Hindering resistance to EGFR tyrosine kinase inhibitors (TKIs) is a major challenge in oncology. Many NSCLC patients show high sensitivity to EGFR TKIs but under drug pressure, drug-resistant clones emerge, and new therapeutic strategies are needed to avoid treatment failure. Sp1 is a well-recognized transcription factor implicated in cancer progression and here we want to demonstrate that concurrent inhibition of EGFR kinase domain and Sp1 may hinder proliferation of cancer stem cells (CSCs), impair cell migration, improve immune response and enhance cell toxicity in NSCLCs.

We used H1975, HCC827, PC9 and PC9 osimertinib resistant (PC9/OR) cell lines growth in adhesion or as tumor-spheres (TS). Cells were screened for Sp1 levels before and after 1µM osimertinib treatment. Cell toxicity assay was performed using osimertinib (0.1-1 µM) alone or in combination with Sp1 inhibitor Mithramycin A (Mit A). We integrated our data with cBioPortal analysis for SP1 gene CNV expression in lung tumors and with computational studies to identify new Sp1 inhibitors. In silico methods along with HDOCK software were used to identify new compounds that show lower toxicity than Mit A but with similar ability to impair DNA binding activity of Sp1. Wound healing and TS assays were also conducted to detect cell migration ability and stem-like cells survival after 72h treatment. Furthermore, modulation of the immunosuppressive receptor CD47 in treated and untreated cells were also assessed along with screening of PDL-1 levels in exosomes isolated from sensitive and OR cells.

Treatment with osimertinib 1 µM for 72h was able to strongly reduce cell viability and increase cell death markers in NSCLC cells. However, concomitant increase of Sp1 levels was also observed in treated cells along with activation of EMT, CSC signaling and CD47 expression. Similar results were obtained by western blot analysis of nuclear extracts and protein content of exosomes released from PC9 and PC9/OR cells. We found the highest Sp1 nuclear localization along with the highest PDL-1 exosomes levels in resistant cells. Bioinformatic analysis of SP1 gene CNVs revealed a lower overall survival (OS) in tumors with SP1 amplification. Interestingly, combined treatment using osimertinib along with Mit A was able to improve cell toxicity, reduce cell migration and inhibit the TS formation. We also found that drugs combination caused a reduction of CD47 levels on NSCLC cells surface. In addition, our computational studies showed that Hederasaponin B, similar to Mit A, binds to the GGGCGG DNA box with high affinity thus representing a new potential inhibitor of Sp1 activity and reported to be a well-tolerated compound.

Co-targeting of EGFR and Sp1 may inhibit migration and survival of resistant cells with stem-like phenotype. In addition, such therapeutic strategy may hinder immune evasion and may be a tool for preventing treatment failure in NSCLC patients.