Lysosomal trapping of palbociclib and its functional implications

Abstract

Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.

Cellular Senescence: Defining a Path Forward

“…From a therapeutic viewpoint, the enlarged lysosomal compartment offers an increased capacity to trap drugs that can be protonated, such as the selective CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib. This capacity reduces their effective concentration in the cytosol and nucleus but is counteracted by the slow release of the drugs from the lysosomes, thereby increasing drug exposure time (Llanos et al, 2019). Another senescence trait, related to lysosomal malfunction, is the intra-lysosomal accumulation of lipofuscin aggresomes (see ''Protein Damage'' and ''Lipid Damage'') (as reviewed in Gorgoulis et al, 2018).…”

Cell size homeostasis is maintained by a circuitry involving a CDK4-determined target size that programs the cell size-dependent activation of p38

“…To reduce CDK4 activity, we exposed cells to palbociclib, a chemical inhibitor of CDK4/6. At concentrations that exceed 1µM, palbociclib promotes cell cycle arrest (Llanos et al, 2019). For our experiments, we carefully optimized a palbociclib concentration range that decreases CDK4 activity but does not result in cell cycle arrest ( Fig.…”

Ribociclib-associated vortex keratopathy

“…8 In this study, the chemically related ribociclib was thought to similarly demonstrate lysosomotropic properties. 8 We hypothesize that this process of lysosomal sequestration posits a potential mechanism by which ribociclib may lead to the development of vortex keratopathy.…”

“…7 A recent study by Llanos et al has demonstrated that palbociclib, another small-molecule inhibitor of CDK4/6, demonstrates the property of lysosomal trapping. 8 The prolonged release of palbociclib from lysosomes may play a key role in its length of inhibition of CDK4/6 and ability to induce sustained cellular senescence. 8 In this study, the chemically related ribociclib was thought to similarly demonstrate lysosomotropic properties.…”

“…8 The prolonged release of palbociclib from lysosomes may play a key role in its length of inhibition of CDK4/6 and ability to induce sustained cellular senescence. 8 In this study, the chemically related ribociclib was thought to similarly demonstrate lysosomotropic properties. 8 We hypothesize that this process of lysosomal sequestration posits a potential mechanism by which ribociclib may lead to the development of vortex keratopathy.…”