PROX1-dependent epigenetic regulation of phenotypic plasticity during colorectal cancer metastasis

PROJECT SUMMARY Metastasis is the leading cause of solid tumor-associated death, yet our understanding of how metastasis initiating cells acquire the capacity to and colonize distant organs has remained limited, hampering development of effective treatment strategies. Large genome sequencing efforts have failed to identify metastasis-specific driver mutations in colorectal cancer (CRC), suggesting that non-genetic phenotypic plasticity is a unique feature of metastasis. We have developed an u biospecimen platform of surgically resected patient normal colon, primary CRC and metastatic tumors from 31 patients, profiled with single cell RNA-sequencing (scRNA-seq) and processed for organoid derivation for functional studies. We show that metastatic evolution across patients is characterized by reprogramming into a conserved fetal intestinal state, followed by lineage transdifferentiation into non-canonical squamous and neuroendocrine cell states that are associated with therapy resistance in patients. My preliminary data demonstrate PROX1, a transcriptional co- repressor upregulated in the fetal intestinal state, is an enforcer of intestinal lineage identity in primary tumor organoids, and its functional loss de-represses non-canonical programs during metastasis. Furthermore, PROX1 has been described to interact with subunits of nucleosome remodeling and deacetylase (NuRD) complex (LSD1 and HDAC1/2) in primary CRC to repress gene programs through epigenetic mechanisms; however, this level epigenetic regulation has yet to be explored in CRC metastasis. I hypothesize that PROX1 constrains intestinal lineage identity in CRC through repressive epigenetic mechanisms through the NuRD complex, while PROX1 dysregulation in metastasis licenses non-canonical lineage expression. I have successfully generated matched organoid pairs modified to downregulate or overexpress PROX1 to assess how PROX1 perturbation differentially alters the transcriptional and epigenetic landscape of primary and metastasis-derived organoids. In Aim 1, I will leverage these matched organoid pairs to assess how PROX1 perturbation differentially alters the transcriptional landscape of primary and metastasis-derived organoids in vitro and in vivo through mouse orthotopic intrahepatic xenograft assays. In Aim 2, I will determine the PROX1- dependent epigenetic mechanisms that restrict non-canonical differentiation and enforce intestinal lineage identity through PROX1 and NurD subunit ChIP-seq studies and chromatin landscape characterization with PROX1 perturbation. I will validate NuRD subunit interaction essentiality in enforcing a transcriptional repression through pharmacologic inhibition studies in the context of PROX1-proficient organoids. Addressing these gaps in our knowledge is of significant clinical importance will aid our understanding of metastasis to inform the development of novel treatment strategies. The work and training plan outlined in this proposal will be completed in the laboratory of Dr. Karuna Ganesh with the co-advisement of Dr. Scott Lowe at Memorial Sloan Kettering Cancer Center and will prepare me for a career as an independent physician-scientist.