Cruz, Leilani, Heidt, Analeah and Rowell, Emily (2017) Postdoc fellowship application for the Damon Runyon Cancer Research Fellowship entitled "Identifying novel pathways responsible for epigenetic modifications in aged regulatory T cells in the tumor microenvironment". Damon Runyon Cancer Research Fellowship.

Abstract

Included in the this OAK submission are the required fellowship application documents. In addition to a research proposal, letters of collaboration, a training plan and biosketches for GNF investigators are included. Summary of the research proposal is as follows:

The process of immunoscenence leads to weaken adaptive immunity. Regulatory T cells (Tregs) are crucial members of the adaptive response, whose main purpose is to maintain immunological tolerance and restrain effector T cell activity. During ageing, numbers of nTreg cells are increased as well as altered immunological response. Many functional studies indicate that epigenetic changes have a huge influence on the ageing process as the epigenetic landscape greatly changes in response to exogenous and endogenous factors. The immune system is imminent for the detection and elimination of malignant cells, through immune surveillance. However, immune evasion can occur with recruitment of immunosuppressive Tregs at the tumor microenvironment (TME). As ageing is a dominant risk factor for developing cancer, it is critical to understand the connection between mechanisms of ageing and the role of tumor-infiltrating Tregs in anti-tumor response through potential epigenetic reprogramming.
Firstly, we will survey the altered epigenetic landscape of young (6-8 weeks) and aged (50-80 weeks) Tregs isolated from healthy and tumor-bearing C57/BL6 mice through Assay for Transpose-Accessible Chromatin with high-throughput sequencing (ATAC)-seq by analyzing the open chromatin regions crossed referenced with published ChIP-seq and our internal generated RNA-seq analysis. We also address whether iTregs also contain similar age-related changes in epigenetic modulation. Isolated naïve T cells will be polarized towards an iTreg fate, followed by ATAC-seq. Additional response metrics such as tumor growth rate measurements, analysis of tumor infiltrating Treg cell activation, proliferation, cytokine production, presence of anti-tumor antigen specific T cells, and in response to checkpoint inhibition will allow us to functional follow up genomic mechanisms and cross reference our analysis to human data.
Secondly, we will identify novel genes and transcriptional regulatory elements specific in aged Treg cell function by exploring possible mechanisms through the addition of small molecule inhibitors as well as specific CRISPR-cas9 targeting of enzymatic proteins that modulate epigenetic architecture in Tregs. Utilizing a GNF internal library of epigenetic modifying compounds spanning multiple enzyme classes we will screen available compounds followed by examination of biological changes in the Treg cell. Lastly, we will develop a pooled CRISPR-Cas9 to identify and target specific enzymes to avoid non-selective effects of small molecule inhibitors. We will transduce naïve CD4+ T cells from young and old Foxp3GFP reporter mice with defined pools of guide RNA (gRNA) against enzymatic proteins that have the ability to catalyze the addition or removal of end groups on specific gene loci necessary for functional Treg conversion. Measurement of GFP frequency will dictate total Treg cell conversion by flow cytometry.
Our research aims to identify the underlying epigenetic mechanisms that control Treg suppressive function, conversion into iTregs and more specifically, the impact of ageing on these processes. Taken together, our findings will greatly contribute to the development of small-peptide immunotherapy targeting tumor-infiltrating Tregs in treating cancer patients.

Item Type:	Article
Date Deposited:	26 Apr 2017 00:45
Last Modified:	26 Apr 2017 00:45
URI:	https://oak.novartis.com/id/eprint/32319