Trilaciclib

G1T28 is a potent and selective CDK4/6 inhibitor that inhibits the phosphorylation of RB and induces an exclusive, reversible G1 arrest. In vitro and in vivo, G1T28 protects RB competent cells from damage by chemotherapy as assessed by gamma-H2A.X (γH2AX) and apoptosis through caspase 3/7 activation.^[1]

HS68, WM2664 and A2058 cells are treated with 300 nM G1T28 or DMSO (0.1%), for 4, 8, 16 or 24 hours. Whole cell extracts are prepared using 1× radioimmunoprecipitation assay buffer (RIPA) containing 1× HALT protease and phosphatase inhibitors. Total protein concentration is determined by using the bicinchoninic acid (BCA) Protein Assay Kit, according to manufacturer's instructions. Fifteen micrograms of protein is heat denatured for 10 minutes at 70 ℃ and resolved by Novex NuPAGE SDS-PAGE gel system and transferred to 0.45 μm nitrocellulose membrane by electroblotting. Membranes are blocked in LiCor Membrane Blocking Buffer and incubated overnight with rabbit anti-pRb (Ser807/811) antibody at a 1:1,000 dilution and mouse anti-MAPK antibody at a 1:2,000 dilution, as a loading control. Secondary antibodies (LiCor) are Goat anti- rabbit (680RD) and Goat antimouse (800CW) at a 1:15,000 dilution. Blots are incubated for one hour, washed and imaged using LiCor ImageStudio software.

In vivo, G1T28 regulates the proliferation of HSPCs in both mouse and canine bone marrow, in a reversible, doseand time-dependent manner. Pretreatment of mice with G1T28 allows a faster recovery of complete blood counts (CBCs) following chemotherapy. In addition, G1T28 does not protect RB deficient tumors from chemotherapy but, instead, adds to the anti-tumor effect.^[1]