O-GlcNAcylation promotes Palbociclib resistance in breast cancer by regulating MITF nuclear translocation [RNA-seq]

Cyclin-dependent kinases 4 and 6 (CDK4/6) are essential drivers of the cell cycle and are also critical for the initiation and progression of diverse malignancies. Pharmacological inhibitors targeting CDK4/6 have demonstrated significant activity against various tumor types such as breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i) (such as palbociclib) remains an immense obstacle in clinical and the underlying mechanisms have not been fully understood. Using quantitative high-throughput combinational screen (qHTCS) and genomic sequencing, we report that the Microphthalmia-associated transcription factor (MITF), was significantly elevated in palbociclib-resistance cells. Inhibition of MITF can enhance the therapeutic efficacy of Palbociclib and surmount Palbociclib resistance both in vitro and in vivo. Mechanistically, we found that O-GlcNAc transferase (OGT) modifies MITF with O-GlcNAcylation at Serine 49 (Ser49) within its nuclear localization signal (NLS), thereby promoting MITF binding to importin α/ β and facilitating its nuclear transportation, which is crucial in regulating senescence. Significantly, clinical studies also confirm that MITF was elevated in palbociclib-resistance patients. Collectively, these results reveal a previously unrecognized mechanism by which MITF-mediated palbociclib resistance, and provide valuable insights for the development of innovative therapeutic strategies in future clinical contexts. To investigate the mechanism of Palbociclib resistance in breast cancer cell lines, we first generated Palbociclib-resistance (PR) cell line MCF-7 PR. Then we compare different genes expression between MCF-7 wild-type (WT) and MCF-7 PR cells by RNA seq. Then we generated MITF-depleted cell line by shRNA (MCF-7 PR shMITF), and compare different genes expression between MCF-7 PR and MCF-7 PR shMITF