Guillermina Lozano - US grants

The p53 oncogene is present in elevated levels in many tumor cells. In tissue culture, p53 cooperates with an activated ras gene to transform rat fibroblasts. Furthermore, p53 is a necessary cellular component of SV40 T antigen mediated transformation. Recently, p53 was found to bind a cellular protein, hsc70. The role of p53, whether it be a critical factor in mediating transformation or whether it be a consequence of the transformed state and simply needed for growth, remains unclear. Therefore, in order to study the effects of overexpression of p53 and its interaction with T antigen and hsc70, several p53 constructs will be introduced into mouse embryos. One of these constructs encodes a mutant p53 cDNA with an in frame linker at amino acid 215 which no longers binds T antigen, but does bind hsc70 and has an increased transformation potential in tissue culture compared to its parent cDNA. The level of expression of p53 mRNA and protein and the tissue specificity will be determined. In addition, p53 transgenic mice will be mated with mice expressing T antigen to determine the effects of p53 gene product on the development of brain tumors in these transgenic mice. The levels of hsc70 in tissues expressing p53 and the interaction of these proteins will be examined in vivo using transgenic mice.

The p53 oncogene is present in elevated levels in many tumor cells. In tissue culture, p53 cooperates with an activated ras gene to transform rat fibroblasts. Furthermore, p53 is a necessary cellular component of SV40 T antigen mediated transformation. Recently, p53 was found to bind a cellular protein, hsc70. The role of p53, whether it be a critical factor in mediating transformation or whether it be a consequence of the transformed state and simply needed for growth, remains unclear. Therefore, in order to study the effects of overexpression of p53 and its interaction with T antigen and hsc70, several p53 constructs will be introduced into mouse embryos. One of these constructs encodes a mutant p53 cDNA with an in frame linker at amino acid 215 which no longers binds T antigen, but does bind hsc70 and has an increased transformation potential in tissue culture compared to its parent cDNA. The level of expression of p53 mRNA and protein and the tissue specificity will be determined. In addition, p53 transgenic mice will be mated with mice expressing T antigen to determine the effects of p53 gene product on the development of brain tumors in these transgenic mice. The levels of hsc70 in tissues expressing p53 and the interaction of these proteins will be examined in vivo using transgenic mice.

This proposal will determine whether cell cycle position and phosphorylation are critical determinants for activation of latent p53, and will measure the activation of its transcriptional targets. The Specific Aim include 1) addressing the physiologic relevance by measurement of phosphorylation during the cell cycle, determination of DNA damaging agents that activate p53 via phosphorylation, and determination of which targets are activated by phosphorylated p 53, 2) study of the effect of C-terminal phosphorylation and intramolecular interactions on modulation of p53 activity, 3) addressing the mechanism of transactivation by measurement of binding affinities to natural p53- binding sites on DNA and the effect of adjacent sequences and binding proteins, and 4) discrimination of cellular conditions that distinguish p53 two effect of growth arrest and apoptosis.

The main functions of this core are the detection and functional characterization of p53 mutation in sarcoma kindreds. These analyses are essential for the aims of projects 1, 4, and 5. Dr. Strong (project 1) uses the data obtained from this core to characterize excess cancer risk in kindreds of childhood sarcoma patients, to assess the relative contribution of p53 germline mutations to childhood sarcoma, and to identify paternal origin of de novo mutations. Additionally, since our discovery of non-p53 cancer prone kindreds, she will characterize cancer risk in these kindreds as well. Her ability to do these analyses is dependents of establishing which individuals carry p53 germline mutations. The data obtained by this core are also essential for Dr. Lozano's aim (project 4) to map the locus or loci responsible for cancer predisposition in non p53 cancer prone kindreds since the identification of additional families and individuals strengthens the power needed for linkage analysis. To determiner where there is a distinct quantitative microsatellite instability phenotype associated with p53 mutations (project 5, Dr. Siciliano), it is clearly essential to identify the patients with germline p53 mutations. The specific aims of this core are: 1) to sequence the p53 coding exons and flanking splice junctions from the probands of the soft tissue sarcoma and osteosarcoma cohorts to identify the presence or absence of germline p53 mutations 2) to examine those samples in which a p53 mutation is not identified by (1) above by southern analysis to exclude possible gross deletions of one of the p53 alleles 3) to examine LOH in tumors and immortalized fibroblasts from LFS patients with and without p53 mutations 4) to develop SSCP and ASO methods to screen family members upon identification of the specific p53 mutation and to screen o6ther known tumor suppressor genes in non p53 kindreds 5) to perform functional analyses of p53 mutations using the yeast functional assay and transient transfection experiments in tissue culture cells to document functional differences between mutations. The function of this core will eventually evolve to include mutational analyses of the other gene or genes that predispose to cancer (see project 4 and core E).

Mutation of p53, inherited in some individuals with Li-Fraumeni Syndrome (LFS), is a critical event in the elaboration of many tumors of diverse origin. Recent data, however, suggest that other genetic alterations also result in the cancer predisposition typical for LFS. The mapping of this locus should yield insight into other genetic events that lead to the genesis of diverse tumor. In addition, since 82% of LFS patients inheriting mutations of p53 inherit a missense mutation in p53, we have created a mouse model containing an arg to his substitution at amino acid 172 of the endogenous p53 gene. This mutation corresponds to the hot spot mutation at amino acid 175 altered in 6% of human tumors. Comparison of p53 missense and null alleles in the mouse will yield valuable insight into the in vivo differences between p53 mutants. Additional genetic events that may modify the ability of p53 to function are suggested by experiments using another mouse model (CE/J) in which heterozygosity or homozygosity at the p53 locus results in embryo lethality. This modifier of p53, mop 1, will also be mappe4d in this study.

DESCRIPTION: (adapted verbatim from the investigator's abstract) The development of mouse models containing a p53 null allele has been invaluable in deciphering the importance of the p53 tumor suppressor in tumorigenesis. However, greater than 80% of p53 alterations are misense mutations in the DNA binding domain and as such, the p53 null mouse does no represent the majority of mutations that occur. The hypothesis to be tested in this proposal is that missense mutations represent a dominant-negative or gain-of-function phenotype in vivo. We have generated a mouse containing an arg-to-his substitution in the endogenous p53 gene corresponding to one of six hotspot mutations (amino acid 175) in human tumors. This mouse also contains the deletion of a G nucleotide at the splice acceptor site of exon 3 and expresses the mutant protein at levels similar to wild type p53. Another mouse containing the arg-to-his mutation expressed at higher levels will also be made. In order to test the dominant-negative and gain-of-function nature of this mutation, we will compare the p53R172H mutations in the presence or absence of a p53 null allele for timing and spectrum of tumor development. Another mutation found in human tumors at this amino acid alters arginine 175 to proline. This mutant retains the ability to arrest growth in G1, but not to induce apoptosis when assayed in tissue culture. ES cells containing this point mutation have been identified and a mouse model will be developed. This model will be crucial in understanding the relative contribution of p53 growth arrest and apoptotic functions to tumorigenesis in vivo. The p53R172H and p53R172P mice will also be mated with mdm2 heterozygous mice to monitor effects on embryo lethality due to loss of mdm2. The possibility of delaying lethality of mdm2 null mice will afford the opportunity to examine the importance of the p53/MDM2 interaction in later development. These mouse models will yield insight into differences between p53 missense mutations in the timing and spectrum of tumor development.

DESCRIPTION (provided by applicant): The p53 tumor suppressor pathway is a complex network of signals that fluctuate to regulate cell proliferation and death. Mdm2 and Mdm4 are crucial inhibitors of p53 and are often present at high levels in tumors thus dampening p53 activity eliminating the need for p53 mutations. Mdm2 also has p53-independent functions that promote chromosomal instability. Biochemical data indicated that Mdm2 and Mdm4 also bind p73, a p53 related protein. We have identified physiologically important interactions of Mdm2 with p73 in vivo. Thus, our overarching hypothesis is that the oncogenic functions of the Mdm proteins are a combined effect of inhibiting p53 and p73 functions. Furthermore, because normal cells, unlike tumor cells, do not tolerate high levels of Mdm2, we also hypothesize that tumor-specific changes alter cell physiology to tolerate high Mdm2 levels. To test these hypotheses, we will decipher the intricate relationship between p73 and Mdm2 by studying the phenotype of Mdm2-/-p73-/- mice. In addition, we will cross tumor prone Mdm2 transgenic and SNP309 mice (with higher than normal Mdm2 levels) with p73 mice to examine effects of concomitant p73 loss on chromosomal aberrations and tumor phenotypes. In aim 2, we will determine the physiological relevance of the Mdm4/p73 interactions, and we will generate p73 heterozygous Mdm4 transgenic mice to examine the importance of this combination on a tumor phenotype. Lastly, we will identify factors that allow tumor (but not normal) cells to tolerate high Mdm2 levels using genetic screens.

DESCRIPTION (provided by applicant): The main objective of this training program in The Molecular Genetics of Cancer is to prepare predoctoral (4) and postdoctoral (5) trainees for careers in cancer research. The training program is based at the world renowned M. D. Anderson Cancer Center. Faculty are collegial and interactive, and belong to five departments, one clinical, with cross appointments in other clinical departments. The interests of the faculty include the study of tumor suppressors (p53, PTEN, WT1, Brca1), genetic modifiers of the cancer phenotype, inherited cancer syndromes, the DNA damage response, DNA recombination and repair, telomere biology, genomic instability, cell regulation, chromatin modification in cancers, and regulators of cell proliferation and apoptosis. The faculty use genetic and biochemical assays, animal models and human samples to understand the normal and abnormal mechanisms that govern cell proliferation and death. Trainees will choose a research mentor of their choice, and submit their applications to open training grant positions. If highly qualified, they will be selected and supported for a maximum of three years with annual review. Trainees will attend classes and receive instruction in the responsible conduct of research. They will attend seminars, journal clubs, and the annual program retreat. They will learn presentation and grant writing skills. They will be encouraged to attend and present at national meetings. The faculty and the interactive environment of MDACC and the Texas Medical Center will stimulate trainees to achieve a life long commitment to Making Cancer History. RELEVANCE: An estimated 565,000 Americans will die of cancer this year. The complexity and heterogeneity of the cancer phenotype, the existence of multiple pathways that control cell proliferation and death suggest that cancer is an individual disease. We have much to learn.

DESCRIPTION (provided by applicant): The p53 gene itself or other important components of the p53 pathway are altered in the genesis of most human cancers. In response to DNA damage signals or inappropriate oncogene activation, p53 levels increase and result in arrest of the cell cycle or induction of apoptosis, p53 surveillance thus prevents inappropriate DNA replication and cell division. Most studies have focused on understanding the mechanisms of apoptosis by p53 while the ability of p53 to induce the cell cycle arrest program has largely been ignored. During the previous funding period, we generated a mouse containing an arg-to-pro mutation at p53 amino acid 172, which distinguishes these pathways. Cells homozygous for the p53(515c) allele are unable to induce apoptosis, yet retain a partial cell cycle arrest pathway. Lymphomas and sarcomas develop in p53(515c) homozygous mice with much later latency than p53-null mice suggesting the importance of cell cycle arrest in tumor suppression. Importantly, tumors that develop in p53(515c)/(515c) mice remain diploid suggesting that the activities of this mutant p53 maintain genomic stability. The generation of these mice and cells from these mice will allow us to decipher the mechanism of genomic stability and the importance of this pathway in the genesis of tumors with other molecular defects. Specifically we will: 1) determine the molecular changes that cooperate with p53(515c)/(515c) in tumorigenesis; 2) examine survival and genomic stability in tumors from p53(515c)/- mice; 3) determine the importance of the cell cycle inhibitor and p53 target p21 in maintaining genomic stability and identify other targets of p53 important in arresting the cell cycle; 4) determine the ability of p53(515c)/(515c) mutant to inhibit c-myc induced tumors; and 5) determine the importance of p53(515c)/(515c) in delaying breast carcinomas in a different tumor prone strain of mice. This unique model will further our understanding of the role of p53 in cell cycle arrest and in maintaining genome stability.

Mutation of p53, inherited in some individuals with Li-Fraumeni syndrome (LFS), is a critical event in the[unreadable] elaboration of many tumors of diverse origin. Most mutations of p53 are single nucleotide changes that alter[unreadable] critical amino acids in the DNA-binding domain rather than alterations that delete the p53 gene. The[unreadable] prevalence of p53 missense mutations coupled with in vitro data has led to the hypothesis that mutant p53[unreadable] has additional properties that make it more detrimental for proper cell cycle regulation and more[unreadable] advantageous to the dividing cell. To test this hypothesis in vivo and model human LFS more accurately, we[unreadable] have generated mouse models inheriting the p53R172H mutation (corresponding to the p53R175H hotspot[unreadable] in human cancers). This mutation disrupts the conformation of p53 resulting in a protein that is tumorigenic in[unreadable] cooperation with ras, and readily immortalizes cells. Mice with the p53R172H mutation have gained[unreadable] additional tumorigenic properties and also exhibit a dominant-negative phenotype. Metastasis is rampant in[unreadable] p53R172H/+ mice as opposed to mice lacking one p53 allele. Additional experiments are needed to[unreadable] ascertain the events leading to tumorigenesis, metastasis, stability of the mutant p53, and the dominantnegative[unreadable] nature of the mutation. To examine the ability of p53R172H to cooperate with Brcal deletion,[unreadable] another common alteration that leads to breast cancer, crosses with Brca1+/- mice in a background[unreadable] sensitive to beast cancer will be performed. An important question that will be addressed in these studies is[unreadable] what other molecular changes, examined by CGH and Affymetric arrays, cooperate with mutant p53 in the[unreadable] genesis of different kinds of cancers. Lastly, since the type of p53 missense mutation may also contribute to[unreadable] different phenotypes, another p53 missense mutation will be generated in mice. The p53R245W mutation[unreadable] represents a DMA contact mutant that alters an arginine amino acid involved in direct contact to DNA.[unreadable] p53R245W is also transcriptionally inactive, but cannot immortalize cells. The p53R245W mutation in[unreadable] another hot spot mutation inherited in LFS patients. Comparison of the two classes of p53 mutants[unreadable] (conformation versus contact) should yield insights into the role of these mutants in tumorigenesis. Our[unreadable] studies suggest that treatment of a patient will have to be tailored not only to the kind of tumor that develops,[unreadable] but to the specific p53 missense mutation identified in the tumor as well.[unreadable] The research outlined in this application is directly relevant to public health in that it proposes to study a[unreadable] gene, p53, that is often altered in different kinds of cancers. The study aims to understand the nature of p53[unreadable] mutations and of other changes that cooperate with this defect. These studies may identify novel therapeutic[unreadable] targets.[unreadable]

The Genomics Facility (GF) began offering high-throughput microarray services to users in September 2001. Its goal is to provide high quality, reasonably-priced, gene-profiling technology platforms to meet the research needs of NCI-funded investigators. The GF provides investigators with access to personnel who are highly-experienced in a variety of genomic technologies as well as access to the methodologies themselves. It avoids expensive duplication of personnel, facilities and equipment required for application of these constantly-evolving methodologies. The GF provides a full range of services, from consultation on experimental design to gene expression analyses to data interpretation via close integration with the Department of Bioinformatics. The GF is housed in 700 sq. ft. of laboratory space and computer office space in the state-of-the-art Basic Science Research Building - North Campus. The GF provides DNA and RNA analysis services for several microarray applications, including gene expression, single nucleotide polymorphism (SNP), Comparative Genomic Hybridization (CGH), Chromatin Immunoprecipitation (ChIP on Chip) and microRNA profiling. Major equipment includes Affymetrix GeneChip system, Axon Instruments GenePix4000B scanner, NimbleGen Hybridization System, Affymetrix High-throughput Array Station, Genomics Solutions Flexys arrayer, Agilent BioAnalyzer, and high-throughput PCR machines. The laboratory has 3 faculty (including Director and Co-Director) and 3 support personnel. Oversight is provided by a multidisciplinary Oversight Committee of 8 senior leaders. Online access to the GF is provided via the Laboratory Information Management System (LIMS). LIMS enables users to track the status of their projects and service requests on-line. Since its launch in September 2005, 81 projects and 300 requests for services have been submitted on LIMS serving 63 principal investigators and 142 total users. Funding is proposed to come from the Cancer Center Support Grant (30%), users' fees (55%), and institutional resources (15%). During its last 5 years, the GF received 50%-65% financial support from user fees serving 102 investigators from 17 of the 21 CCSG programs. 74% of the users had peer-reviewed funding and accounted for 85% of utilization. Users of the facility have published important papers in tumor biology using genomics approaches, development of genomics technology, bioinformatics and biostatistics of genomics analysis. Future plans include development, production and implementation of new microarray platforms as requested by the investigators and expansion of services on the existing platforms including methylation assay on the NimbleGen platform and microRNA profiling on the Affymetrix platform

Project Summary While recent tumor sequencing efforts have provided a catalog of mutations that occur across many different tumor types, the challenge now is to understand which mutations directly contribute to tumorigenesis. It has also become clear that tissue and cellular context contribute a great deal to the ability of a mutation to cause cancer, making it essential to study these effects in robust and relevant model systems. Despite arising from the same organ, the two most frequent cancers of the pancreas are distinct, with different incidences and natural histories, as well as unique mutation patterns. Pancreatic ductal adenocarcinomas (PDACs) have nearly penetrant gain-of-function mutation in the KRAS oncogene, and loss-of-function mutations in the TP53 tumor suppressor gene. As these are amongst the most frequent mutations in human cancers, robust mouse models have been built for this disease. Pancreatic neuroendocrine tumors (PanNETs), on the other hand, have frequent loss of function mutations in the epigenetic regulators MEN1, DAXX and ATRX. These were the first tumors identified to have mutations in DAXX and ATRX, and as such our understanding of how these mutations contribute to cancer is limited. DAXX and ATRX interact and function as a chaperone for the histone variant H3.3, specifically loading H3.3 into heterochromatic regions of DNA, including telomeres and centromeres. This proposal aims to better understand the physiological consequences and molecular mechanism downstream of DAXX loss that contribute to tumorigenesis and to address the provocative question of why tumorigenesis in the endocrine pancreas is dramatically different than in the exocrine pancreas. Using our recently developed conditional Daxx allele in the mouse, we will use genome-wide ChIP- sequencing experiments for both Daxx and H3.3 to define the specific role of Daxx as an epigenetic regulator in the pancreas. We will also use this new allele to build a relevant mouse tumor model of Daxx mutant PanNETs. We will determine if Daxx loss alone, or in cooperation with Men1 loss (lesions that co-occur in a subset of human tumors), can promote tumorigenesis. Additionally, we will conduct a CRISPR/Cas9 screen to identify novel cooperating lesions and mechanisms in vivo. As PanNETs are rare, having a robust and faithful model become even more valuable for understanding the pathogenesis of the disease and to identify novel vulnerabilities and therapeutic targets. Combined, this work aims to uncover the potential cell-type specific differences that impact tumorigenesis in the pancreas to advance our understanding of the molecular mechanisms through which Daxx loss contributes to PanNETs and to develop novel models and reagents for future studies.

PROJECT SUMMARY/ABSTRACT This request is for upgrading an existing A1 NIKON confocal with a LUNV Laser Launch and DU-G detector unit. The A1 confocal is currently located in the BSRB Microscopy Facility, in the Genetics Department at UT-MD Anderson Cancer Center, under the leadership of Dr. Lozano. The BSRB Microscopy Facility was founded in 1998 and expanded its capabilities to cope with high demand to its current configuration. Our current A1 confocal was acquired in 2011 and it has been upgraded over the years and currently is integrated into a NIKON Ti microscope body equipped with motorized stage and CO2 stage-top incubator. Our system is composed of 7 laser lines: 405nm, 561nm and 647 nm are solid state lasers, and 453nm, 477nm, 488nm and 514nm are delivered by an Argon Ion Gas laser. Although the system is under service contract, the multi-line argon laser is no longer being supported by NIKON. A replacement option is available for the 488nm laser only with a solid state single-line laser, but since the existing laser launch only supports four lasers, we will not be able to replace the 453nm and 514nm lines, which we currently use. Thus, in order to keep our current functionality, we need to replace our existing laser launch with a new one capable of running at least 6 single- line solid state lasers. This new laser launch would also provide bright illumination with improved stability and reduced maintenance. Although our investigators are experts in good sample preparation, many of the projects involve samples that are limited in brightness for various reasons. Examples include: Dr. Behringer's group (major user) works with a variety of endogenous expressed fluorescent proteins (requiring multiple laser lines including 453nm and 514nm), that are sometimes weakly expressed, to study defects in reporter mice reproductive tract development; Dr. McCrea's group works with 3D image rendering of dendrites (about 2 mm thick) and performs co-localization analysis of two novel protein complexes PdLim5:delta catenin and Mag- 1:delta catenin within dendritic processes. He also plans to do image-based Fuorescence Resonance Energy Transfer (FRET) studies to look at the activity and spatial distribution of RhoA, Cdc42 and Rac1 (by utilizing established GTPase activity FRET biosensors) at the dendrites as part of his recent NIH R01 submission. Dr. Andrew Gladden (junior faculty) currently looks at FRET signals of established vinculin tension sensor (VinTS) at the focal adhesions, which are very small in size and restricted to the base of the cell. Dr. Galdden also does multi-color live cell imaging of three-dimensional lumen and organotypic cultures. The majority of the projects cited in this application require confocal sectioning microscopy with optimal spatial resolution, multiple excitation lines covering the 405nm-640nm spectral range (including 453nm and 514nm lasers), high depth penetration as provided by laser excitation, high detection efficiency, and improved sensitivity. Therefore the acquisition of solid-state lasers and hybrid GaAsP detectors are necessary to provide adequate excitation illumination and to improve quantum efficiency and the quality of our data.

PROJECT SUMMARY: GENETICALLY ENGINEERED MOUSE FACILITY (GEMF) The MD Anderson Genetically Engineered Mouse Facility (GEMF) provides a wide range of mouse genetic engineering services to investigators at a reasonable cost. Compensated services provided include transgenic mouse generation (using pronuclear and blastocyst injection), gene targeting (using CRISPR/Cas9, transposon-mediated transgenesis, the electroporation of embryonic stem [ES] cells, and TARGATT@ protocols), archiving of mouse embryos and sperm, generation of ES cells from investigators' own mouse lines, and rederivation of mouse lines. In addition, consultation for assessing how genetically engineered animals can contribute to PIs' projects and grant support are provided at no cost. A service unique to the GEMF is the inclusion of a Mouse Resource Facility to provide reagents (e.g., superovulation hormones, ES cell media), DNA plasmids (required for generating DNA constructs for gene targeting and pronuclear injection), and Cre, LacZ, GFP, and p53 transgenic mice (commonly used for the detection and/or generation of conditional mutations in mice and for probing tumor phenotypes). Support provided through CCSG Development Funds was essential for developing the new GEMF service of in vitro transcription of sgRNA. The facility has been in service since 1988 and was started by Dr. Guillermina Lozano, who was appointed director in 2013, and is currently managed by Dr. Jan Parker-Thornburg, the co-director, who is a recipient of an R50 award. The facility has an annual budget of $775,887 (74% for costs for highly expert personnel), $342,039 (44%) of which is supported with CCSG funding. During the present grant period, the institution invested $292,586 in funding for capital equipment, including replacement microscopes, automated injection systems, and a multi-functional electroporation system that can be used both for ES cells and for CRISPR/Cas9 embryo electroporation. Services provided have been fairly evenly distributed among investigators at MD Anderson; 155 cancer center members representing all 16 CCSG programs have used GEMF services over the past 6 years. In grant Yr42, peer-reviewed funding?supported usage accounted for 95% of all usage, and CCSG funds are requested to cover 41% of total expenses in Yr44 ($316,563). The animals and materials produced by the GEMF have contributed significantly to high-impact science at MD Anderson that has resulted in more than 201 publications, including 69 (34%) in journals with IF >10 and 146 (73%) in journals with IF >5. The GEMF's specific aims are: Aim 1. To produce the genetically engineered animal models required by MD Anderson investigators for their studies in cancer research using both traditional and state-of-the-art techniques, based on the need of the faculty. Aim 2. To provide services to MD Anderson investigators to derive and archive mouse models. Aim 3. To use investigator-produced mouse embryos for the generation of unique ES cell lines. Aim 4. To provide expertise and training in the generation, care, and handling of GEM models.

PROJECT SUMMARY Breast cancer is a deadly disease and new strategies are needed to fulfill the goals of treatment and eradication. Recent development of epigenetic-based inhibitors offers new avenues of potential therapeutics for breast and other human cancers. Our work using a new mouse model of breast cancer that parallels the aberrant expression of TRIM24 in all breast cancer sub-types will be used to gain a deeper understanding of tumor development and treatment. Epigenetic regulators are frequent targets of aberrant regulation, amplification or mutation in all human cancers. Histone ?writers?, ?erasers? and ?readers? are epigenetic regulators that catalyze addition, removal and/or interaction, respectively, with post-translational modifications (PTMs) of histones or other modified proteins, with subsequent regulatory outcomes for gene expression. Our laboratory discovered Tripartite Motif Protein 24 (TRIM24) as a histone reader and showed that TRIM24: (i) ubiquitinated p53 and siRNA-depletion of TRIM24 led to p53-dependent apoptosis of embryonic stem cells and breast cancer-derived cells (MCF7), (ii) recruited estrogen receptor to chromatin by PHD/bromodomain reading of a unique signature of histone PTMs (H3K4me0; H3K23ac) to co-regulate estrogen-dependent transcription, (iii) induced transformation of immortalized human mammary epithelial cells (iHMECs) by altering metabolism and up-regulating c-Myc expression when ectopically expressed, and (iv) a small molecule inhibitor of the TRIM24 bromodomain disrupts chromatin interactions in vitro. Importantly, we found that aberrant expression of TRIM24 negatively correlates with breast cancer patient survival. TRIM24 is over expressed in all sub-types of breast cancer and is highest in basal breast cancers. We developed a mouse model of TRIM24-expressing breast cancers by conditional over-expression of a Trim24 transgene in mammary epithelia. We saw that aberrant, tissue-specific expression of TRIM24 is sufficient for tumor initiation, development and progression to highly heterogeneous mammary carcinomas. We hypothesize that our proposed, multi-faceted studies, including mouse models, cultured cells and in vitro analyses will uncover how aberrant expression of TRIM24 drives heterogeneous tumor development in mammary/breast epithelia, and that our findings will further development of epigenetic-based therapeutics to treat breast cancers. Our long-term goal is to leverage a deep mechanistic understanding of TRIM24 functions toward innovative therapeutic approaches to treat breast and other cancers in humans.