Glutamine antagonist DRP-104 suppresses tumor growth and enhances response to checkpoint blockade in KEAP1 mutant lung cancer
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE237592 | GEO | 2024/04/03
REPOSITORIES: GEO
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Project description:Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a pro-survival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a previous study of KEAP1 mutations observed in lung cancer, we classified 40% of the mutations as ‘superbinders’ (superbinders). These mutants bind and ubiquitylate NRF2 but do not promote NRF2 degradation. Here, we further investigated the molecular mechanism(s) driving the superbinder phenotype. BioID-based quantitative proteomic analysis of the R320Q and R470C superbinder mutations revealed increased co-complexed NRF2 without significant alteration to other KEAP1-associated proteins, including CUL3, VCP, and several ubiquitin receptors within the proteasome lid. Dynamic simulation modeling and limited proteolysis analyses suggest that superbinder mutations stabilize residues in KEAP1 that contact NRF2. In cells, KEAP1 R320Q and R470C mutants co-localize with NRF2, p62/SQSTM1 and polyubiquitin in spherical clusters that rapidly fuse and dissolve; KEAP1-NRF2 localization to these clusters requires p62. Expression of R320Q and R470C in lung cancer cells provided resistance to the reactive oxygen species-inducing drug bleomycin. We present a model wherein superbinder mutations alter the conformational dynamics of the KEAP1-NRF2 complex to alter the cycling of KEAP1 between open and closed conformations, thus inhibiting NRF2 degradation.
2018-11-07 | PXD008411 | Pride
Project description:Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the antioxidant program downstream from the nuclear factor, erythroid 2-like 2 (NRF2) transcription factor and correlates with poor prognosis1–3. We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis4. To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a Keap1-mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation5, as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.
2020-04-13 | GSE145945 | GEO
Project description:Immune cells were isolated from the lungs of mice bearing KEAP1 mutant or wildtype tumors. We found that in KEAP1 mutant tumors there is a reduction in CD103 DCs and early activated T cells as well as an increase in exhausted CD8 T cells in comparison to WT controls.
2023-11-27 | GSE241481 | GEO
Project description:Immune cells were isolated from the lungs of mice bearing KEAP1 mutant or wildtype tumors. We found that in KEAP1 mutant tumors there is a reduction in CD103 DCs and early activated T cells as well as an increase in exhausted CD8 T cells in comparison to WT controls.
2023-11-27 | GSE241480 | GEO
Project description:Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer. A factor that contributes to the poor prognosis of the disease is the complex tumor microenvironment (TME). The PDAC TME is composed of excessive fibrosis and desmoplasia, which creates a harsh environment resulting in hypoxia and altered nutrient availability. To promote survival and proliferation in this environment, PDAC cells can reprogram glutamine (Gln) metabolism. Previous studies have demonstrated that PDAC cells use Gln to support proliferation and redox balance. However, earlier attempts to inhibit Gln metabolism using glutaminase inhibitors resulted in rapid metabolic reprogramming and ultimately therapeutic resistance. Here, we hypothesized that using a Gln analogue, such as 6-Diazo-5-oxo-L-norleucine (DON), could broadly target Gln metabolism in PDAC and prevent rapid adaptation. Indeed, DON treatment led to a significant decrease in PDAC proliferation in a dose-dependent manner, as well as a profound reduction of various metabolites involved in central carbon and nucleotide metabolism, suggesting that DON creates a metabolic crisis. In addition, we observed a significant decrease in tumor growth in various in vivo models (syngeneic, immunodeficient) using DRP-104 (sirpiglenastat), a novel pro-drug version of DON that was designed to circumvent DON associated GI toxicity and allow the therapeutic exploration of broad Gln antagonism. Mechanistically, we found that ERK signaling is increased as a compensatory mechanism through the increased activity of receptor tyrosine kinases (RTKs). Combinatorial treatment of DRP-104 and Trametinib (MEK1/2) inhibitor led to a significant increase in survival in a syngeneic model PDAC. Taken together, these pre-clinical results suggest that broadly targeting Gln metabolism could provide a new therapeutic avenue for PDAC and that the combination with an ERK signaling pathway inhibitor could further improve the therapeutic outcome.
2023-08-01 | GSE236225 | GEO
Project description:O-GlcNAcylation is an essential, nutrient-sensitive post-translational modification, but its biochemical and phenotypic effects remain incompletely understood. To address this knowledge gap, we investigated the global transcriptional response to perturbations in O-GlcNAcylation. Unexpectedly, many transcriptional effects of O-GlcNAc transferase (OGT) inhibition were due to the activation of NRF2, the master regulator of redox stress tolerance. Moreover, we found that a signature of low OGT activity strongly correlates with NRF2 activation in multiple tumor expression datasets. Guided by this information, we identified KEAP1 (also known as KLHL19), the primary negative regulator of NRF2, as a direct substrate of OGT. We show that O-GlcNAcylation of KEAP1 at serine 104 is required for the efficient ubiquitination and degradation of NRF2. Interestingly, O-GlcNAc levels and NRF2 activation co-vary in response to glucose fluctuations, indicating that KEAP1 O-GlcNAcylation links nutrient sensing to downstream stress resistance. Our results reveal a novel regulatory connection between nutrient-sensitive glycosylation and NRF2 signaling, and provide a blueprint for future approaches to discover functionally important O-GlcNAcylation events on other KLHL family proteins in various experimental and disease contexts.
2017-06-26 | GSE81740 | GEO
Project description:To compare hepatic gene expression in conditional Keap1 knockout (Alb-Cre:Keap1(flox/-)) and genetic control mice. Disruption of Keap1-mediated repression of Nrf2 signaling was expected to result in increased expression of Nrf2-regulated genes. Keywords: comparative expression profiling
2008-05-01 | GSE11287 | GEO
Project description:104 early-stage (IA and IB) non-small cell lung cancers were evaluated with the Illumina Human HT-12 v4 BeadChip.
2015-06-01 | E-MTAB-3448 | biostudies-arrayexpress
Project description:We analyzed the esophagus of Keap1 conditional knockout mice. In these mice, there were both Keap1-normal and Keap1-deleted cells in the esophageal epithelium. To compare the gene expression of these cells, we conducted single-cell RNA-seq for the mice esophagus.
2023-02-19 | GSE197558 | GEO
Project description:Considerable advances have been made in lung cancer therapies, but there is still an unmet clinical need to improve survival for lung cancer patients. Immunotherapies have improved survival, although only 20-30% of patients respond to these treatments. Interestingly, cancers with mutations in KEAP1, the negative regulator of the NRF2 cytoprotective pathway, are resistant to immune checkpoint inhibition and correlate with decreased immune cell infiltration. NRF2 is known for promoting an anti-inflammatory phenotype when activated in immune cells, but the study of NRF2 activation in cancer cells has not been adequately assessed. The objective of this study was to determine how lung cancer cells with constitutive NRF2 activity interact with the immune microenvironment to promote cancer progression. To assess this, we generated CRISPR-edited mouse lung cancer cell lines by knocking out KEAP1 or NFE2L2 genes. We also utilized publicly available single cell data through the Gene Expression Omnibus to investigate tumor/immune cell interactions. We show here that KEAP1-mutant cancers promote immunosuppression of the tumor microenvironment. Our data suggests KEAP1-deletion is sufficient to alter the secretion of cytokines, increase expression of immune checkpoint markers on cancer cells, and alter recruitment and differential polarization of immunosuppressive macrophages that ultimately lead to T cell suppression.
2024-03-20 | GSE250166 | GEO