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 a/ ß 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. Overall design: 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