Strikingly, Cpd-1 and Cpd-2 failed to accumulate in the lysosomes, despite the fact that their intracellular concentration was comparable to that of ribociclib (Fig

Strikingly, Cpd-1 and Cpd-2 failed to accumulate in the lysosomes, despite the fact that their intracellular concentration was comparable to that of ribociclib (Fig. together with their associated kinase partners, the cyclin-dependent kinases CDK4 and CDK6, drive cell cycle progression by phosphorylating the retinoblastoma protein, RB1, and RB1-related p107 and p130 proteins. During early G1 phase of the cell cycle, RB1 exists in a hypophosphorylated state and constrains cell proliferation by binding to and inhibiting the activity of E2F transcription factors. Phosphorylation of RB1 by cyclin DCCDK4/6 and later by cyclin ECCDK2 kinases functionally inactivates RB1, resulting in derepression of the E2F activity. This, in turn, allows progression of cells into the DNA synthesis phase (S phase) (gene (encoding cyclin D1) takes place in up to 20% of breast cancers, while cyclin D1 protein is usually overexpressed in more than 50% of cases (oncogene (gene, which explains their lack of response. However, several TNBC cell lines displayed resistance to CDK4/6 inhibition in the absence of any obvious abnormalities in the RB1 pathway. We verified that these cell lines were also resistant to treatment with two other FDA-approved CDK4/6 inhibitors, namely ribociclib and abemaciclib (fig. S1B). Open in a separate windows Fig. 1 Sequestration of palbociclib into tumor cell lysosomes mediates resistance to chemical CDK4/6 inhibition.(A) Fraction of bromodeoxyuridine (BrdU)Cpositive cells treated with palbociclib (PALBO) (1 M) or dimethyl sulfoxide (DMSO) for 24 hours (means SD, = 3). (B) Portion of BrdU-positive cells transfected with anti-CDK4/CDK6 or control siRNA for 48 hours (means SD, = 3; HCC1954, = 2). (C and Mst1 D) Microscopic analysis of HCC1806 cells treated with palbociclib (1 M) or DMSO for 24 hours and stained with LysoTracker Green (LTR-green) (C), or HC-030031 treated with palbociclib or palbo/bafilomycin A1 (BAF) (100 nM) for 24 hours (D). PALBO auto., palbociclib autofluorescence. Level bars, 20 m. (E) Portion of BrdU-positive cells treated with palbociclib (1 M) and/or bafilomycin A1 (10nM-SUM149, 25nM-HCC1806/SUM149, 50nM-CAL120) or DMSO for 24 hours (means SD, = 3, two-sided test). (F) TNBC cells transfected with anti-ATP6AP1 or control siRNAs for 36 hours, stained with LysoSensor HC-030031 Green, and analyzed HC-030031 by fluorescence-activated cell sorting (FACS). (G) BrdU-positive portion of ATP6AP1-depleted and control cells treated with palbociclib (1 M) or DMSO for 24 hours (means SD, = 3, two-sided test). (H) Portion of BrdU-positive cells treated with palbociclib (1 M) and/or NH4Cl (50 mM) or DMSO for 24 hours (means SD, = 3, two-sided test). (I) Portion of BrdU-positive cells treated with palbociclib, ribociclib (RIBO), abemaciclib (ABEMA) (1 M), and/or bafilomycin A1 (25 nM) for HC-030031 24 hours (means SD, = 3, two-sided test). (J) HC-030031 Portion of BrdU-positive cells in nontargeting single-guide RNA (snt) or = 3, two-sided test). To evaluate the requirement for CDK4 and CDK6 in these resistant TNBC cells, we depleted CDK4 and CDK6 using two impartial sets of small interfering RNAs (siRNAs). Very unexpectedly, three of the CDK4/6 inhibitorCresistant TNBC cell lines (HCC1806, SUM149, and SUM159) showed a nearly total proliferative arrest following CDK4/6 depletion (Fig. 1B and fig. S1C). A CRISPR screen for essential genes in a fourth cell collection (CAL120) also revealed that these cells depend on CDK4 for proliferation (R.J. and M.B., unpublished observations). We made a similar observation in basal-like, HER2-positive HCC1954 cells. These cells were resistant to treatment with all three CDK4/6 inhibitors, while depletion of CDK4/6 arrested their proliferation (Fig. 1B and fig. S1, C and D). Hence, these TNBC cell lines, like hormone receptorCpositive breast cancer cells, critically require CDK4 and CDK6 for their proliferation, and yet, they are resistant to treatment with all available CDK4/6 inhibitors. To explain these findings, we hypothesized that in resistant breast malignancy cells, palbociclib fails to reach its targets (CDK4 and CDK6) in the nucleus. We required advantage of our observation that palbociclib has autofluorescent properties, and we followed the localization of this compound in TNBC cells by light microscopy. Live-cell imaging of TNBC (and basal-like HCC1954 cells) revealed that upon palbociclib treatment, this compound accumulates in discrete structures in tumor cells cytoplasm. Costaining of cells with LysoTracker Green, a green fluorescent dye that staining lysosomes, revealed a complete overlap with palbociclib staining (Fig. 1C and fig. S2A). We next asked whether inhibition of lysosomal acidification by blocking the lysosomal proton pump would trigger the release of palbociclib from lysosomes. To test this, we treated cells with bafilomycin A1, a specific inhibitor of the proton pump vacuolar (H+)Cadenosine triphosphatase (ATPase) (v-ATPase), which mediates acidification of intracellular compartments (TNBC HCC1806 and SUM159 cells.