RESUMO
Various lines of investigation support a signaling interphase shared by receptor tyrosine kinases and the DNA damage response. However, the underlying network nodes and their contribution to the maintenance of DNA integrity remain unknown. We explored MET-related metabolic pathways in which interruption compromises proper resolution of DNA damage. Discovery metabolomics combined with transcriptomics identified changes in pathways relevant to DNA repair following MET inhibition (METi). METi by tepotinib was associated with the formation of γH2AX foci and with significant alterations in major metabolic circuits such as glycolysis, gluconeogenesis, and purine, pyrimidine, amino acid, and lipid metabolism. 5'-Phosphoribosyl-N-formylglycinamide, a de novo purine synthesis pathway metabolite, was consistently decreased in in vitro and in vivo MET-dependent models, and METi-related depletion of dNTPs was observed. METi instigated the downregulation of critical purine synthesis enzymes including phosphoribosylglycinamide formyltransferase, which catalyzes 5'-phosphoribosyl-N-formylglycinamide synthesis. Genes encoding these enzymes are regulated through E2F1, whose levels decrease upon METi in MET-driven cells and xenografts. Transient E2F1 overexpression prevented dNTP depletion and the concomitant METi-associated DNA damage in MET-driven cells. We conclude that DNA damage following METi results from dNTP reduction via downregulation of E2F1 and a consequent decline of de novo purine synthesis. SIGNIFICANCE: Maintenance of genome stability prevents disease and affiliates with growth factor receptor tyrosine kinases. We identified de novo purine synthesis as a pathway in which key enzymatic players are regulated through MET receptor and whose depletion via MET targeting explains MET inhibition-associated formation of DNA double-strand breaks. The mechanistic importance of MET inhibition-dependent E2F1 downregulation for interference with DNA integrity has translational implications for MET-targeting-based treatment of malignancies.
Assuntos
Dano ao DNA , Fator de Transcrição E2F1 , Proteínas Proto-Oncogênicas c-met , Purinas , Dano ao DNA/efeitos dos fármacos , Purinas/biossíntese , Purinas/metabolismo , Animais , Camundongos , Humanos , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F1/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Reparo do DNA/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Transdução de Sinais/efeitos dos fármacosRESUMO
The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability.
Assuntos
Proteína Quinase Ativada por DNA , Proteínas Serina-Treonina Quinases , Humanos , Proteínas de Ciclo Celular/genética , DNA/metabolismo , Dano ao DNA , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Mitose/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
PURPOSE: Oncogene addiction provides important therapeutic opportunities for precision oncology treatment strategies. To date the cellular circuitries associated with driving oncoproteins, which eventually establish the phenotypic manifestation of oncogene addiction, remain largely unexplored. Data suggest the DNA damage response (DDR) as a central signaling network that intersects with pathways associated with deregulated addicting oncoproteins with kinase activity in cancer cells. EXPERIMENTAL: DESIGN: We employed a targeted mass spectrometry approach to systematically explore alterations in 116 phosphosites related to oncogene signaling and its intersection with the DDR following inhibition of the addicting oncogene alone or in combination with irradiation in MET-, EGFR-, ALK- or BRAF (V600)-positive cancer models. An NSCLC tissue pipeline combining patient-derived xenografts (PDXs) and ex vivo patient organotypic cultures has been established for treatment responsiveness assessment. RESULTS: We identified an 'oncogene addiction phosphorylation signature' (OAPS) consisting of 8 protein phosphorylations (ACLY S455, IF4B S422, IF4G1 S1231, LIMA1 S490, MYCN S62, NCBP1 S22, P3C2A S259 and TERF2 S365) that are significantly suppressed upon targeted oncogene inhibition solely in addicted cell line models and patient tissues. We show that the OAPS is present in patient tissues and the OAPS-derived score strongly correlates with the ex vivo responses to targeted treatments. CONCLUSIONS: We propose a score derived from OAPS as a quantitative measure to evaluate oncogene addiction of cancer cell samples. This work underlines the importance of protein phosphorylation assessment for patient stratification in precision oncology and corresponding identification of tumor subtypes sensitive to inhibition of a particular oncogene.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Vício Oncogênico , Medicina de Precisão , Fosforilação , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Mutação , Proteínas do CitoesqueletoRESUMO
Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.