Several selective CDK4/6 inhibitors are in clinical trials for non-small cell 
lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III 
Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential 
cellular activity between palbociclib and the structurally related ribociclib 
(LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based 
chemoproteomics approach in H157 cells and primary tumor samples, we here report 
distinct proteome-wide target profiles of these two drug candidates in LUSQ, 
which encompass novel protein and, for palbociclib only, lipid kinases. In 
addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. 
Palbociclib interacted with several kinases not targeted by ribociclib, such as 
casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib 
engaged several lipid kinases, most notably, PIK3CD and PIP4K2A/B/C. 
Accordingly, we observed modulation of autophagy and inhibition of AKT signaling 
by palbociclib but not ribociclib.