Role And Mechanism Of Lorlatinib Therapy Sensitizing Melanoma To Ferroptosis

Background: Melanoma represents one of the most metastasizing and drug resistant solid tumors.Although targeted therapy and immunotherapy have improved the prognosis of some melanoma patients,drug resistance and non-response limit their further application in the treatment of malignant melanoma.Hence,an immediate call needs to be made to explore and develop a new potential melanoma treatment strategy.Ferroptosis is a new form of regulated cell death characterized by the excessive accumulation of iron-dependent lipid peroxides,which is distinct from the traditional form of cell death and plays an important role in tumor development and treatment.Melanoma cells with dedifferentiated phenotype,high invasiveness,or drug resistant show the susceptibility to ferroptosis.In addition,ferroptosis also contribute to immunotherapy and radiotherapy mediated antitumor effect.These suggest targeting ferroptosis may be a new therapeutic strategy in melanoma.However,multiple mechanisms mediate the resistance of melanoma to ferroptosis,promoting the melanoma development and metastasis.These resistance mechanisms of ferroptosis limit the application of therapeutic strategy for targeting ferroptosis in melanoma.Combination therapy is an effective strategy to enhance the therapeutic effects and solve the resistance.This study intends to explore the ferroptosis sensitizing Food and Drug Administration(FDA)-approved antineoplastic agents and the specific mechanism.Objectives:(1)To screen the potential FDA-approved antineoplastic agents that can sensitize ferroptosis in melanoma;(2)To verify the effect of lorlatinib sensitizing ferroptosis in melanoma;(3)To elucidate the specific mechanism of lorlatinib sensitizing ferroptosis;(4)To evaluate the antitumor effect of lorlatinib sensitizing ferroptosis in preclinical animal models;(5)To explore the clinical value of the target of lorlatinib sensitizing ferroptosis in melanoma;Methods: Large-scale drug combination screening was established,which based on the combination of FDA-approved antineoplastic agents and RSL3,a ferroptosis inducer by inhibiting glutathione peroxidase 4(GPX4),and the synergistic effect was evaluated by two-drug interaction index(CDI),and drug combination index(CI)to screen the drugs that can sensitize cell death of melanoma.The effects of loratinib combined with RSL3 on the survival of melanoma cells in different concentrations and at different times were examined by light microscope and CCK8.The forms of cell death after treatment of loratinib combined with RSL3 were determined through measuring the rescue effect of different cell death inhibitors by CCK8,MDA by kit,lipid ROS by flow cytometry,CHAC1 and PTGS2 by q RT-PCR,mitochondrial change by electron microscopy.RNA-seq,lipidomics,bioinformatics,and CRISPR/Cas9 gene knockout technology were used to elucidate the specific mechanism of loratinib sensitizing ferroptosis in melanoma ferroptosis.The synergistic effect and potential clinical value of the loratinib were detected and assessed on mouse tumor bearing model and the melanoma patient public database.Results:(1)Based on the drug combination screening platform,it was found that loratinib can synergistically induce melanoma cell death with GPX4 inhibition in a time-and dose-dependent manner;(2)Ferroptosis inhibitors rescued Loratinib combined with GPX4 inhibition induced melanoma cell death,and this combination can upregulate the ferroptosis markers,including malondialdehyde,lipid reactive oxygen species,cha C glutathione specific gammaglutamylcyclotransferase 1(CHAC1)and prostaglandin-endoperoxide synthase 2(PTGS2)in memanoma cells,and ferroptotic mitochondrial changes were also observed.All results confirmed that loratinib sensitized melanoma ferroptosis rather than other forms of death;(3)RNA-seq,lipomics,bioinformatics and CRISPR/Cas9 gene knockout techniques were performed and confirmed that lauratinib can mediate the downregulation of PI3K/AKT/m TOR signaling pathway by targeting the inhibition of IGF1 R,and then inhibited the maturation of sterol regulatory element binding transcription factor 1(SREBF1)into the nucleus,following the down-regulation of Stearoyl-Co A desaturase(SCD),ultimately sensitizing melanoma ferroptosis;(4)Lauratinib combined with GPX4 inhibition promoted melanoma ferroptosis and inhibited tumor growth in preclinical animal models;(5)IGF1R,SREBF1 and SCD were highly expressed in melanoma.The expression of IGF1 R and SREBF1 was positively correlated with SCD.Melanoma patients with both low expression of GPX4 and IGF1 R exhibited the better prognosis.Conclusions: In this study,we found that loratinib can sensitizing melanoma ferroptosis by inhibiting PI3K/AKT/m TOR/SREBF1 pathway and SCD through IGF1 R.Moreover,the melanoma patients with the both low expression of GPX4 and IGF1 R exhibited the improved prognosis.In conclusion,our study will lay a foundation for the basic and translational research of ferroptosis in melanoma.Figures 24,Tables 18,References 92...