PROJECT 3 – REGULATORY RNAS AS CANCER DRIVERS AND DEPENDENCIES PROJECT SUMMARY Long non-coding RNAs (lncRNAs) represent a large and relatively understudied class of RNA molecules that have great potential to provide novel opportunities to influence gene regulation and cancer biology. The long- term goal of Project 3 is to identify lncRNAs that are over-expressed in basal-like triple negative breast cancer (B/TNBC) and basal-like pancreatic ductal adenocarcinoma (B/PDAC), define their roles in the disease, and develop approaches to manipulate their expression in vivo to impact cancer progression and metastasis. MALAT1 lncRNA has been shown to be upregulated in over 20 different cancer types and high expression of MALAT1 has been shown to be associated with the propensity to metastasize, poor patient outcome, and drug resistance. In Aim 1, a series of studies are proposed to determine the role of human MALAT1 lncRNA, taking advantage of a recently established innovative patient-derived breast tumor organoid BioBank containing organoids from various tumor sub-types as well as normal breast organoids. RNA antisense purification of MALAT1 followed by mass spectrometry (RAP-MS) will be carried out to identify common and tumor-specific MALAT1-associated proteins with a specific focus on proteins involved in transcription and/or pre-mRNA splicing. To probe the role of MALAT1 in cancer progression, ASO-mediated knockdown (KD) will be carried out to assess alterations in gene expression and pre-mRNA splicing, as well as impact on tumor growth and metastasis in patient-derived organoid xenograft (PDO-X) models +/- standard-of-care drugs. In Aims 2 and 3, a series of studies are proposed to determine the function of several recently identified lncRNAs that are significantly upregulated in both B/TNBC and B/PDAC. Cellular fractionation and a CRISPR screen will identify those lncRNAs that are chromatin-associated and essential. Chromatin Isolation by RNA Purification (ChIRP) coupled to deep sequencing and RAP-MS will be used to identify genes that these lncRNAs regulate, and critical protein interactors. Candidate lncRNA function in proliferation, migration, invasion will be assessed upon ASO KD and over-expression studies. The impact of lncRNA loss on gene expression, pre-mRNA splicing, and chromatin structure, as well as therapy response, will be evaluated. PDO-X models will be used to study tumor growth and metastasis in vivo and to assess the impact of lncRNA KD on tumor progression. These studies will define the functions of several newly identified chromatin-associated lncRNAs that are over-expressed in both B/TNBC and B/PDAC, and determine their role(s) in gene expression as well as their potential as therapeutic targets. Together, the proposed studies in this Project will address the function of a set of nucleic acid therapeutic targets and nucleic acid antisense-based therapies to impact cancer progression and metastasis.