Protein-altering point mutations are uncommon in prostate cancer. The overall and protein-altering mutation rate of primary prostate cancer is among the lowest reported, approximately an order of magnitude lower than other cancers. Consistent with this, recurrent protein-altering mutations are rare in prostate cancer. Mutations in AR, PTEN, and AKT1 are among the most common; these occur rarely in primary prostate cancer, with reported frequency around 1%. The SPOP gene (Speckle-type POZ Protein) encodes for the substrate-recognition component of a Cullin3- based E3-ubiquitin ligase. Mutations in SPOP in prostate cancer were recently reported in two systematic sequencing studies. We have identified the presence of recurrent mutations in SPOP in 6-13% of human prostate cancers in multiple independent patient cohorts (Barbieri et al., Nature Genetics 2012). Recurrent missense mutations were found exclusively in the structurally-defined substrate-binding cleft of SPOP, and structural analysis suggests that these mutations will inactivate SPOP function by disrupting SPOP-substrate interaction. Further, we found that loss of SPOP function in prostate cell lines resulted in increased invasion, and altered gene expression; evidence of this expression signature was identified in primary tumors harboring SPOP mutation. Importantly, all SPOP mutations occurred in tumors that were negative for ERG rearrangement and PTEN deletion; these tumors displayed characteristic somatic copy number aberrations. Taken together, these findings support a distinct molecular class of prostate cancer. The overall goal of this proposal is to define the role of SPOP mutations in prostate cancer and elucidate the biology of SPOP mutant prostate cancer as a distinct molecular subclass. We propose three specific Aims that together will examine the substrate specificity of SPOP mutant proteins, test the common SPOP mutations in an in vivo model of prostate cancer, identify cooperating and mutually exclusive genetic events, and examine the impact of SPOP mutation on prostate cancer patients. We will use a combination of biochemical, structural biology, in vivo and molecular biology approaches to study these events in prostate cancer models systems, while also employing large cohorts of primary and metastatic human prostate cancer samples to integrate genetic and epidemiologic analyses. Furthermore, we have assembled an outstanding team of co- investigators with complementary skills and resources to execute these proposed studies.