Targeting the cancer neo-genome for destruction with CRISPR-Cas enzymes

项目总结/文摘

Project Summary/Abstract Advances in understanding the cancer genome have led to the development of novel therapeutics that target distinct alterations in protein products of cancer DNA. The resulting targeted therapies, together with protein-targeting immunotherapies, have led to significant advances in cancer patient survival. However, not all patients benefit from these new therapies and instead many cancers continue to be treated with DNA double- strand break (DSB)-generating radio- and chemo- therapies. Nucleic acid targeting approaches such as the CRISPR-Cas9 system now enable investigation of a new modality for experimental cancer therapeutics: targeting DNA directly rather than its protein products. This proposal is aimed at the initial investigation and evaluation of this modality. Hypothesis: Targeted induction of DNA DSBs at multiple rearrangement junctions, specific to the cancer genome, can lead to cancer-cell specific cytotoxicity and spare damage to healthy tissue. Specific Aim 1. Characterize the landscape of cancer-specific Cas9-targetable genomic sites across cancers and cancer cell lines. In this aim, a computational pipeline will be developed to characterize the spectrum of Cas9-targetable genome alterations across the Cancer Cell Line Encyclopedia and Pan-Cancer Analysis of Whole Genomes databases of over 300 cancer cell lines and 2500 cancer genomes. Sequence features that may affect target effectiveness in cancer therapy will be characterized, including target clonality, target copy number, and the distribution of targetable genomic alterations across cancer types. This pipeline will become a publicly available tool to generate lists of Cas9-targetable rearrangement breakpoints for use in future studies. Aim 2. Develop methods to induce cytotoxicity in cancer cells using Cas9 targeted to cancer-specific DNA rearrangement breakpoints. In a series of proof-of-principle experiments, this aim will assess whether Cas9 can induce targeted cell death through targeting DNA breaks to either single highly amplified sites or several unique sites in the cancer neo-genome and whether this generates toxicity in healthy tissue. Aim 3. Identify protein factors responsible for resistance or sensitivity to Cas9-mediated DNA damage: This aim will comprise a CRISPR knockout screen to discover proteins that affect cell sensitivity to multiple Cas9- induced DNA breaks. This proposal will provide training in cancer genomics and targeted therapeutics under the guidance of Professor Matthew Meyerson at Dana-Farber Cancer Institute, who is experienced in both fields. In addition to experimental and computational research, the applicant will present results at conferences, train future scientists, and interact with colleagues and mentors in the cancer genomics, genome editing, and cancer therapeutics fields. The planned research is intended to culminate in publication in peer-reviewed journals. Ultimately, this training should prepare the applicant for a career as research faculty at a United States research institution.