ARTICLE ABSTRACTCyclin-dependent kinase 4/6 (CDK4/6)-specific inhibitors, such as palbociclib, have shown clinical efficacy, but primary or secondary resistance has emerged as a problem. To develop more effective therapeutic approaches, investigation is needed into the mechanisms of resistance or adaption. Here, it is demonstrated that CDK2 compensates for loss of CDK4 activity to rescue palbociclib-arrested breast cancer cells, suggesting that inhibition of both kinases is required to achieve durable response. In addition, a novel strategy is described to inhibit tyrosine phosphorylation of p27Kip1 (CDKN1B) and simultaneously inhibit both CDK2 and CDK4. p27Kip1 is a required assembly factor for cyclin–CDK4 complexes, but it must be phosphorylated on residue Y88 to open or activate the complex. The Brk-SH3 peptide, ALT, blocks p27 Y88 phosphorylation, inhibiting CDK4. Nonphosphorylated p27 is no longer a target for ubiquitin-mediated degradation and this stabilized p27 now also inhibits CDK2 activity. Thus, ALT induction inhibits both the kinase that drives proliferation (CDK4) and the kinase that mediates resistance (CDK2), causing a potent and long-lasting cell-cycle arrest. ALT arrests growth of all breast cancer subgroups and synergizes with palbociclib to increase cellular senescence and to cause tumor regression in breast cancer xenograft models. The use of ALT demonstrates that both CDK4 and CDK2 need to be inhibited if long-term efficacy is to be achieved and represents a novel modality to inhibit breast cancer cells.Implications: Modulating tyrosine phosphorylation of p27 impacts both proliferative (CDK4) and resistance (CDK2) mechanisms in breast cancer and suggests that phospho-p27 status may serve as a biomarker for patients that are responsive to CDK4/6 inhibition. Mol Cancer Res; 16(3); 361–77. ©2018 AACR.