PROJECT SUMMARY/ ABSTRACT Alterations in the PI3K pathway occur in 40-60% of ER+ breast cancer or AR+ breast cancer, representing the most common genomic alteration in such tumors, and indicating that the PI3K signaling pathway plays an important role in the tumorigenesis of hormone-dependent tumors. There is important bidirectional regulatory crosstalk between PI3K and ER or AR signaling in breast and prostate cancers respectively, leading to tumors that adapt and survive when either single pathway is pharmacologically inhibited. Mechanistically, we demonstrated that PI3K inhibition activates ER activity to drive the growth of in ER+/PIK3CA mutant tumors, through regulation of the histone methyltransferase KMT2D. KMT2D is phosphorylated by the PI3K effectors AKT1/SGK1, which inhibits its recruitment to chromatin and its role as a coactivator at ER target genes in breast cancer. Upon PI3K inhibition, this inhibitory phosphorylation is lost, allowing KMT2D to drive ER-dependent transcription. We hypothesized that KMT2D could be a common mechanism in controlling nuclear hormone receptor function, PI3K pathway crosstalk, and ER and AR luminal cell differentiation in breast and prostate models respectively. Preliminary data have shown that KMT2D is required for ER and AR transcriptional activity upon PI3K inhibition in breast and prostate cancers respectively. Furthermore, KMT2D loss sensitizes cancer cells to PI3K/AKT inhibition in cells, tumors, and patient derived organoids. We now aim to characterize the epigenetic and transcriptional role of KMT2D as a key modulator of AR/ER nuclear receptor activity in cells and organoids using bulk epigenomic and single cell sequencing (Aim 1). We have also identified the lysine methyltransferase SMYD2 as a novel level of regulator of KMT2D and ER/AR activity. We now plan to elucidate the role of SMYD2-catalyzed-mediated methylation on KMT2D activity and cofactor associations in breast and prostate cancer models (Aim 2). Additional preliminary data demonstrate that SMYD2 loss can sensitize tumors further to PI3K/AKT inhibition. To this end, we aim to determine the role that the genetic manipulation or pharmacological inhibition of SMYD2 has in the therapeutic response to PI3K/AKT inhibitors in breast and prostate cancer (Aim 3). Altogether, this proposal is benefiting from i) a multidisciplinary team of collaborators who are experts in breast and prostate cancer research, protein methylation, and epigenetics, ii) unique patient resources and reagents, iii) robust preliminary data propelled by at least of 7 years momentum as a leader in the field of nuclear receptor regulation which will be critical to design new and improved therapies for hormone- dependent tumors.