Pilot Projects Awarded

As part of its charter, the Chicago Region Physical Sciences Oncology Center offers pilot project funding for up to two projects each year to researchers for innovative projects that are closely aligned with the central research mission of the Center. Selected projects are funded for one year up to $100,000 direct costs.

Chromatin-dependent Nuclear Dynamics
Principal Investigator: Jason Brickner, PhD

The spatial organization of the genome reflects and, in some cases, contributes to genome function. My group has shown that the spatial positioning of genes within the nucleus and their clustering together plays a critical role in both promoting transcription and in facilitating epigenetic poising. These roles are conserved between budding yeast and humans and factors involved are critical drivers of several types of leukemia.

Using the yeast system, we have recently developed methods for monitoring and quantifying the biophysical behavior of individual genes in living cells and we find that gene positioning to the nuclear pore complex is associated with dramatic change in the spatial constraint imposed on the sub-diffusive behavior of these genes. This change in the anomalous diffusion is a measure of the effects of both interchromosomal interactions and interactions between genes and the nuclear pore, providing a way to quantify the contributions of each. Using yeast molecular genetics, we can dissect each of these effects to better understand the contributions of mRNA production, interaction with the NPC and interchromosomal clustering on the anomalous diffusion of individual genes in a living nucleus. The proposed work will use both conditional inactivation and conditional recruitment of critical chromatin regulators to perturb local and global chromatin structure to assess the role of chromatin structure in regulating the biophysical behavior of genes in the nucleus.

Project Dates:  May 1, 2018 – April 30, 2019

Define 3D Chromatin Reorganization that Predisposes to Genomic Instability in
T-cell Leukemia
Principal Investigator: Fotini Gounari, PhD

Genome instability results from the illegitimate repair of DNA breaks and is linked to oncogenesis and therapy resistance. Genotoxic stress, as well as the biological processes of transcription and replication combined with chromosome looping, results in DNA breaks. Distinct DNA repair mechanisms are activated in a cell cycle- dependent manner to resolve specific types of breaks. Preserving genome integrity requires the precise coordination of the intertwined processes of transcription, replication and chromosome looping with DNA repair, however, we know little about the regulating mechanisms. We have generated a mouse model of T-cell leukemia that allows us to study epigenetic processes, including chromosome looping events, involved in promoting genomic instability. In this model, stabilizing β-catenin in thymocytes (CAT) causes genomically unstable leukemia that strictly depends on the presence of Tcf-1, the DNA binding partner of β-catenin. CAT leukemias have multiple chromosome defects including recurrent translocations of the T-cell receptor alpha (Tcra) to the Myc-Pvt1 locus resulting from the illegitimate repair of two distinct types of DNA double-strand breaks (DSBs). The breaks in the Tcra site are catalyzed by Rag at the G0/G1 phase of the cell cycle however Myc/Pvt1 is a fragile site (FS) that sustains Rag-independent DSBs that are temporally separated from the Tcra breaks. Chromosome defects frequently occur at FSs that regularly sustain DSBs and their location depends on the local chromatin conformation. β-Catenin activation likely modifies the local chromatin conformation as a result of dramatically increasing H3 acetylation around Tcf-1 binding sites. Acetylation mediated conformation changes are likely facilitated by the understudied property of Tcf-1 to bend the DNA. Such conformation changes may impact the distribution of FSs. On this basis, we hypothesize that aberrant β-catenin, in coordination with Tcf- 1, modifies local chromosome looping and the distribution of FSs and thereby predisposes cells to genomic instability and transformation. Thus, we propose to determine the cooperative effect of aberrant β-catenin and Tcf-1 on chromatin conformation (Aim 1) and on the distribution of FSs (Aim 2). Findings from the proposed studies are expected to promote our understanding on the mechanisms underlying genomic instability.

Project Dates:  May 1, 2018 – April 30, 2019

Defining the Molecular Consequences of Isochromosomes on Chromosome Segregation and Gene Transcription
Principal Investigators: Jaehyuk Choi, MD, PhD; Daniel Foltz, PhD

The goal of this project is to define the effects of a chromosomal rearrangement that is highly recurrent in cancer on centromeres, centromere chromatin, and gene expression. Isochromosomes are present in ~10% of cancers. Isochromosomes are a result of p arm deletions followed by p-arm fusions resulting in a symmetrical chromosome that has two q-arms, two centromeres, and two shortened p-arms. Despite their high prevalence in cancer, our knowledge of these chromosomes is limited due to technological limitations. Because of their stereotypical chromosomal rearrangements, isochromosomes present unique challenges to the cell. Due to the presence of two centromeres, stable retention of isochromosomes requires either unified activity of the two centromeres or inactivation of one of the two centromeres. Furthermore, the stereotypical rearrangements in isochromosomes by their nature will impact chromatin and gene transcription. There will be losses, gains, and rearrangements of gene loci, enhancers, insulators, and promoters. Furthermore, the impact of an inactivated or unified centromere on transcription of the remaining p-arm elements remains unclear. Interestingly, we found isochromosomes in 85% of cutaneous T cell lymphomas (CTCL), a cancer of the mature skin-homing CD4+ T cell. This is a much higher rate than previously reported for any other cancer. The high prevalence of isochromosomes in this disease makes CTCL the perfect model system to study the effects of isochromosomes on chromosome segregation and gene transcription. In addition, new technologies utilized in this study make it possible to individually sequence a single iso17q chromosome. Successful completion of this proposal will elucidate the effect of linear rearrangements on 3-D chromatin structure and centromere biology. While our experiments will be focused on CTCL, we believe our findings will be of general importance to cancer biology.

Project Dates:  May 1, 2017 – April 30, 2018


Identification of Chromatin Regulators that Contribute to Abnormal Nuclear Shape in Cancer
Principal Investigator: Tanmay Lele, PhD

Nuclear shapes are abnormal in a large number of cancers, and become progressively abnormal during tumor progression. Changes in the nucleus occur early in cancer development and nuclear surface abnormalities correlate with clinical prognosis. The nucleus of the cancer cell has been used for decades as a prognostic/diagnostic marker for grading many types of cancers including breast cancer, prostate cancer and lung cancer. Yet, we know little about how the nucleus becomes abnormal in cancer, and whether these abnormalities drive cancer progression. The field has primarily focused on cytoskeletal forces exerted on the nucleus through the LINC complex on the ‘soft’ nucleus (as a result of depletion of lamin proteins) as an explanation for abnormal cancer nuclear shapes. However, intranuclear forces, generated for example from chromatin remodeling, are also likely to be important but have not been studied. Here we will infect GFP- laminA expressing MCF10A cells with a library of guide RNAs to disrupt chromatin regulators Cas9. We will use Brg1, a chromatin-remodeling enzyme and lamin A/C as a positive control. We will screen for eccentrically shaped nuclei or nuclei with irregular shapes with a high throughput, high content imaging scanner at 40 X resolution. This study will identify candidates that are frequently altered in cancer which will be further evaluated for mechanistic studies in the future. An understanding of these mechanisms may help develop new and more targeted therapies. This pilot project will identify chromatin regulators that cause abnormal nuclear shapes upon knockdown.

Project Dates:  May 1, 2017 – April 30, 2018


Cytokine-Induced Transcriptional Memory
Principal Investigator: Jason Brickner, PhD

My lab has discovered a conserved epigenetic mechanism by which the experiences of cells can be “remembered”, leading to a change in gene expression in the future. Using yeast and human cells, we have defined the molecular mechanism that controls this phenomenon, called transcriptional memory. This work identified a number of proteins that are both essential for transcriptional memory and implicated in driving oncogenesis. However, the scope and physiological significance of transcriptional memory in mammals remains unknown. Here, we propose to test the hypothesis that IL-1β, a pro-inflammatory cytokine that increases with aging, induces transcriptional memory through the mechanism we have defined. Using cutting edge technologies, we will quantify gene expression and map the genome-wide distribution of chromatin changes and protein binding before, during and following treatment with IL-1β. This will provide the first test of this hypothesis and may provide important new insights into the interplay between aging, inflammation and cancer.

Project Dates: May 1, 2016 – April 30, 2017


Characterizing The Oncogenic Role Of The Insulator Protein Ctcf In T Cell Leukemia
Principal Investigator: Panagiotis Ntziachristos

Acute lymphoblastic leukemia (ALL) is a highly aggressive blood cancer afflicting 6000 new pediatric and adult cases in the United States annually. While many patients are cured with chemotherapy, subsets of high risk patients have poor outcomes, and acute and late toxicities are often debilitating. Overall up to 25% pediatric ALL patients fail frontline treatment and face dismal prognosis. We have generated cumulative evidence suggesting that ALL is an “epigenetic” disease, linked to mutations of numerous members of the epigenetic machinery (e.g. chromatin remodelers, histone modulators and enzymes that affect DNA methylation) or their binding sites onto the genome. A lot of the genetic changes are located in the noncoding parts of the genome, areas that have been largely understudied especially as the scientific community is focused on gene mutations and generation of new chimeric proteins affecting the coding part of the genome. We hypothesize that genetic engineering of the non-coding part of the cancer genome can be a new therapeutic strategy through altering the binding of  transcription factors and their associated chromatin modifying partners and disrupting oncogene-specific nuclear architecture and transcriptional circuitries. This application addresses this key question: What is the functional connection and the critical elements between the non-coding genome, aberrant 3D organization in the nucleus and leukemia initiation and progression? We propose to characterize and delete critical binding sites of a specific chromatin protein, the insulator factor CTCF, in the genome in my effort to answer these questions. We will also determine the effect of repressive histone modifications and DNA methylation on chromatin structure, especially as oncogenes, like NOTCH1, antagonize heterochromatin to activate their targets. Findings from these studies are pertinent to other proteins, genetic alterations and types of cancer as the mechanisms employed by oncogenes are universal. Moreover, we will generate state of the art tools and experimental platforms that can benefit the cancer community.

Project Dates: May 1, 2016 – April 30, 2017


Dynamic Nucleosome Landscape During Epithelial-Mesenchymal Transition
Principal Investigators: Xiazhong Alec Wang, PhD; Ji-Ping Wang, PhD

Epithelial-mesenchymal transition (EMT) plays a critical role in carcinoma metastasis. Emerging evidence shows that EMT is regulated by epigenetic mechanisms such as DNA methylation and histone modifications. However, nucleosome positioning, a key aspect of the epigenetic landscape, has not been examined during EMT. We have recently developed a chemical biology approach to determine genome-wide nucleosome positions in mammalian cells. Thus far, this chemical mapping strategy has enabled us to reveal new features of nucleosome organization in embryonic stem cells. Here we propose to use the newly established chemical mapping strategy to investigate dynamic changes of nucleosome positions in a mouse mammary epithelial cell line following EMT induction. Delineating dynamic nucleosome positioning during EMT is an essential step to better understand the role of epigenetic regulation in metastasis.

Project Dates: May 1, 2016 – April 30, 2017


3D Chromatin Reorganization during Prostate Cancer Progression
Principal Investigator: Debabrata Chakravarti, PhD
Collaborator: Vadim Backman, PhD

Androgen and its receptor, AR, which is a member of the human nuclear receptor (NRs) superfamily of transcription factors, play critical roles in hormone (androgen) sensitive prostate cancer (PC) and castrate resistant prostate cancers (CRPC) that kill thousands of people world-wide. Moreover anti-androgen resistance of CRPC is a major problem in therapeutic intervention of prostate cancer. We posit that a better understanding of NR signaling pathway and its integration in epigenomic signaling and chromatin reorganization are keys for development of future understanding and therapeutics of this deadly disease. Higher order chromatin organization is critical for gene expression and other chromatin-based processes and its misregulation contributes to multitude of human diseases including cancer. 4D-nucleome analysis being developed in the laboratory of Dr. Vadim Backman, the collaborator of the PI Dr. Chakravarti, will allow us to visualize and quantify higher order chromatin structure in nanoscale in 3D and more importantly dynamically in live prostate cancer cells. We hypothesize that hormone sensitive, castrate resistant, and anti-androgen-resistant prostate cancer cells display distinct 3D-chromatin organization and propose to develop key signatures that will allow differentiation of prostate cancer based on chromatin reorganization at various stages of cancer progression, hormone sensitivity, and therapy resistant. Results from this pilot project will be used to expand the concept in other hormone-responsive cancers (breast, ovary). The results of the project will also enable us to develop submission of a multi-PI R01/P01 application. This proposed work uniquely combines extensive expertise of Dr. Chakravarti in cancer biology, epigenomics and transcription (Cancer biologist) and Dr. Backman in developing cutting edge biophysical tools (Physical scientist) to probe nuclear/chromatin dynamics in living cells representing various stages of prostate cancer progression. We hope that the reviewers will find that this proposal is highly relevant to PSOC mission as it uses cutting edge physical sciences technologies to advance knowledge in oncogenesis and cancer progression.

Project Dates:  October 1, 2015 – September 20, 2016


Quantitative Analysis of Temporal Histone Modification Changes Underlying Leukemogenesis
Investigator: Alex Ruthenburg, PhD

This project will perform rigorous analytical chemistry on cancer cell chromatin, with the goal of quantifying key hallmarks of chromatin organization, histone posttranslational modifications, for the first time in in leukemia with locus precision. My lab has pioneered the “analytical chemical biology” of using barcoded semisynthetic nucleosomes to calibrate chromatin immunoprecipitation (ChIP), a central technique of the chromatin field, on an absolute scale. This internal-standard calibrated (ICeChIP) technology resolves many of the most severe drawbacks in ChIP that render it qualitative, flakey, and difficult to compare amongst experiments. More importantly, ICeChIP affords direct quantification of histone modification and variant amounts revealing an unprecedented level of sub-nucleosomal chromatin structure can be queried on a biologically meaningful scale. For instance, we can measure the average symmetry of histone marks within high-occupancy nucleosomes (whether both copies or one copy of a histone bears a given mark). Although there has been a good deal of conventional ChIP produced from cancer cells, such absolute measurements with this level of architectural detail have never been made in any cancer cell. In Aim 1 we propose to use our powerful new method to understand and quantify the progression of leukemias at the chromatin-level where histone modification changes are clearly causal. Specifically, we will perform an ICeChIP timecourse on MLL fusion transduced mouse bone marrow hematopoietic progenitors (in collaboration with Greg Wang’s lab at UNC) to track and quantify the distributions and changed in several of these marks as a function of time. This more basic science question has the potential for almost immediate diagnostic impact, as there are a number of cancer therapeutic drugs that target histone modifications already in the clinic with many more in the late stages of the development pipeline. The second aim will use ICeChIP-seq, to quantify several important chromatin hallmarks of leukemia that are notionally targeted by therapeutics to in patient cells to directly measure their efficacy where it is likely to matter most—at a small set of loci. This aim will also measure active enhancer function demarcated by chromatin signatures of active enhancers as a proxy for 3D-architectural changes amply addressed in other PSOC projects that provide complementary information.

Project Dates:  October 1, 2015 – September 20, 2016