PTC-209 is a potent and selective BMI-1 inhibitor.

PTC-209 effectively reduces the frequency of functional colorectal cancer cancer-initiating cells (CICs) in vivo. Administered at a dosage of 60 mg/kg/day subcutaneously (s.c.), PTC-209 significantly inhibits the production of BMI-1 in tumor tissues and halts the growth of pre-established tumors in mice with primary human colorectal cancer xenografts, as well as in xenografts from human colorectal cancer cell lines LIM1215 or HCT116.

PTC-209 irreversibly inhibits the growth of colorectal cancer initiating cells (CIC) by targeting BMI-1, resulting in the destruction of these cells. It suppresses UTR-mediated reporter gene expression and endogenous BMI-1 expression in human colorectal HCT116 cells and human fibrosarcoma HT1080 tumor cells. Furthermore, PTC-209 reduces the growth of rectal tumor cells in a BMI-1 dependent manner.

Untranslated region-mediated luciferase reporter expression: HEK293 cells are transfected with a GEMS reporter vector that contains the luciferase open-reading frame flanked by and under post-transcriptional control of the BMI-1 5′ and 3′ UTRs. The resulting stable cells (F8) are treated with PTC-209 or vehicle control overnight, and then luciferase reporter activity is determined using Bright-Glo assays. The assays are run in triplicate for each point, and the percentage of inhibition was calculated against vehicle control.

To determine whether pretreatment with the inhibitor affects tumor cell growth, cells are plated with the inhibitor for 4 d in vitro and plated in limiting doses in vitro without adding further inhibitor. Trypan blue exclusion is used to count viable cells. The in vitro sphere-initiating cell frequency is calculated after inhibitor treatment by evaluating the number of wells containing spheres. For the experiments where LDAs are set up following recovery of PTC-209 treated cells, 6-well plates were seeded with 1E6 cells per well and incubated overnight. Cells are subsequently treated for 4 d in triplicate with either DMSO vehicle or PTC-209 (0.01, 0.1, 1 and 10 μM). Drug treatments are washed off and 4 mL fresh suspension medium added to all wells. To assess cell viability following the 4 d treatment window, cells are trypsinized and counted at 0, 24, 72 and 120 h after removal of the drug. Long-lasting effects of the drug treatment on sphere-forming ability are assessed by plating LDAs (50,000, 10,000, 1,000,100, 10 and 1 cell per well) using the cells obtained 120 h after the 4-d drug treatment.(Only for Reference)