/2 days off. Tumor measurement is done with digital calipers. Tumor volume is calculated using the equation L×W2/2 where L is the longest dimension and W is the dimension perpendicular to W. Mice are followed until at least one tumor per mouse reached 1000 mm3 or 90 days from start of treatment, whichever comes first[1].

• Brit j pharmacol (2022). PMID:36495259

EC50: 0.37 μM for activating p53; 0.47 μM for inhibiting NF-κB

CBL0137 (hydrochloride) is a curaxin that activates p53 and inhibits NF-κB.

The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in almost all tumors, making them attractive targets for therapeutic activation and inhibition, respectively.

In vitro: CBL0137 was identified as a metabolically stable curaxin activating p53 and inhibiting NF-κB. CBL0137 could functionally inactivate chromatin transcription complex, resulting in the effects on p53 and NF-κB and promoting cancer cell death [1]. It was also found that CBL0137 alone was a potent inducer of apoptosis in pancreatic cancer cell lines and was toxic not only for proliferating bulk tumor cells, but also for pancreatic cancer stem cells [2].

In vivo: In mice, CBL0137 was effective against orthotopic gemcitabine resistant PANC-1 model and patient derived xenografts, in which CBL0137 anti-tumor effect related with overexpression of FACT. Moreover, the combination effects of CBL0137 and gemcitabine might be explained by the ability of CBL0137 to inhibit several transcriptional programs induced by gemcitabine, including NF-kappaB response and expression of ribonucleotide reductase [2].

Clinical trial: A phase 1 trial of CBL0137 in patients with metastatic or unresectable advanced solid neoplasm and a study of IV CBL0137 in previously treated hematological subjects are crrently recruiting patients [https://clinicaltrials.gov/ct2/results term=CBL0137&Search=Search].

References:
1. A. V. Gasparian, C. A. Burkhart, A. A. Purmal, et al. Curaxins: Anticancer compounds that simultaneously suppress NF-κB and activate p53 by targeting FACT. Sci.Transl.Med. 3(95), (2011).
2. C. Burkhart, D. Fleyshman, R. Kohrn, et al. Curaxin CBL0137 eradicates drug resistant cancer stem cells and potentiates efficacy of gemcitabine in preclinical models of pancreatic cancer. Oncotarget 5(22), 11038-11053 (2014).