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Targeting Pancreatic Cancer Metabolic Dependencies through Glutamine Antagonism

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE236225 | GEO | 2023/08/01

REPOSITORIES: GEO

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Project description:Glutamine (Gln) metabolism has been reported to play an essential role in cancer. However, a comprehensive analysis of its role in lung adenocarcinoma is still unavailable. This study established a novel system of quantification of Gln metabolism to predict the prognosis and immunotherapy efficacy in lung cancer. Further, the Gln metabolism in tumor microenvironment (TME) was characterized and the Gln metabolism-related genes were identified for targeted therapy. We comprehensively evaluated the patterns of Gln metabolism in 513 patients diagnosed with lung adenocarcinoma (LUAD) based on 73 Gln metabolism-related genes. Based on differentially expressed genes (DEGs), a risk model was constructed using Cox regression and Lasso regression analysis. The prognostic efficacy of the model was validated using an individual LUAD cohort form Shandong Provincial Hospital, an integrated LUAD cohort from GEO and pan-cancer cohorts from TCGA databases. Five independent immunotherapy cohorts were used to validate the model performance in predicting immunotherapy efficacy. Next, a series of single-cell sequencing analyses were used to characterize Gln metabolism in TME. Finally, single-cell sequencing analysis, transcriptome sequencing, and a series of in vitro experiments were used to explore the role of EPHB2 in LUAD. Patients with LUAD were eventually divided into low- and high-risk groups. Patients in low-risk group were characterized by low levels of Gln metabolism, survival advantage, “hot” immune phenotype and benefit from immunotherapy. Compared with other cells, tumor cells in TME exhibited the most active Gln metabolism. Among immune cells, tumor-infiltrating T cells exhibited the most active levels of Gln metabolism, especially CD8 T cell exhaustion and Treg suppression. EPHB2, a key gene in the model, was shown to promote LUAD cell proliferation, invasion and migration, and regulated the Gln metabolic pathway. Finally, we found that EPHB2 was highly expressed in macrophages, especially M2 macrophages. It may be involved in the M2 polarization of macrophages and mediate the negative regulation of M2 macrophages in NK cells. This study revealed that the Gln metabolism-based model played a significant role in predicting prognosis and immunotherapy efficacy in lung cancer. We further characterized the Gln metabolism of TME and investigated the Gln metabolism-related gene EPHB2 to provide a theoretical framework for anti-tumor strategy targeting Gln metabolism.

Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.

Project description:Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers with no targeted or personalized therapeutics. PDAC cells reprogram their transcriptional and metabolic state to sustain their energetic needs in the nutrient-poor tumor microenvironment. Identifying molecular regulators of PDAC growth is a critical step towards an improved understanding of the disease and new therapeutic approaches. We employed in vivo CRISPR screening in an orthotopic PDAC model to identify transcription factors (TFs) and chromatin regulators (CRs) whose inhibition promotes aggressive growth of PDAC cells in vivo. The screening identified a previously uncharacterized ISL LIM homeobox 2 (ISL2) gene as a candidate tumor suppressor among some anticipated growth modulators. We confirmed that depletion of ISL2 leads to enhanced cell proliferation and tumor growth in vitro and in vivo. Conversely, the exogenous expression of ISL2 or CRISPR-mediated epigenetic upregulation of the endogenous loci led to reduced cell proliferation, supporting the hypothesis that ISL2 is a candidate tumor suppressor. Importantly, we find that ISL2 is a nuclear, chromatin-associated transcription factor that is epigenetically silenced through DNA methylation in a significant fraction of PDAC tumors. Critically, higher DNA methylation of ISL2 or its reduced expression correlates with poor patient survival. Mechanistically, ISL2 binds to thousands of genes, and its depletion increases antioxidant capacity, oxidative phosphorylation (OXPHOS) and fatty acid metabolism. Critically, ISL2 depleted cells have heightened stemness potential, and we find PPAR as a critical modulator of cell proliferation in ISL2 depleted cells. Notably, ISL2 depleted cells are sensitive to small molecule inhibitors of mitochondrial complex I in vitro and in vivo. Collectively, these findings nominate ISL2 as a putative tumor suppressor candidate whose inactivation leads to an aggressive PDAC growth through increased mitochondrial metabolism and antioxidant capacity, which creates a potentially exploitable therapeutic vulnerability.

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Targeting Pancreatic Cancer Metabolic Dependencies through Glutamine Antagonism

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