PROJECT SUMMARY Platinum drugs such as cisplatin serve as a mainstay of cancer therapy, but resistance limits their curative potential. Through a kinome-wide RNAi screen, we identified microtubule-associated serine/threonine kinase 1 (MAST1) as a main cisplatin resistance driver in human cancers and identified lestaurtinib as a promising MAST1 inhibitor. MAST1 was found to reactivate the MEK1 pathway, conferring cisplatin resistance. However, we do not know how this pivotal factor, MAST1, is activated, expressed, and associated with oncogenic mutations. Through a proteomics study and mutational analysis, we identified aurora kinase B (AURKB) as an upstream kinase that phosphorylates MAST1 at S1258, critical for MAST1 activation. Our transcription factor study revealed that the transcription factor CEBPb is responsible for MAST1 gene expression, and wildtype p53 inhibits the activity of CEBPb to induce MAST1. In line with this observation, TCGA analysis revealed that MAST1 expression is high in lung cancer patients with mutant p53. These data suggest that AURKB and p53/CEBPb may regulate MAST1 in transcription-independent and -dependent manners. Recently, cancer has been indicated as a metabolic disease and altered unique metabolic properties of cancer cells make cancer metabolism an attractive target in treating cancers. However, the link between MAST1 and cellular metabolism has never been explored. Through real-time cell metabolic analysis and metabolomic profiling, we found that MAST1 elevates glucose uptake and controls redox metabolism when cells are exposed to cisplatin. We noticed that MAST1 is involved in the gene induction of glucose transporter 1 (GLUT1) and peroxiredoxin 2 (PRX2). Furthermore, MAST1 directly phosphorylates c-myc at S62 and may stabilize c-myc, the potential transcription regulator of GLUT1 and PRX2. These preliminary data suggest that MAST1 may play a critical role in the metabolic reprogramming of cancer cells. To further benefit from the MAST1 therapy, we performed a kinase inhibitor synergy screen and identified the combination of lestaurtinib and ponatinib as an optimal combinatorial strategy to overcome cisplatin resistance. Moreover, lestaurtinib with BAY-876 (GLUT1 inhibitor) synergistically enhanced cisplatin sensitivity in various cancer cells. Our central hypothesis is that MAST1 modulated by AURKB and CEBPb provides metabolic advantages for cancer cells to confer cisplatin resistance; MAST1 signaling axis linked to AURKB/CEBPb and GLUT1/PRX2 represents a promising cisplatin-sensitizing target. Three specific aims are proposed: (1) To determine how MAST1 is regulated by AURKB and p53/CEBPb; (2) To decipher the mechanism of how MAST1-c-myc-GLUT1 and PRX2 signaling confers metabolic advantages; (3) To evaluate the optimal MAST1-based combinatorial strategy in treating cisplatin-resistant cancers.