Dialogue between genomic instability and metabolism in diseases

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Aim 1: Investigate the synergistic relationship between genomic instability and redox homeostasis to define precision medicine. Our metabolic CRISPR screen revealed multiple genes required for survival upon ATM inhibition. In this aim, we will investigate the extent to which DNA repair deficiency affects redox homeostasis and metabolism in disease. Our preliminary findings revealed that lung (NSCLC) and breast cancer cells (TNBC) depleted for KEAP1 exhibited elevated cell death when exposed to ATM inhibitors. Ectopic expression of KEAP1 in KEAP1-mutant cells will confirm the requirement for KEAP1. These observations are also confirmed using KEAP1-knockout cells in which ATM was silenced with short hairpin RNA interference. To extend these findings, we will treat lung and breast cancer cells with two ATM inhibitors currently under clinical trials (AZD0156 and AZD1390) in combination with two FDA-approved KEAP1-Nrf2 inhibitors (dimethyl fumarate (DMF) and oltipraz) and assess cell death as well as the requirement for both ATM and KEAP1 for cell survival. We have established a collaboration with AstraZeneca (UK) to use these ATM inhibitors in mouse models. We will generate xenografts utilizing breast cancer cells (MDA-MB-231 and MCF7) lacking KEAP1 in the presence or absence of the ATM inhibitor. If a synergistic interaction is demonstrated between ATM and KEAP1-Nrf2, we will treat xenograft mouse models generated with parental cells with a combination of ATM inhibitor (AZD1390) and the KEAP1-Nrf2 inhibitor (DMF). We will further validate the effect of this drug combination on patient-derived xenografts in mice, measuring tumor burden, metastatic rate, and overall survival in each group to determine the clinical relevance of combination treatment. These results will establish whether ATM and KEAP1 comprise a previously unknown synthetic lethality. Because KEAP1 is known as a substrate for an E3 ubiquitin ligase complex and has a high affinity for Nrf2, we will examine the effect of ATM inhibition on KEAP1-Nrf2 interaction and its relevance to oxidative stress and cell survival, we will examine levels of DNA damage and DNA damage response (DDR) in cells deficient for KEAP1 upon ATM inhibition. These findings will provide insight into how ATM controls KEAP1 activity to yield resistance to therapy, possibly opening up a new understanding of the role of DNA repair and redox homeostasis in human disease. While the current data provide clear evidence for the role of KEAP1 in cell death induced by ATM inhibition, we will expand this research to determine the degree to which other hits identified in our metabolic CRISPR screen affect response to ATM inhibition. Our study will open avenues of research towards identifying new players in ATM-mediated DNA damage response and metabolism. Aim 2: Elucidate how genomic stability controls energetic metabolism using a PARylation-deficient cell model and a metabolism-centered CRISPR library. The premise from the above aim indicates that a metabolic CRISPR library could be harnessed to identify redox homeostasis genes and pathways exhibiting previously undescribed dialogues with genome repair pathways. In this aim, we utilized a similar approach to access how genomic instability controls metabolic homeostasis. DNA repair-deficient cells exhibit significant alterations in mitochondrial biogenesis genes and impaired energy metabolism due to higher levels of PARylation following DNA damage. In fact, NAD+ consumption by PARP activation upon DNA damage may lead to depletion of the NAD+ pool and subsequent metabolic reprogramming. This pathway is particularly prominent in neurological and age-related diseases in which genomic instability couples with impaired bioenergetics to promote cellular damage. Notably, concomitant deregulation of mitochondrial homeostasis and increased genomic instability during aging creates an environment prone to the development of a wide range of neurodegenerative disorders, including Alzheimer's (AD) and Parkinson's diseases (PD). For instance, we have previously demonstrated that murine cells harboring genomic instability exhibit alterations in mitochondrial energetic metabolic regulators PGC1-alpha, TFB2M, TFAM, and oxidative phosphorylation (OXPHOS) subunits. To elucidate the mechanism of energetic metabolic impairment caused by genomic instability, we have conducted a metabolic CRISPR screen of 3,000 metabolic genes (as described above) in cells exposed to the PARP1/2 inhibitor Olaparib. We are pursuing this aim by analyzing metabolic perturbations underlying mechanism of resistance to PARP inhibitors. If successful, our study will provide a comprehensive mechanism of how genomic instability leads to reprogramming of energetic metabolism, possibly opening a new understanding of some age-related human conditions such as cancer and neurological diseases.