EACR25-2269

Immunomodulatory effects of HuR inhibition in EGFR-TKI resistant NSCLC cells

M. Casciello1, A. Nigro2, A. Cascone2, V. Pagliara3, A. Napoli4, M. Bevilaqua4, F. Sabbatino3, V. Casolaro4, C. Stellato4, J. Dal Col4
1University of Salerno, Baronissi (SA), Italy, Specialization School in Clinical Pathology and Clinical Biochemistry, Baronissi, Italy
2University of Salerno, Specialization School in Clinical Pathology and Clinical Biochemistry, Baronissi, Italy
3University of Salerno, Oncology Unit, Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana, Baronissi, Italy
4University of Salerno, Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana, Baronissi, Italy
Introduction:

Immunotherapy has produced positive results in patients with advanced non-small cell lung cancer (NSCLC), except for those bearing a mutant epidermal growth factor receptor (EGFR). In particular, once resistance to EGFR-tyrosine kinase inhibitors (TKIs) is established, EGFR-mutant NSCLC patients do not benefit from therapy with immune checkpoint inhibitors (ICIs) despite PD-L1 expression. Acquired resistance to EGFR-TKI is associated with an immune suppressive phenotype, involving downregulation of class I HLA antigens expression and increased secretion of interleukin(IL)-6, IL-8 and transforming growth factor-(TGF)β. These factors are all targets of the RNA-binding protein (RBP) HuR, which regulates mRNA stability/translation of bound transcripts. Given the mounting role of HuR in cancer immune evasion, we investigated its function in modulating two mechanisms contributing to the poor response to ICIs in EGFR-TKI resistant cells: cytokine secretion and class I HLA expression.

Material and method:

EGFR-TKI-resistant cell lines (HCC827GR/PC9GR/H1975OR) were generated by treating HCC827/PC9/H1975 cells with Gefitinib or Osimertinib. PC9- and H1975-HuR-KO were generated by CRISPR/Cas9 technology. KH-3 and SRI-42127 commercially available HuR inhibitors were used. Protein expression was analyzed by immunoblotting, confocal microscopy, flow cytometry. Cytokine secretion on cell supernatants was analyzed by ELISA assay and flow cytometry-based multiplex immunoassays.

Result and discussion:

Confocal microscopy analysis identified increased cytoplasmic HuR, a feature of functional activation, in EGFR-TKI-resistant cells compared to parental cells. Levels of HuR targets IL-6 and IL-8 were significantly increased in EGFR-TKI-resistant cells respect to parental cells (p<0.01), while their levels were reduced in PC9- and H1975-HuR-KO cells (p< 0.05). Furthermore, the loss of HuR impaired in vitro acquisition of resistance to both gefitinib and osimertinib in PC9 and H1975 cells, together with a selective decrease (>80%; p< 0.01) of IL-8 secretion. HuR inhibitor SRI-42127 (2.5µM, 24 hours) reduced only IL-8 release from both PC9GR and H1975OR, whereas the inhibitor KH-3 significantly reduced, in a dose-dependent manner, the levels of both IL-6 and IL-8 in H1975OR cells. In addition, flow cytometric analysis showed PD-L1 upregulation and class I HLA antigens downregulation in H1975OR compared to H1975 cells. Intriguingly, pharmacological HuR inhibition induced a 2 fold increase of class I HLA antigens in both cell lines suggesting an improvement in antigen presentation.

Conclusion:

Our results indicate that HuR contributes to EGFR-TKIs resistance acquisition and modulates IL-8 and class I HLA expression, which are determinants of poor response to ICIs. These studies support the evaluation of HuR inhibitors to restore the EGFR-TKI responsiveness to immunotherapy. Study supported by AIRC