(A) Scheme depicting the generation of syngeneic Trp53-null preclinical models. Donor mammary epithelium from Trp53-null BALB/c mice was transplanted in situ into cleared mammary fat pads of wild-type recipient hosts, resulting in the derivation of heterogeneous, Trp53-null, transplantable mammary tumors representative of the different intrinsic molecular subtypes of human breast cancer. (B) Top: Representative images of H&E staining and IHC staining of F4/80 and S100A8 in Trp53-null models used in this study. Scale bars: 50 μm. Bottom: Quantification of IHC staining. Three to 6 representative ×20 images were analyzed for each tumor. (C) Schematic of animal treatment. Freshly dissociated tumor cells were injected into the fourth mammary fat pad of BALB/c mice. Mice were randomized and treatment was initiated when tumors reach a volume of 70–150 mm³. Mice were treated with either vehicle or zotatifin every 3 days until ethical endpoint. (D) Tumor growth curves of BALB/c mice treated with either vehicle or zotatifin. n = 6 for 2225L-LM2 and n = 5 for all other models in each treatment arm. Data are presented as mean ± SEM and were analyzed using 2-way ANOVA with Bonferroni’s multiple-comparison test. (E) Left: Representative images of IHC staining of BrdU in ethical endpoint 2153L tumor tissues. Scale bar: 50 μm. Right: Quantification of IHC staining. Five representative ×20 images were analyzed for each tumor. n = 5 biological replicates. Data are presented as mean ± SEM and were analyzed using 2-tailed, unpaired Student’s t test. (F) Left: Representative images of cell cycle distribution of 2153L cells that were cultured in complete medium and treated with vehicle or 40 nM zotatifin for 48 hours. Right: Quantification of the cell cycle phases from 3 independent experiments. Data are presented as mean ± SD and were analyzed using 2-tailed, unpaired Student’s t test.