Mats Ljungman - US grants

It has become increasingly clear in recent years that induction of apoptosis is of great importance for the success of radiation-and chemotherapy. However, many tumor cells acquire resistance to the induction of apoptosis by up-regulation of the apoptosis antagonists Bcl-2 and Bcl-XL. Thus, an important target for therapeutic intervention would be to inactivate or lower the cellular levels of Bcl-2 and Bcl-XL. We recently reported that the short chain fatty acid butyrate can lower the cellular level of Bcl-XL in human fibroblasts and human breast cancer cells. Furthermore, we showed that butyrate acting in synergy with radiation, UV light and cisplatin to induce apoptosis. The goal of this proposal is to explore whether butyrate may act in synergy with radiation, chemotherapy or hormone withdrawal to induce prostate cancer cells to undergo apoptosis. In specific aim #1 we will address whether butyrate may act in synergy with ionizing radiation to preferentially induce apoptosis in prostate cancer cells compared to normal prostate cells. Preliminary results suggest that the prostate cancer cell line PC-3 is affected by butyrate at concentrations about ten-fold than what affected normal human fibroblasts. In addition, we show that butyrate significantly reduces the cellular level of Bcl-XL in PC-3 cells. In specific aim #2 we will explore whether butyrate may act in synergy with chemotherapy agents to induce apoptosis in prostate cancer cancers. Finally, butyrate will in specific aim #3 be used in combination with hormone withdrawal to explore whether butyrate act in synergy with hormone withdrawal to induce prostate cells to undergo apoptosis. These studies will define whether butyrate may be use in combination with traditional anti-cancer protocols for the treatment of prostate cancer.

We have recently shown that blockage of RNA polymerase II is sufficient to cause the induction of p53 in mammalian cells and may be a common mechanism by which p53 is stabilized following various cellular stresses. The cellular proteins MDM2 and JNK are known to bind p53 and stimulate p53 degradation. Recent studies show that both the and C-terminal domains of p53 are required for efficient degradation of p53. We have obtained preliminary results suggesting that ser315 of p53 is important in the regulation of p53 stability. Firstly, phosphorylation of ser315 enhanced the degradation of p53 in vitro. Secondly, inhibition of the ser315 kinases Cdc2 and Cdk2 via inhibition of the CDK-activating kinase (CAK), caused a stabilization of p53 in cells. Thirdly, we have found a phosphatase, hCDC14, which binds p53 and specifically dephosphorylates the ser315 site of p53 in vitro. In this proposal, we will further explore the potential role of ser315 phosphorylation in the regulation of p53 degradation and how blockage of RNA polymerase II may attenuate ser315 phosphorylation. In specific aim number 1, we will utilize a cell-free extract system established in the lab to study the degradation of p53 in vitro. Recombinant wild-type p53 will be phosphorylated specifically on ser315 and its stability in cell extracts will be assessed. Preliminary results suggest that phosphorylation of ser315 enhances degradation in vitro. We will also assess the stability of recombinant p53 mutated at the 315 site, or containing deletions of the C- or N-terminal domains. In specific aim number 2, we will investigate the role of the Cdk- activating kinase (CAK), Cdk2 and Cdc2 in the regulation of p53 stability. CAK is a component of the RNA polymerase II holoenzyme, and as such, it may be a sensor for RNA polymerase II elongation. We hypothesize that under normal conditions CAK will activate Cdc2 and Cdk2 which in turn will phosphorylate p53 on ser315. Following inhibition of RNA polymerase II elongation, the activity of CAK is attenuated leading to increased stability of p53 by abrogation of ser315 phosphorylation of p53. We will use dominant negative CAK, Cdc2 and Cdk2 to specifically inhibit these enzymes in cells and study whether inhibition of these kinases causes an increased stability of p53. Finally, in specific aim number 3, we will explore the role of the novel human p53 phosphatase, hCDC14, in the regulation of p53 stability. Preliminary studies have shown that hCDC14 binds p53 and specifically dephosphorylates ser315 of p53. We will express wild-type or mutant CDC14 in MCF7 cells and study what effect it will have on basal and induced levels of p53.

DESCRIPTION (provided by applicant) The 11th Annual Midwest DNA Repair Symposium (AMDRS) will take place at the campus of the University of Michigan in Ann Arbor, Michigan, May 16-17, 2009. This symposia series started in Ann Arbor in 1999 with the intention of bringing together scientists and students from the Midwest region on a yearly basis to present and discuss cutting edge DNA repair research. DNA repair is a fundamental cellular process critical to the maintenance of genome integrity and human health whose dysfunction has been linked with many inherited and acquired diseases, especially cancer. The investigators are pleased that this symposia series has been very successful and that it has shown significant growth in the last three years. One key to the success of these symposia is that the investigators have been able to keep the costs low. This has been possible because the symposia have been organized by individuals at their home institutions and that each year funding has been secured to cover some of the key expenses. In times when researchers have restricted funding, the AMDRS has always been a meeting to which principal investigators have been able to take their graduate and postdoctoral fellows for exciting interactions and for the opportunity to present their work. By once again organizing this symposium in Ann Arbor, they signal the start of a second 10-year cycle of these symposia. The three main goals of the AMDRS, to be continued at the 11th AMDRS are: (1) To provide a venue for scientists, principally from Midwestern States, to present and discuss the current research in the field of DNA damage, repair and mutagenesis, and to provide opportunities to foster scientific collaborations. (2) To provide students, postdoctoral fellows, and beginning independent investigators the opportunity to p resent their work to peers and to interact with leaders in the field. (3) To provide an exceptional value of these symposia by keeping them affordable.

DESCRIPTION (provided by applicant): We recently reported that ATDC (also known as TRIM29) is highly expressed in pancreatic cancers and that it exhibits oncogenic properties by promoting cell proliferation via the WNT/b-catenin pathway (Cancer Cell 15:207, 2009). Our preliminary results also show that ATDC protects pancreatic cancer cells against radiation and chemotherapy. The properties of growth promotion and resistance to therapy suggest that ATDC is an important therapeutic target. To better understand the function of ATDC and its role in resistance, we explored what proteins ATDC interacts with before and/or after exposure to ionizing radiation. Using immunoprecipitation assays we found that ATDC interacts with poly(ADP-ribose)polymerase1 (PARP1) and that this interaction is substantially increased following exposure to ionizing radiation. To investigate whether this interaction is dependent on the catalytic activity of PARP, we treated cells with PARP inhibitors and found surprisingly that this treatment lead to a marked loss of the cellular protein levels of ATDC. Furthermore, knockdown of Parp1 expression by siRNA resulted in reduced levels of ATDC indicating that PARP1 stimulates the expression of ATDC in pancreatic cancer cells. Importantly, our preliminary results suggest that PARP inhibition results in reduced proliferation and increased sensitivity to ionizing radiation of pancreatic cancer cells. We hypothesize that ATDC is a promising novel therapeutic target in pancreatic cancer because its inactivation may lead to both reduced tumor growth and sensitization to radiation therapy. Furthermore, we hypothesize that PARP inhibitors will target ATDC and selectively inhibit growth and sensitize ATDC-expressing pancreatic cancer tumors. PUBLIC HEALTH RELEVANCE: Pancreatic cancer has the worst outcome of any cancer and kills more than 30,000 people in the US annually. We recently found that the protein ATDC is expressed at abnormally high levels in pancreatic cancer and this overexpression promotes the growth and resistance of pancreatic tumors. Unexpectedly, we found that a class of chemotherapeutic drugs currently in clinical use reduces ATDC levels in pancreatic cancer cells and make them more sensitive to radiation. In this R21 proposal we will explore the mechanisms of how these drugs reduce ATDC levels and whether they will target ATDC in pancreatic tumors in laboratory mice.

DESCRIPTION (provided by applicant): The development of innovative high throughput genome-wide methodologies and technologies such as RNA-Seq and ChIP-Seq have made it possible to identify and analyze all coding and non-coding RNAs as well as map epigenetic marks and transcription factor binding sites. However, RNA-Seq is typically performed using total RNA isolated from cells or tissues and such analysis will generate signatures of steady-state levels of RNA but will not inform on whether particular gene expression alterations are due to altered transcription and/or altered RNA stability. Recently, a number of different approaches have been developed to estimate nascent transcription of genomic sequences such as GRO-Seq, NET-Seq and by metabolic labeling and isolated of nascent RNA. We will in this R01 application further develop and validate two approaches that we believe add important new capabilities to existing techniques for the comprehensive and high throughput exploration of gene expression signatures in human cells. BruChase-Seq is based on bromouridine pulse-chase labeling coupled to deep sequencing directly measuring the kinetics of synthesis and degradation of all primary and mature mRNAs and non-coding RNAs as well as determines splicing kinetics of all intron sequences. BrUV-Seq introduces random transcription-blocking lesions by UV light prior to BrU-labeling, identifying transcription start sites (TSS), putative enhancer elements and by stabilizing transcripts that might be labile if completely transcribed allowing their more sensitive detection. Each of these advances has the potential to greatly expand current genomic annotations by assigning additional functional assessments to known genes, showing how the parts of genes act independently, and identifying cryptic intergenic elements.

DESCRIPTION (provided by applicant): The Environmental Mutagen Society (EMS) is the primary scientific society fostering research on the basic mechanisms of DNA repair, mutagenesis and environmental epigenetics, and application of this knowledge to understanding human health effects from exposure to environmental toxins. Studies of DNA repair, mutagenesis and epigenetics by EMS scientists are integrated with research on inherited and acquired genetic alterations that predispose individuals to cancer, premature aging, and other diseases. Fundamental understanding of the mechanisms and consequences of cellular, tissue, whole organism, and human population responses to genotoxic agents is crucial to informed regulatory decision-making with respect to environmental health hazards. The integration of multi-disciplinary basic and applied research is essential to this process. Such integration remains the central focus of the EMS, and the annual meetings are key to the dissemination of information and fostering the necessary interactions. In addition, the meetings provide an important forum for students and new and early stage investigators to present their research and to interact with leading scientists in their field in a setting that encourages open exchange o ideas. The 43rd Annual Meeting of the EMS will be held in Bellevue, Washington from September 8-12, 2012. The theme is EMS: The Next Generation referring to emerging next generation scientific areas and technologies in our field as well as enticing students and young investigators to become the next generation of EMS members. The scientific program encompasses the full range of scientific interests within EMS and is comprised of symposia, workshops, plenary lectures, and poster and platform sessions. EMS Annual Meetings bring together academic, industrial and governmental scientists interested in how environmental factors lead to genotoxic and epigenetic outcomes. The dual objectives of this conference grant application are to request funds for Travel Awards for students and new and early stage investigators to attend the Annual Meeting and partial support for travel expenses of keynote and symposia speakers. Travel Awards assist in offsetting the financial burden that might otherwise prevent students and new investigators from attending. Support to defray travel costs for invited non-member speakers enables EMS to provide the greatest value at lower costs to attendees. The inclusion of non-member speakers provides the cross fertilization of research and ideas which is necessary for a world-class meeting. EMS member speakers cover their expenses for participation in the conference, thus expenses for their participation are not requested in this application. PUBLIC HEALTH RELEVANCE: EMS is a multifaceted Society whose members have expertise in multiple disciplines covering all aspects of basic and applied research on mechanisms and consequences of responses to genotoxic agents. Integration of these disciplines remains the central focus of the EMS, and the Annual Meetings are keys to the dissemination of information and to foster interactions between researchers and individuals who must make informed regulatory decisions with respect to environmental health hazards. The meetings also provide a forum in which students, postdoctoral fellows, and early career investigators can share information with established scientists through formal scientific sessions and informal interactions.

DESCRIPTION (provided by applicant): Exposure of cells to ionizing radiation (IR) activates DNA damage responses leading to a comprehensive reprogramming of gene expression by regulation of both transcriptional and post- transcriptional events. The mechanisms regulating transcriptional and post-transcriptional events are not fully elucidated and a better knowledge about these mechanisms could help improve radiation therapy in the clinic. We will in this revised R21 grant proposal use the newly developed BrU-Seq technique to study these mechanisms in detail. In Specific Aim #1, the global effects of IR on the synthesis and stability of all RNAs in human fibroblasts will be explored. Preliminary experiments show that the stress kinase ATM is required for increased synthesis and stability of certain mRNAs such as BTG2 in human fibroblasts and the hypothesis to be tested is that there may be many more genes affected at the level of synthesis and/or stability in an ATM-dependent way following exposure to IR. In Specific Aim #2, the effects of IR on alternative splicing will be interrogated. Preliminry results show that intron retention is common among processed mRNAs in human fibroblasts and our hypothesis is that since a large number of splicing factors are substrates for activated ATM, IR may affect the splicing code signature in human cells in an ATM-dependent manner. In Specific Aim #3 the effects of IR on the activation of transcription start sites (TSS) and enhancer elements in the genome will be studied using a technique involving UV-irradiation prior to the BrU pulse-labeling to introduce random transcription-blocking lesions in the genome. Preliminary results show that with this technique, all transcription start sites and potentially enhancer elements can be mapped in the genome. The hypothesis to be tested is that IR may affect the selection of TSS and enhancer elements and that ATM may be involved in regulating these alterations. The major innovation of this R21 proposal is the use of the novel BrU-Seq technique for global exploration of how IR affects the transcriptome, the RNA stabilome, the splicing code and selection of TSS and enhancer elements. The analysis will not only include mRNAs but also non-coding RNAs such as microRNAs. These studies may have a great impact on not only our understanding of the mechanisms of altered gene and microRNA regulation following exposure to IR but also, this new technique will have general applications for studying mechanisms of gene expression in other settings.

DESCRIPTION (provided by applicant): The Environmental Mutagen Society (EMS) is the primary scientific society fostering research on the basic mechanisms of DNA repair, mutagenesis, and environmental epigenetics and application of this knowledge to understanding human health effects from exposure to environmental toxins. Studies of DNA repair, mutagenesis, and epigenetics by EMS scientists are integrated with research on inherited and acquired genetic alterations that predispose individuals to cancer, premature aging, and other diseases. Fundamental understanding of the mechanisms and consequences of cellular, tissue, whole organism, and human population responses to genotoxic agents is crucial to informed regulatory decision-making with respect to environmental health hazards. The integration of multi-disciplinary basic and applied research is essential to this process. Such integration remains the central focus of the EMS, and the annual meetings are keys to the dissemination of information and fostering the necessary interactions. In addition, the meetings provide an important forum for students and new/early stage investigators to present their research and to interact with leading scientists in their field in a setting that encourages open exchange of ideas The 44th Annual Meeting will be held in Monterey, California from September 21-25, 2013. The theme is Embracing the Science of the Future through Cross-Disciplinary Research, encompassing areas of current research that converge on the issues of environmental exposure and how cells and organisms respond to such challenges. The scientific program encompasses the full range of scientific interests within the Society and is comprised of symposia, workshops, plenary lectures, and poster sessions. EMS Annual Meetings bring together academic, industrial, and governmental scientists interested in how environmental factors lead to genotoxic and epigenetic outcomes. The dual objectives of this conference grant application are to request funds for Travel Awards for students and new and early stage investigators to attend the Annual Meeting and partial support for travel expenses of nonmember keynote and symposia speakers. Travel Awards assist in offsetting the financial burden that might otherwise prevent students and new/early stage investigators from attending. Support to defray travel costs for invited nonmember speakers enables the Society to provide the greatest value at lower costs to attendees. The inclusion of nonmember speakers provides the cross-fertilization of research and ideas which is necessary for a world-class meeting. EMS member speakers cover their expenses for participation in the conference, thus expenses for their participation are not requested in this application.

? DESCRIPTION (provided by applicant): Travel Awards for 4th Asian Conference on Environmental Mutagens Abstract We request funding to support travel awards for 10 students and early career investigators to attend the 4th Asian Conference on Environmental Mutagens (ACEM) to be held December 10- 12, 2014, in Kolkata, India, organized by organized by the CSIR-Indian Institute of Chemical Biology. The ACEM are held every two to three years in different Asian countries, bringing together about 300 scientists from academia, industry, and government interested in basic mechanisms of environmental mutagens and their impact on human health. The International Association of Environmental Mutagenesis and Genomics Societies (IAEMGS) is submitting this grant as it coordinates the activities of the environmental mutagenesis sister organizations worldwide. World renowned invited speakers have been selected for keynote addresses for the meeting, themed Gene-Environment Interplay in Human Health. The symposium, platform, and poster presentations will represent cutting edge research in the field of environmental mutagenesis and genomics, including cutting edge research in the basic science of DNA damage and repair, cellular stress responses, epigenetics, transcription, replication, cell cycle arrest, and apoptosis. Furthermore, the influence of the environment on the aging process and the induction of cancer and other diseases will be thoroughly covered with discussions on how mutagenic agents can be better identified and detected, what risk to human health they pose, how the environmental levels of these agents could be best regulated and controlled, and how to best harmonize regulatory decisions globally. An awards committee selected by the organizers representing different geographical and scientific areas will select 10 graduate students and emerging independent investigators on the merits of their submitted abstracts and other supporting materials. This R13 grant funding will increase the number of travel awards that can be made and will have career defining impact. The early career scientists who will be selected for awards will most likely be among the leaders in our field in the near future.

ABSTRACT The ENCODE project has provided a tremendous resource for scientists with a treasure trove of data from a large set of different cell types describing RNA expression (RNA-seq), epigenetic signatures (ChIP-seq), chromatin structure (DNase-seq, ATAC-seq, Hi-C, ChIA-PET) and binding patterns of specific proteins to both DNA and RNA (ChIP-seq, CLIP-seq, RIP-seq). We have recently developed a set of techniques that are based on the specific labeling of nascent RNA with bromouridine (Bru) followed by lysis, capturing of the Bru-labeled RNA using specific antibodies and deep sequencing. Bru-seq captures the nascent RNA transcriptome, a signature of ongoing transcription in the genome where the relative rates of transcription of all genes can be obtained. In BruChase-seq, cells are labeled with Bru and then chased in uridine for different periods of time to allow for the determination of the relative stability of all transcripts are assessed. Finally, BruUV-seq allows for the capturing of RNA species that normally are rapidly turned over by the RNA exosome and can be used to map active enhancer elements producing eRNA genome-wide. In this UM1 grant application we are proposing to use these three Bru-seq techniques to obtain critical novel information on the nascent RNA transcriptome, RNA stabilome and active enhancome that will complement and enrich existing ENCODE data. We are proposing to focus our mapping efforts on two specific Aims: Aim 1: Obtain nascent RNA transcriptome, RNA stabilome and active enhancome data for human and mouse ENCODE cell lines. Aim 2: Obtain dynamic signatures of nascent transcription, RNA stability and activity of enhancer elements genome-wide following cellular exposure to environmental perturbations. The mapping efforts proposed in this UM1 grant application are seeking to identify novel candidate regulatory elements genome-wide that will complement and extend existing ENCODE data. Furthermore, dynamic studies will be initiated to assess the activities of regulatory elements during selected cell transitions and specific cellular responses. GOALS: The main goal of this proposal is to generate comprehensive novel data sets using our newly developed Bru-seq analysis platform that will complement and extend existing ENCODE data. The emphasis will be on mapping candidate functional elements in the human genome.

PROJECT SUMMARY/ABSTRACT (EXPERIMENTAL IRRADIATION) The Experimental Irradiation (EI) Shared Resource (SR) provides access to state-of-the-art equipment and expert consultation for University of Michigan Comprehensive Cancer Center (UMCCC) members undertaking radiation therapy studies in mouse models and cell lines. First established in 1991, this SR has been approved and funded continuously by the NCI CCSG since its inception. At the last CCSG renewal, the EI SR received a merit rating of ?Outstanding.? The services of this SR are critical to members interested in radiation biology and radiation therapy, as well as those for whom radiation serves as a research tool.

The RNA exosome is a RNA degradation machinery that plays a critical role in RNA quality control by degrading erroneous or incorrectly spliced RNAs. In many cancers, such as pancreatic cancer, the splicing process is compromised, resulting in the generation of large amounts of aberrant transcripts. We hypothesize that cancer cells are uniquely dependent on RNA exosome activity to remove excess amounts of aberrant transcripts and that the RNA exosome therefore may be an impactful new target for therapeutic intervention for pancreatic cancer. We have developed a set of first-in-class small molecule inhibitors of the RNA exosome and will in this proposal refine these compounds and assess their tumor- targeting efficacy for pancreatic cancer in three specific Aims. In Aim 1 a lead optimization campaign will be initiated of our first-in-class RNA exosome inhibitors. The refined compounds will be evaluated for their RNA exosome-targeting activities using both in vitro and cell-based RNA exosome activity assays and we will identify their cellular targets. In Aim 2, the potencies of the 10 top RNA exosome inhibitors will be assessed by MTT and clonogenic survival assays to select the top three compounds for in vivo studies. These top three compounds will then be used in combination with either ionizing radiation or gemcitabine to assess synergy. Inducible shRNA and auxin-inducible protein degradation technologies will be used to inactivate the RNA exosome and these systems will be compared with the top three inhibitors to explore fundamental mechanisms of action of the RNA exosome in transcriptional and post-transcriptional events. In Aim 3, we will test the 3 most promising compounds in a PDX orthotopic model for pancreatic cancer either alone or in combination regimens with gemcitabine and nab-paclitaxel. This is a very innovative proposal exploring a novel cancer therapeutic target and highly focused on the development of new impactful treatments for pancreatic cancer. Upon the successful completion of this grant period, we intend to perform IND-enabling studies of the best candidate compounds.

ABSTRACT Chromosome rearrangements are common in cancers and typically occur early in carcinogenesis. They drive tumor development by altering the expression or function of oncogenes and tumor suppressor genes by copy number alterations, formation of oncogenic fusions or by alterations of DNA elements responsible for regulating cancer genes. Despite being a well-known feature of cancer, discovered over hundred years ago, this common cancer hallmark has not yet been therapeutically exploited. Now, for the first time, technological advancements in whole genome sequencing and the development of CRISPR technologies for precision targeting makes it possible to identify and target chromosome rearrangement junctions (CRJs) in tumor cells. In addition, recent breakthroughs in in vivo delivery of CRIPR reagents involving nanoparticles will open new avenues into clinical utilization. In this R21 application, we present a precision CRISPR approach that aims at targeting cancer-specific CRJs as an untapped Achilles heel common to all tumors. This CRISPR-based precision approach consists of pairs of CRJ-targeting guide RNAs (gRNAs) that bring together two parts of a dCas9-conjugated endonuclease, Fok1, leading to its activation and the induction of lethal DNA double strand breaks (DSBs) specifically in cancer cells. Regardless of the tumor-driving mechanism, the presence of CRJs will be exploited to selectively force these cancer cells to die. We have obtained proof-of-principle of the successful targeting of CRJs with this approach in both colon and bladder cancer cell lines. We have assembled a multi- disciplinary team with expertise in CRISPR technology and design, lipid nanoparticle delivery systems and bladder cancer mouse models. With this team in place and with strong evidence of efficient targeting of CRJs with Fok1-dCas9 in cancer cell lines, we are now primed to develop and test our approach in pre-clinical cancer models using recently developed breakthrough lipid nanoparticle delivery technologies. If successfully implemented, this paradigm-shifting precision- targeting cancer therapeutic platform could be transformative in designing uniform treatments for all cancer types irrespective of the biology of the tumor.