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1.
bioRxiv ; 2024 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-38746175

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges for effective treatment, with systemic chemotherapy often proving inadequate due to poor drug delivery and the tumor's immunosuppressive microenvironment. Engineered bacteria present a novel approach to target PDAC, leveraging their ability to colonize tumors and deliver therapeutic payloads. Here, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce the pore-forming Theta toxin (Nis-Theta) and evaluated its efficacy in a preclinical model of PDAC. Probiotic administration resulted in selective colonization of tumor tissue, leading to improved overall survival compared to standard chemotherapy. Moreover, this strain exhibited cytotoxic effects on both primary and distant tumor lesions while sparing normal tissues. Importantly, treatment also modulated the tumor microenvironment by increasing anti-tumor immune cell populations and reducing immunosuppressive markers. These findings demonstrate the potential of engineered probiotic bacteria as a safe and effective therapeutic approach for PDAC, offering promise for improved patient outcomes.

2.
bioRxiv ; 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-38105998

RESUMO

Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.

3.
bioRxiv ; 2023 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-36993718

RESUMO

To identify novel drivers of malignancy in pancreatic ductal adenocarcinoma (PDAC), we employed regulatory network analysis, which calculates the activity of transcription factors and other regulatory proteins based on the integrated expression of their positive and negative target genes. We generated a regulatory network for the malignant epithelial cells of human PDAC using gene expression data from a set of 197 laser capture microdissected human PDAC samples and 45 low-grade precursors, for which we had matched histopathological, clinical, and epidemiological annotation. We then identified the most highly activated and repressed regulatory proteins (e.g. master regulators or MRs) associated with four malignancy phenotypes: precursors vs. PDAC (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Integrating across these phenotypes, the top MR of PDAC malignancy was found to be BMAL2, a member of the PAS family of bHLH transcription factors. Although the canonical function of BMAL2 is linked to the circadian rhythm protein CLOCK, annotation of BMAL2 target genes highlighted a potential role in hypoxia response. We previously demonstrated that PDAC is hypovascularized and hypoperfused, and here show that PDAC from the genetically engineered KPC model exists in a state of extreme hypoxia, with a partial oxygen pressure of <1mmHg. Given the close homology of BMAL2 to HIF1ß (ARNT) and its potential to heterodimerize with HIF1A and HIF2A, we investigated whether BMAL2 plays a role in the hypoxic response of PDAC. Indeed, BMAL2 controlled numerous hypoxia response genes and could be inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its association with RAS activity. Knockout of BMAL2 in four human PDAC cell lines led to defects in growth and invasion in the setting of hypoxia. Strikingly, BMAL2 null cells failed to induce glycolysis upon exposure to severe hypoxia and this was associated with a loss of expression of the glycolytic enzyme LDHA. Moreover, HIF1A was no longer stabilized under hypoxia in BMAL2 knockout cells. By contrast, HIF2A was hyper-stabilized under hypoxia, indicating a dysregulation of hypoxia metabolism in response to BMAL2 loss. We conclude that BMAL2 is a master regulator of hypoxic metabolism in PDAC, serving as a molecular switch between the disparate metabolic roles of HIF1A- and HIF2A-dependent hypoxia responses.

4.
Nat Metab ; 4(6): 724-738, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35726024

RESUMO

Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.


Assuntos
Ácido Aspártico , Neoplasias Pancreáticas , Linhagem Celular Tumoral , Humanos , Hipóxia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética
5.
Nat Commun ; 12(1): 4860, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381026

RESUMO

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.


Assuntos
Aspartato Aminotransferase Citoplasmática/antagonistas & inibidores , Ferroptose , Neoplasias Pancreáticas/metabolismo , Animais , Antioxidantes/farmacologia , Aspartato Aminotransferase Citoplasmática/genética , Aspartato Aminotransferase Citoplasmática/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Cistina/metabolismo , Ferroptose/efeitos dos fármacos , Glutationa/biossíntese , Humanos , Ferro/metabolismo , Camundongos , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/patologia
6.
Science ; 368(6486): 85-89, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241947

RESUMO

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Cisteína/deficiência , Ferroptose , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Transportador 1 de Aminoácidos Catiônicos/genética , Linhagem Celular Tumoral , Cistationina gama-Liase/administração & dosagem , Cistationina gama-Liase/farmacologia , Cistina/metabolismo , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Deleção de Genes , Humanos , Camundongos , Camundongos Mutantes
7.
Cell ; 168(5): 878-889.e29, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28235199

RESUMO

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.


Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/química , Animais , Antineoplásicos/química , Calorimetria , Linhagem Celular , Fibroblastos/metabolismo , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transdução de Sinais , Bibliotecas de Moléculas Pequenas
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