Project Summary The concept of synthetic lethality has been validated clinically with the use of PARP inhibitors in treating breast cancers with loss-of-function mutations in BRCA1/2. Nevertheless, only 5-10% of breast tumors are caused by inherited mutations in BRCA1/2, highlighting a need to identify new synthetic lethal interactions that can be exploited clinically. In this proposal, we capitalize on our recent discovery of a new synthetic lethal interaction that is exposed by a cancer-specific genetic alteration. Centrosomes are microtubule-organizing centers that catalyze the assembly of the mitotic spindle during cell division. Centrosome duplication is tightly coupled to cell cycle progression and controlled by the master regulatory kinase (Polo-like kinase 4) PLK4. Chemical inhibition of PLK4 activity leads to cell division in the absence of centrosome duplication, producing centrosome-less cells that exhibit delayed mitotic spindle assembly. Although most cancer cells can proliferate in the absence of centrosomes, we recently discovered that inhibition of PLK4 leads to centrosome depletion that selectively triggers mitotic catastrophe in cancer cells overexpressing TRIM37. This has therapeutic relevance as the TRIM37 chromosomal locus is amplified in 50- 60% of neuroblastomas and ~10% of breast cancers. In addition, amplification of this region is associated with aggressive cancers with highly rearranged and unstable genomes. In this application, we will define how TRIM37 overexpression promotes tumorigenesis and increases the vulnerability to PLK4 inhibitors. We will also test the effectiveness of PLK4 inhibition in achieving selective killing of human cancer organoids with TRIM37 amplification. Aim 1 will determine how TRIM37 overexpression increases the sensitivity to PLK4 inhibitors and test if TRIM37-driven centrosome dysfunction contributes to tumorigenesis by promoting mitotic errors. The 17q23 amplicon contains ~30 genes, including TRIM37 and the TP53-antagonizing phosphatase PPM1D. The oncogenic properties of PPM1D overexpression have been validated in several models. Selective PPM1D inhibitors have been developed, but how PPM1D cooperates with other genes encoded in the 17q23 amplicon remains unknown. In Aim 2, we determine if PPM1D overexpression promotes TRIM37-mediated genomic instability and test if PPM1D inhibition can potentiate the action of PLK4 inhibitors in these tumors. Finally, Aim 3 will examine if TRIM37 overexpression confers increased sensitivity to PLK4 inhibition in breast and neuroblastoma tumor organoids. Understanding the role of TRIM37 in tumorigenesis will help define how its overexpression drives the development of aggressive cancers and provide a rationale for the use of PLK4i in the treatment of cancers with genomic amplification of TRIM37.