Ramin Shiekhattar - US grants

? DESCRIPTION (provided by applicant) Integrator is a multi-protein complex stably associated with the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Integrator contains a catalytic RNA endonuclease subunit (INTS11) with high degree of homology to cleavage and polyadenylation specificity factor 73 (CPSF73), the enzyme responsible for cleavage and termination of messenger RNA (mRNA) genes. We showed that Integrator occupies the small nuclear RNA genes (snRNA) and that its catalytic activity was required for the 3'-end processing of these non- polyadenylated transcripts. We have expanded our functional studies during the course of this grant funding to uncover a role for Integrator at protein-coding genes and distal enhancers. In the proposed studies we intend to illuminate the molecular mechanism underlying Integrator function at enhancers and their respective protein-coding promoters through the following specific aims: Aim1) We will investigate the molecular mechanisms by which Integrator regulates transcriptional elongation (RNAPII pause release) at protein-coding genes. Aim 2) We will elucidate the role of Integrator in enhancer RNA biogenesis and enhancer function. Aim 3) We will address the physical and functional association of Integrator and different phosphorylation states of the CTD in regulation of transcriptional activation.

DESCRIPTION (provided by applicant): Abstract We have recently identified a class of long ncRNA termed ncRNA-activating (ncRNA-a), which can activate the expression of neighboring protein-coding genes at long distances. Our current data suggest that that ncRNA-a function by associating with the co-activator complex, Mediator, to promote DNA looping. The association between ncRNA-a and the Mediator complex will be investigated to understand how specificity for target selection is achieved and to pinpoint the contribution of RNA to transcriptional activation. The three Aims are designed to provide a thorough molecular understanding of the activating ncRNAs and their association with the Mediator complex. Aim 1 describes experiments that examine the molecular basis for ncRNA-a nuclear localization and transcriptional activation. Aim 2 describes experiments to delineate the molecular basis of ncRNA-a and the Mediator complex interaction. We will determine whether ncRNA-a is critical for the recruitment of Mediator to its target sites and perform structure/function experiments to assess the importance of ncRNA-a/Mediator interaction in transcriptional activation. Aim 3 will assess the molecular basis by which ncRNA-a activate the CDK8 kinase activity. Importantly, recurrent mutations in Med12 subunit of Mediator complex, which is critical for kinase activity, result in development of multiple human cognitive syndromes and a benign smooth muscle neoplasm. Therefore, we will test the hypothesis that such mutations affect the kinase activity of Mediator by disrupting its association with the ncRNA-a.

Project Summary/Abstract Activating mutations in KRAS, BRAF and EGFR are the underling cause of a large number of deadly human cancers. A single missense amino acid mutation in these genes causes cancers of diverse origins including a large number of pancreatic, lung, skin, and colorectal cancers. The general signaling pathway affected by these mutations, the so-called RAS-RAF-MEK-ERK pathway (ERK signaling), is a major target of drug design by pharmaceutical companies. However, regardless of the molecular target (activated BRAF or EGFR, for example), this approach has been hindered due to the emergence of drug resistance in nearly all cases. To tackle this important human disease there is a need for new groundbreaking unconventional approach to cancer treatment. We propose to establish a potentially transformative approach to treating these cancers, which we refer to as Enhancer RNA Therapy. Indeed, we believe this plan will be applicable to a wide spectrum of human diseases. Our proposal hinges on our discovery of the functional importance of enhancer RNAs (eRNAs); noncoding RNAs that are transcribed from tissue and disease-specific enhancers. Enhancers are DNA elements that govern spatial and temporal regulation of gene expression during development and disease. Aberrant regulation of enhancers is considered to be a key event in the genesis and progression of cancer. We aim to target disease-induced eRNAs to silence cancer-causing genes as a transformative treatment in cancers induced by activating mutations of KRAS, BRAF and EGFR. We will develop an atlas of disease-induced enhancers in multiple cancers with activating mutations in KRAS and BRAF. We will then functionally characterize these cancer-induced enhancers using genetic and functional approaches. Finally, in collaboration with Isis Pharmaceuticals, we will develop derivatized anti-sense oligonucleotides (ASOs) that will neutralize the oncogenic eRNAs in vivo allowing for tissue specific inhibition of pathogenic gene expression in cancer. We therefore propose to investigate the feasibility of 'Enhancer RNA Therapy' using cancers with activating mutations in ERK signaling pathway as a model system.

PROJECT SUMMARY: CANCER EPIGENETICS RESEARCH PROGRAM The Sylvester Comprehensive Cancer Center (Sylvester) Cancer Epigenetics (CE) Research Program, established in 2014, comprises 18 investigators from seven departments. Co-led by Ramin Shiekhattar, PhD, and Maria Figueroa, MD, the program aims to incorporate basic and clinical research in the field of cancer epigenetics to pinpoint epigenetic factors that contribute to cancer risk, initiation, progression, and treatment response, taking into consideration the specific needs present within Sylvester?s catchment area. To achieve these objectives, the CE program encompasses three specific aims: 1) Elucidate the oncogenic molecular mechanisms associated with epigenetic regulators that are mutated in cancer; 2) Define the enhancer and transcriptional reprogramming that is imposed through aberrant signal transduction cascades in cancer; and 3) Validate epigenetic targets for therapeutic intervention and biomarker development in pre-clinical and clinical studies. Currently, program members receive $5.5M in annual direct peer-reviewed funding, including $2.2M from the NCI and $1.5M from other NIH Institutes. CE?s research efforts rely heavily on Sylvester?s shared resources, notably the Onco-Genomics, Flow Cytometry, and Biostatistics and Bioinformatics Shared Resources. The CE program fosters robust intra- and inter-programmatic collaborations as well as inter- institutional collaborations to leverage the depth and breadth of member expertise in basic, translational, and clinical research and develop innovative approaches to ameliorate the impact of cancer in Sylvester?s catchment area, a four-county area known as South Florida. For instance, CE works collaboratively with the Tumor Biology Research Program to understand the interplay between oncogenic signaling pathways and epigenetic deregulation in cancer, and with the Cancer Control Research Program to understand how specific environmental and socio-economic disparities impact the epigenome and influence cancer initiation and progression. These collaborative efforts are supported by a rapidly expanding portfolio of peer-reviewed funding, including an NCI Pioneer Award, multi-PI R01 grants, a recently awarded Leukemia and Lymphoma Society (LLS) Scholar Award, and an LLS Specialized Center of Research program project grant. Several investigator-initiated studies are also emerging from CE program discoveries. Furthermore, CE members have published 143 cancer-relevant papers since 2014; 24% represent intra-programmatic, 29% represent inter- programmatic, and 77% reflect multi-institutional collaborations. Some of CE?s major contributions include 1) dissecting the role of DNA methylation and hydroxymethylation in the hematological malignancies; 2) targeting histone readers, writers, and erasers in AML; and 3) defining the role of BAP1 and ASXL1 in uveal melanoma and their contribution to histone methylation and ubiquitination; and 4) discovering of enhancer RNAs (eRNAs) and targeting eRNAs in cancer to affect pathogenic gene expression and cellular growth.