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Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition

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Science Advances  17 Jun 2020:
Vol. 6, no. 25, eabb2210
DOI: 10.1126/sciadv.abb2210
  • Fig. 1 Sequestration of palbociclib into tumor cell lysosomes mediates resistance to chemical CDK4/6 inhibition.

    (A) Fraction of bromodeoxyuridine (BrdU)–positive cells treated with palbociclib (PALBO) (1 μM) or dimethyl sulfoxide (DMSO) for 24 hours (means ± SD, n = 3). (B) Fraction of BrdU-positive cells transfected with anti-CDK4/CDK6 or control siRNA for 48 hours (means ± SD, n = 3; HCC1954, n = 2). (C and D) Microscopic analysis of HCC1806 cells treated with palbociclib (1 μM) or DMSO for 24 hours and stained with LysoTracker Green (LTR-green) (C), or treated with palbociclib or palbo/bafilomycin A1 (BAF) (100 nM) for 24 hours (D). PALBO auto., palbociclib autofluorescence. Scale bars, 20 μm. (E) Fraction of BrdU-positive cells treated with palbociclib (1 μM) and/or bafilomycin A1 (10nM-SUM149, 25nM-HCC1806/SUM149, 50nM-CAL120) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (F) TNBC cells transfected with anti-ATP6AP1 or control siRNAs for 36 hours, stained with LysoSensor Green, and analyzed by fluorescence-activated cell sorting (FACS). (G) BrdU-positive fraction of ATP6AP1-depleted and control cells treated with palbociclib (1 μM) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (H) Fraction of BrdU-positive cells treated with palbociclib (1 μM) and/or NH4Cl (50 mM) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (I) Fraction of BrdU-positive cells treated with palbociclib, ribociclib (RIBO), abemaciclib (ABEMA) (1 μM), and/or bafilomycin A1 (25 nM) for 24 hours (means ± SD, n = 3, two-sided t test). (J) Fraction of BrdU-positive cells in nontargeting single-guide RNA (snt) or RB1-deleted cells (ΔRB1) treated with palbociclib (1 μM) and/or bafilomycin A1 (25 nM) for 24 hours (means ± SD, n = 3, two-sided t test).

  • Fig. 2 Increased lysosomal mass in CDK4/6 inhibitor–resistant TNBC cells.

    (A) Schematic representation of possible mechanisms of lysosome-mediated resistance. (B) Palbociclib/LysoTracker Red ratio in immunoprecipitated lysosomes, quantified by mass spectrometry (mean, n = 2, two-sided t test). (C and D) Representative histogram overlay of flow cytometric analysis (C) and median fluorescence intensity (MFI) (D) of TNBC cells stained with LysoTracker Green (means ± SD, n = 5, two-sided t test). (E) Representative electron microscopic images of resistant and sensitive TNBC cells. Arrowheads, lysosomes. Scale bars, 2 μm. (F) Quantification of lysosome number in electron microscopic images per 100 μm2 of cytoplasm (analyzed images: HCC1806, n = 21; SUM159, n = 16; HCC38, n = 21; HCC1143, n = 17). (G) Immunoblot analysis of TFEB protein levels in nuclear fractions. Proliferating cell nuclear antigen served as loading control (dashed line indicates splicing of nonadjacent parts of the same membrane). (H) Flow cytometric analysis of fluorescence intensity in TFEB-depleted (TFEBsi) and control cells stained with LysoTracker Green. (I) Fraction of BrdU-positive cells transfected with control or anti-TFEB siRNA for 48 hours and treated with palbociclib (1 μM) or DMSO for 24 hours (means ± SD, n = 3, two-sided t test). (J) Gene set enrichment analysis (GSEA) for GO (gene ontology) lysosomal lumen category in TNBC PDX tumors displaying intrinsic resistance to treatment with ribociclib versus in ribociclib-sensitive tumors. The response (stable/progressive disease; fig. S3) was from (19). FDR, false discovery rate; NES, normalized enrichment score.

  • Fig. 3 Lysosomotropic compounds restore the sensitivity of TNBC to CDK4/6 inhibitors.

    (A) Cell numbers of in vitro cultured TNBC cells at time 0 and after 6 days of culture in the presence of DMSO, palbociclib (1 μM), and/or lysosomotropic antibiotic azithromycin (AZI; 50 μM) (means ± SD; SUM159, n = 6; HCC1806, n = 4; two-sided t test). (B) Similar assay as in (A); cells were treated with palbociclib (1 μM) and/or anxiolytic/antidepressant compound siramesine (SIRA; SUM159, 3.5 μM, and HCC1806, 2 μM) (means ± SD, n = 3, two-sided t test). (C and D) Short-term ex vivo culture of a patient-derived TNBC tumor (grade 3). Tumor fragments were cultured for 48 hours in the presence of DMSO, palbociclib (1 μM), siramesine (5 μM), or palbociclib (250 nM or 1 μM) and siramesine and pulsed with BrdU (4 hours). (C) Representative images of tumor sections stained for BrdU. Scale bars, 20 μm. (D) Percentage of BrdU-positive tumor cells. (E) Cell numbers of in vitro cultured TNBC cells at time 0 and after 6 days of culture in the presence of palbociclib (250 nM or 1 μM) and/or chloroquine (CQ; SUM159, 25 μM, and HCC1806, 15 μM) or DMSO (means ± SD; SUM159, n = 3; HCC1806, n = 4; two-sided t test). (F) Tumor weights of a TNBC PDX (IDC50) following 4 days of treatment of mice with palbociclib (50 mg/kg) and/or hydroxychloroquine (HCQ; 60 mg/kg) or vehicle only (CTRL). Two-sided t test.

  • Fig. 4 Coinhibition of CDK2 and CDK4/6 arrests proliferation of TNBC cells.

    (A) Immunoblot analysis of CDK2 protein levels in SUM159 and HCC1806 cells following CRISPR-Cas9–mediated knockout of CDK2 and ectopic expression of Flag-tagged analog-sensitive CDK2 (CDK2AS/sg); nt, cells expressing control nontargeting sgRNA. Tubulin served as loading control. (B) Fraction of BrdU-positive control HCC1806 and SUM159 cells (nt) or CDK2 knockout HCC1806 and SUM159 cells expressing analog-sensitive CDK2 (CDK2AS/sg) treated with palbociclib (1 μM) and/or 3MB-PP1 (1 μM; an inhibitor of analog-sensitive kinases) or DMSO (means ± SD, n = 4, two-sided t test). (C) Cell numbers of in vitro cultured control (nt) or CDK2 knockout cells expressing analog-sensitive CDK2 (CDK2AS/sg) at time 0 and after 6 days. Cells were cultured in the presence of palbociclib (250 nM or 1 μM) and/or 3MB-PP1 (1 μM) (means ± SD, n = 3, two-sided t test).

  • Fig. 5 Generation and characterization of novel structurally altered CDK4/6 inhibitors with less basic characteristics.

    (A) Structures of ribociclib and its less basic analogs Cpd-1 and Cpd-2. (B) Top, amount of ribociclib, Cpd-1 and Cpd-2 in immunoprecipitated lysosomes quantified by mass spectrometry. Middle, the abundance of LysoTracker-Red in pulldowns indicating amount of immunoprecipitated lysosomes. Bottom, mass spectrometric quantification of ribociclib, Cpd-1, and Cpd-2 in whole-cell lysates. a.u., arbitrary units. (C) Fraction of BrdU-positive TNBC cells cultured with ribociclib (1 μM), Cpd-1 (50 μM) or Cpd-2 (25 μM), or DMSO. RB1-negative HCC1937 and RB1-positive, CDK4/6-independent HCC70 cells served as control (means ± SD, n ≥ 3, two-sided t test). (D) Cell numbers at time 0 and after 6 days of in vitro culture; cells were treated as in (C) (means ± SD, n ≥ 4, two-sided t test). (E to G) Short-term ex vivo cultures of two TNBC tumors derived from different patients (grades 3 and 2). Tumor fragments were cultured for 48 hours in the presence of DMSO, ribociclib (1 μM), Cpd-1 (50 μM), or Cpd-2 (25 μM) and pulsed with BrdU for 4 hours. (E) Representative images of tumor sections stained for BrdU (TNBC grade 3). Scale bars, 20 μm. (F and G) Percentage of BrdU-positive tumor cells in TNBC grade 3 (F) and grade 2 (G).

  • Fig. 6 Phosphorylation of CDK4 at Thr172 as a predictor of CDK4/6-dependence of TNBC.

    (A) Immunoblot analysis of phospho-Thr172 CDK4 and total CDK4 protein levels in the indicated TNBC cell lines and in the ER+ breast cancer cell line MCF7. Cells were classified as CDK4/6 dependent (cell cycle arrest upon depletion of CDK4/6) or CDK4/6 independent (no cell cycle arrest). The sensitivity (SEN) or resistance (RES) of cells to palbociclib is also indicated. GAPDH, glyceraldehyde-3-phosphate-dehydrogenase (loading control). (B) Phospho-Thr172 CDK4 immunohistochemical staining of xenografts of palbociclib-resistant, CDK4/6-dependent HCC1954 cells and palbociclib-resistant, CDK4/6-independent MDA-MB-468 cells. Scale bars, 50 μm. (C) A similar analysis as in (A), in a panel of TNBC PDXs from the Dana-Farber/Harvard Cancer Center PDX collection. GAPDH, loading control. (D and E) Ex vivo Matrigel culture of a TNBC PDX [17-01 from (C)], treated with palbociclib (1 μM) and/or chloroquine (CQ, 30 μM) or DMSO for 24 hours. (D) The fraction of 5-ethynyl-2’-deoxyuridine (EdU)–positive cells (DMSO, n = 5; palbociclib, n = 7; chloroquine, n = 3; chloroquine/palbociclib, n = 3; two-sided t test). (E) Representative images of cells stained for EdU. Scale bars, 50 μm. (F) GSEA for KEGG lysosome category in ER+/HER2 tumors displaying intrinsic resistance to palbociclib versus in palbociclib-responsive tumors. Gene expression data were from NeoPalAna trial.

Supplementary Materials

  • Supplementary Materials

    Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition

    Anne Fassl, Christopher Brain, Monther Abu-Remaileh, Iga Stukan, Deborah Butter, Piotr Stepien, Avery S. Feit, Johann Bergholz, Wojciech Michowski, Tobias Otto, Qing Sheng, Alice Loo, Walter Michael, Ralph Tiedt, Carmine DeAngelis, Rachel Schiff, Baishan Jiang, Bojana Jovanovic, Karolina Nowak, Maria Ericsson, Michael Cameron, Nathanael Gray, Deborah Dillon, Jean J. Zhao, David M. Sabatini, Rinath Jeselsohn, Myles Brown, Kornelia Polyak, Piotr Sicinski

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