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Time-of-day specificity of anticancer drugs may be mediated by circadian regulation of the cell cycle

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Science Advances  12 Feb 2021:
Vol. 7, no. 7, eabd2645
DOI: 10.1126/sciadv.abd2645
  • Fig. 1 Rhythmic action of anticancer drugs is associated with rhythmic expression of some target genes.

    (A) The heatmap represents anticancer inhibitors (62) exhibiting rhythmic patterns of drug sensitivity from the drug panel (126) tested in U2OS cells. Cells were synchronized by 100 nM dex in staggered fashion, 6 hours apart from each other as shown in fig. S1. Drug treatment was at a single time point 24 hours after the last group was synchronized with dex (thus, at 24, 30, 36, 42, or 48 hours following dex). (B) Anticancer drugs show peaks of IC50 value at different times. Each line graph represents drugs that are most ineffective at the same time of day (30, 36, 42, and 48 hours) following synchronization of endogenous rhythms with dex. (C) Circular plots depict the number of rhythmically acting cancer inhibitors, including those that target rhythmically expressed genes (44; dark orange), of the total tested (126; light orange). (D) The bar graph shows rhythmically expressed genes (in brown) targeted by rhythmically acting anticancer inhibitors. Rhythmicity was determined by PJTKCycle (P < 0.05) and based on ad hoc analysis of RNA sequence data of dex-synchronized U2OS cells across circadian time (see fig. S2A). Gray bars indicate nonrhythmic genes targeted by drugs that act rhythmically (PJTKCycle > 0.05).

  • Fig. 2 Rhythmic action of anticancer drugs is largely dependent on the circadian clock.

    (A) Schematic of the experimental schedule to validate time dependence of candidates from the drug screen. Twenty-four hours after 1-hour dex synchronization (100 nM, light blue bolt), CRISPR scramble control reporter U2OS cells were treated with vehicle or candidate drugs (0.01 to 1 μM, red bolt) at 6-hour intervals over the course of 24 hours and subjected to an Alamar Blue cell proliferation assay at the indicated time points (48 hours later for each sample). (B) Graphs of experimental procedures in (A) to show time-dependent antiproliferative effects of the indicated anticancer drug candidates at various doses (0.01, 0.1, and 1 μM). The candidate drug targets were indicated in the parentheses. PJTKCycle < 0.05 denotes results of JTK-Cycle analysis, which detected a significant 24-hour rhythm in the action of AZD4547, MK1775, triptolide, dinaciclib, NMS-873, raltitrexed, FK866, and 17-AAG (details are in data file S3). Data were normalized to represent the average ± SD; n = 3 for each time point. RFU, relative fluorescence units. (C) Bioluminescence recordings of Bmal1 promoter (pBmal1-dsLuc) luciferase rhythms in CRISPR scramble control reporter U2OS cells (CTL, dark gray) and BMAL1 CRISPR knockout (BMAL1 KO, brown red) are shown. (D) The graphs show compromised rhythms of antiproliferative action of the indicated drugs in BMAL1 null cells. Several doses were tested. (E) Bioluminescence recordings of Per2 promoter (pPer2-dsLuc) luciferase rhythms in RB mutant C33A cell clones (clone 1: light gray green and clone 2: dark gray green) are shown. (F) The graphs show compromised rhythms of antiproliferative action of the indicated drugs in RB mutant C33A cells at various doses. (G) JTK-cycle analysis of rhythm amplitude for the time courses shown in (B), (D), and (F). *P < 0.05, ****P < 0.0001.

  • Fig. 3 Rhythmic effect of HSP90 inhibitors is associated with circadian expression of HSP90 genes.

    (A) Line graphs are replotted on the basis of the primary screening results in Fig. 1 to show that several HSP90 inhibitors are more effective at inhibiting cell proliferation at a specific time of day. The x axis depicts circadian phase as determined by the timing of dex addition (see Materials and Methods). (B) Temporal mRNA expression of the indicated HSP90 isoforms of dex-synchronized U2OS cells across circadian time (15). PJTKCycle < 0.05 indicates significant rhythms of HSP90AA1, HSP90AB1, and HSP90B1 (details are in data file S3). (C) Western blot analysis of the indicated HSP90 isoforms and BMAL1 in control (CTL) and BMAL1 knockout U2OS (BMAL1 KO) cells collected every 6 hours for 24 hours after dex stimulation. (D) Statistical analysis of the Western blot data in (C) showing protein abundance over time in CTL (gray circle) and BMAL1 KO (red brown circle) cells. JTKCycle identified a significant 24-hour rhythm in the expression of HSP90AA1, HSP90AB1, and HSP90B1 in CTL cells. Data were normalized to actin and are represented as means ± SEM from n = 3 independent experiments. (E) mRNA expression of the indicated HSP90 isoforms in control (CTL) and REV-ERB α/β double-knockout U2OS cells (REV-ERBα/β DKO). **P < 0.005; two-way ANOVA with Bonferroni’s multiple comparisons test. Data are shown with the means ± SD; n = 3 to 6 in each genes. (F) Line graphs, depicting means based on the data in fig. S6B, show expression profiles of the indicated HSP90 isoforms in CTL (gray) and REV-ERBα/β DKO (light brown) cells. (G) Bar graphs depict cell viability assays performed 48 hours after 17-AAG treatment in CTL (gray bar) and REV-ERBα/β DKO (light brown bar) cells. ***P < 0.005. Data show means ± SD; n = 6 for all doses.

  • Fig. 4 Isoforms of HSP90 that mediate temporal drug sensitivity affect circadian rhythms and the cell cycle.

    (A) Graphs show time-of-day–dependent cytotoxic effect of 17-AAG delivered to dex-synchronized U2OS cells treated with control (si-CTL) or siRNAs targeting the indicated HSP90 isoforms. JTKCycle detected a significant 24-hour rhythm in 1 μM drug-treated si-CTL and si-HSP90AB1 samples (details are in data file S3). (B) Period analysis of temporal 17-AAG cytotoxicity data shown in (A) using the Biodare2 rhythm analysis program (https://biodare2.ed.ac.uk/). AR, arrhythmic. (C) Western blot analysis to verify efficiency of siRNAs targeting the indicated HSP90 isoforms. (D to G) Bioluminescence recordings of a Bmal1 promoter–luciferase reporter (pBmal1-dsLuc) in U2OS cells treated with vehicle (gray) or 17-AAG at the indicated doses (D) or 48 hours after transfection of control siRNA (si-CTL, gray) or siRNAs targeting the indicated HSP90 isoforms (E to G) (top). Graphs below present period (left) and amplitude (right) analysis results of the top data panels. *P < 0.05, **P < 0.005, and ****P < 0.00001 by Biodare2 analysis. Data show means ± SD; n = 3 for all samples. (H to K) Real-time cell analysis of U2OS cells stably expressing FUCCI cell cycle indicators [RFP (G1), dark pink; GFP (S-G2-M), gray green) 48 hours after transfection of control siRNA (si-CTL) (H) or siRNAs targeting the indicated HSP90 isoforms (I to K) (top). (L to P) DMSO (control) or cell cycle arrest inducers such as thymidine [Thy (1 mM), G1-S boundary arrest] and nocodazole [Noc (100 nM), G2-M arrest] were delivered alone or in combination as indicated to block cell cycles in U2OS cells before timed 17-AAG treatment and cell viability analysis as in Fig. 2A. Data are normalized to represent average ± SD; n = 6 per time point. PJTKCycle detected a significant 24-hour rhythm in control cells treated with 0.01 μM, 0.1 mM, or 1 μM 17-AAG (details are in data file S3).

  • Fig. 5 17-DMAG exerts time-of-day–specific anticancer activity on a mouse melanoma.

    (A) Time-of-day–dependent cytotoxicity of different doses of 17-AAG in dex-synchronized B16 melanoma cells. PJTKCycle < 0.05 indicates significant cytotoxic rhythms at 0.1 and 1 μM 17-AAG. (B) mRNA expression of Hsp90aa1 (red) and Hsp90ab1 (blue) in dex-synchronized B16 melanoma cells. (C) Western blot analysis of CRISPR scramble (CTL) and Bmal1 knockout (Bmal1 KO) B16 cells using indicated antibodies. (D) Bioluminescence recordings of dex-synchronized control CTL (black) and Bmal1 KO (red) cells stably expressing Per2 promoter–driven luciferase reporter (pPer2-dLuc). (E) Seven or 10 days after subcutaneous injection of CTL or Bmal1 KO cells (1 × 106), mice were orally administered 17-DMAG at ZT3 (orange arrow) or ZT15 (green arrow). Black arrowheads denote times of tumor measurement. Red arrows indicate sampling of tumor and liver tissues from mice sacrificed at ZT3 or ZT15 for gene expression analysis. (F and G) Time-dependent effects of 17-DMAG administration on CTL (F) or Bmal1 KO (G) tumor growth in mice; untreated (black circle), treated at ZT3 (orange rectangle), and treated at ZT15 (green triangle). (H) Quantification of relative tumor growth rate calculated from linear regression by fitting a linear equation to the normalized data panels in (E) and (F). *P < 0.05, two-way ANOVA and Tukey’s multiple comparisons test. Data are means ± SEM. (I and J) Hsp90aa1(I) and Hsp90b (J) expression in CTL or Bmal1 KO melanoma and liver tissues from mice as described in (D). *P < 0.05, **P < 0.005, and ***P < 0.0001, one-way ANOVA and Tukey’s multiple comparisons test. Data are means ± SD; n = 3. (K) Schematic depicts circadian regulation of Hsp90 by RORs/Rev-erbs, via the specific response elements (RORE), and its action in the cell cycle to confer temporal specificity to the antimelanoma effects of 17-DMAG. n.s., not significant.

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