RNA sequencing analysis of TNBC PDX treated with MLK3 and pan MLK's inhibitor

Mixed Lineage Kinase 3 (MLK3) is a viable emerging target for neoplastic diseases; however, it is uncertain whether its activators or inhibitors can act as anti-neoplastic agents. Previously, we reported that the kinase activity of MLK3 was significantly higher in triple-negative breast cancer (TNBC) than hormone receptor-positive human breast tumors where estrogen inhibits MLK3 kinase activity and provides a survival advantage to ER+ breast cancer cells. Here, we identified that in TNBC, the higher MLK3 kinase activity paradoxically promotes cell survival via activating the downstream PAK1-NF-kB axis. The MLKs/MLK3 inhibitors, CEP-1347, and URMC-099 induced cell death in TNBC but not in hormone receptor-positive breast cancer cells. The MLKs/MLK3 inhibitors reduced TNBC cell line and patient-derived xenografts’ tumor burden. The MLKs/MLK3 inhibitors also decreased MLK3, PAK1, and NF-kB protein expression and activation, prompting cell death in breast PDXs. The RNA-seq analyses indicated several genes downregulated upon MLKs/MLK3 inhibition in tumors. Significantly NGF/TrkA MAPK pathway was enriched in tumors undergoing tumor reduction by CEP-1347 and URMC-099. The tumors or cell lines that did respond to MLKs/MLK3 inhibitors had higher TrkA expression, and TrkA overexpression in the presence of MLK3 sensitized the TNBC cell line that initially did not respond to MLKs/MLK3 inhibitors. These results suggest that the functions of MLK3 in cancer cells are dependent on downstream targets/signaling, and rationalized decision needs to be made before using its inhibitor in different breast cancer subtypes. Our data also highlight that MLK3 inhibitor warrants further investigation for treating TNBC. 12 samples of 3' RNA-seq. 3 experimental groups, 4 biological replicates per group.