Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) Volcano plots showing the differential drug sensitivities between the near-euploid and highly-aneuploid cancer cell lines, based on the large-scale GDSC6 and PRISM screens9. MPS1-IN-1 and MPI-0479605, the only SAC inhibitors included in each screen, respectively, are highlighted in red. (b) The sensitivity of near-euploid and highly-aneuploid cancer cell lines to the SAC inhibitors MPS1-IN-1 and MPI-0479605 in the GDSC (left) and PRISM (right) screens. ****, p=1e-0.5; n.s., p=0.23; two-tailed t-test. (c) Experimental validation of the response of 5 near-euploid (CAL51, EN, MHHNB11, SW48 and VMCUB1) and 5 highly-aneuploid (MDAMB468, NCIH1693, PANC0813, SH10TC and A101D) cell lines to 72hr exposure to the SAC inhibitor reversine. *, p=0.016, two-tailed Wilcoxon rank-sum test; n=5 cell lines in each group. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range. (d) Comparison of the sensitivity to reversine between near-euploid and highly-aneuploid cancer cell lines subjected to the PRISM cell viability assay, confirming the reduced sensitivity of highly-aneuploid cells to a 120hr exposure to SAC inhibitors. n.s., p>0.05; *, p<0.05; **, p<0.01; two-tailed t-test. (e) An association analysis failed to identify a genomic biomarker of reversine sensitivity. Shown are the top 1000 genomic features identified by our model (see Methods). No feature stands out in terms of importance and/or correlation, and the overall predictive value is poor.

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: Inhibition, Two Tailed Test, Viability Assay, Biomarker Assay

Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) Dose response curves of HCT116/HPT cells (left), or RPE1/RPT cells (right), to MPI-0479605 (120hr). EC50=0.09μM, 0.08μM, 5.02μM and 4.85μM, for HCT116-WT, HCT116-GFP, HPT1 and HPT2, respectively. EC50=0.16μM, 1.48μM, 1.52μM and 3.31μM, for RPE1-GFP, RPT1, RPT3 and RPT4, respectively. *, p<0.05; **, p<0.01; ***, p<0.001; two-tailed t-test. (b) The response of HCT116 and HPT cells (left; n=3), or RPE1 and RPT cells (right; n=4), to siRNA-mediated knockdown of BUB1B, MAD2 or TTK (72hr). Results are normalized to a non-targeting siRNA control. **, p<0.01; ***, p<0.001; two-tailed t-test. (c) Dose response curves of the near-diploid RPE1 clone SS48 and its isogenic aneuploid clones SS51 and SS111, to MPI-0479605 (120hr). EC50=0.02μM, 0.08μM and 0.04μM, for SS48, SS51 and SS111, respectively. *, p<0.05; **, p<0.01; ***, p<0.001; two-tailed t-test; n=3 for near-diploid and n=4 for aneuploid clones. (d) Response of 3 near-diploid and 4 aneuploid RPE1 clones to siRNA-mediated knockdown of BUB1B, MAD2 or TTK (72hr). Results are normalized to a non-targeting siRNA control. **, p<0.01; ***, p<0.001; two-tailed t-test. (e) Proliferation curves of HCT116 and HPT cells cultured in the presence of siRNAs against BUB1B, MAD2, TTK, or a non-targeting control siRNA. (f) Proliferation curves of HCT116 and HPT cells cultured in the presence of MPI-0479605 (250nM) or DMSO control. (g) Representative images of the cells from the experiment described in (f). Scale bar, 100μm. Calculated doubling times for (e) and (f) are presented in Extended Data Fig. 8a. In all plots, data represent the mean ± s.d. unless otherwise noted; n=3 biological replicates in all experiments unless otherwise noted.

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: Two Tailed Test, Clone Assay, Cell Culture

Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) Comparison of the doubling times of HCT116 and HPT cells exposed to siRNAs against BUB1B, MAD2 or TTK. The drug effect of SACi is stronger in the near-diploid HCT116 cells at d5, but is stronger in the highly-aneuploid HPT cells at d21. (b) Comparison of the doubling times of HCT116 and HPT cells exposed to the SAC inhibitors MPI-0479605 or reversine. The drug effect of SACi is stronger in the near-diploid HCT116 cells at d5, but at d21 it becomes stronger in the highly-aneuploid HPT cells. *, p=0.034, p=0.046 and p=0.049 for MPI-0479605 and reversine at d5 and d14, respectively; **, p=0.0015; one-tailed t-test; n=2 independent cell lines. (c) Representative images of cells from the drug experiment (same images as in Fig. 2g), with cell masking performed using the image analysis software ilastik51. Scale bar, 100μm. (d) The relative viability of the aneuploid RPE1 clones, SS111 and SS51, following reversine exposure. The viability effect was normalized to the effect of the drug in the near-diploid RPE1 clone, SS48. The drug effect of SACi is comparable during the first week of drug exposure, but the highly-aneuploid cells become significantly more sensitive with time. *, p=0.045, **, p=0.002, p=0.001 and p=0.005 for the comparisons between d3 and d14, d5 and d14 and d7 and d14, respectively; two-tailed t-test. (e) The relative viability of 5 near-euploid (CAL51, EN, MHHNB11, SW48 and VMCUB1) and 5 highly-aneuploid (MDAMB468, NCIH1693, PANC0813, SH10TC and A101D) cell lines to 72hr and 14d exposure to the SAC inhibitor reversine. *, p=0.012 and p=0.037, for 3d and 14d time points, respectively; two-tailed Wilcoxon rank-sum test.

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: One-tailed Test, Software, Clone Assay, Two Tailed Test

Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) The top 10 results of a Connectivity Map (CMap) query52 of the transcriptional response of HCT116 and HPT cells to the SAC inhibitors, reversine (250nM and 500nM) and MPI-0479605 (250nM). The top connection is “Cell cycle inhibition”, correctly identifying the expected mechanism of action of these compounds. GOF, gain of function; OE, over-expression; KD, knockdown. (b) Functional enrichment of gene sets related to cell cycle regulation. Shown are the gene sets that were significantly more affected by SACi in the highly-aneuploid HPT1 and HPT2 cells than in the nearly-diploid HCT116-WT and HCT116-GFP cells. *, p<0.05, one-tailed Fisher’s exact test. (c) Functional enrichment of gene sets related to cell death. Shown are the gene sets that were significantly more affected by SACi in the highly-aneuploid HPT1 and HPT2 cells than in the nearly-diploid HCT116-WT and HCT116-GFP cells. *, p<0.05, one-tailed Fisher’s exact test. (d) The mitotic index of HCT116 and HPT cells cultured under standard conditions or exposed to the SAC inhibitor reversine (500nM) for 24hr. *, p=0.035; n.s., p=0.17; two-tailed t-test; Error bars, s.d.; n=3 biological replicates. (e) Imaging-based quantification of the prevalence of cell divisions with multipolar spindles in HCT116 and HPT cell lines cultured under standard conditions or treated with reversine (500nM) for 24hr; n=3 biological replicates. Error bars, s.d. (f) The prevalence of premature mitotic exit (cytokinesis failure) in HCT116 and HPT cells exposed to the SAC inhibitor reversine (500nM) for 24hr. *, p=0.047; two-tailed Fisher’s exact test. (g) Representative images of premature mitotic exit in HPT2 cells exposed to reversine (500nM). T=0 defines nuclear envelope breakdown (NEB). Scale bar, 10μm. (h) The prevalence of micronuclei formation in RPE1 and RPT cells cultured under standard conditions or exposed to the SAC inhibitor reversine (500nM) for 24hr. n.s., p>0.05; *, p=0.013 and p=0.015 for the differences between the treated and untreated RPT1 and RPT3 cells, respectively; **, p=0.004; ***, p<0.0002; two-tailed t-test. (i) The prevalence of cell divisions with multipolar spindles in RPE1 and RPT cells cultured under standard conditions or exposed to the SAC inhibitor reversine (500nM) for 24hr. n.s., p>0.05; *, p=0.028; two-tailed t-test. Error bars, s.d. (j) The prevalence of premature mitotic exit (cytokinesis failure) in RPE1 and RPT cells exposed to the SAC inhibitor reversine (500nM) for 24hr. *, p=0.044 and p=0.019 for the comparisons between RPE1 and RPT1 or RPT3, respectively; two-tailed t-test. (k) Chromosomal copy number states of HCT116 and HPT cells at each of the 3 time points that were sequenced by scDNAseq. Differences between the pre-treated (d3) and post-treated (d14+3) populations are highlighted. (l) Chromosomal heterogeneity scores of the HCT116 and HPT cells at each of the 3 time points. Highly-heterogeneous chromosomes in the post-treated populations (d14+3) are highlighted; n=23 chromosomes. (m) Comparison of the chromosomal heterogeneity scores between the near-diploid HCT116 cells and the highly-aneuploid HPT cells. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range; circles, individual chromosomes. ****, p=2e-09; two-tailed t-test.

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: Inhibition, Over Expression, Functional Assay, One-tailed Test, Cell Culture, Two Tailed Test, Imaging

Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) Schematics of gene expression profiling. HCT116 and HPT cells were treated with two SAC inhibitors, reversine (250nM and 500nM) and MPI-0479605 (250nM), global gene expression profiles were generated at 6hr, 24hr and 72hr post-drug exposure, and gene set enrichment analysis was performed to compare the transcriptional effect of SACi. (b) Unsupervised hierarchical clustering of the 4 cell lines based on drug-induced transcriptional changes. (c) Time from mitotic arrest to division following nocodazole (200ng/mL) treatment. **, p<0.01; two-tailed t-test. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range; circles, individual cells. (d) Flow cytometry-based quantification of G2/M phase arrest induced by 48hr exposure to MPI-0479605 (250nM). ****, p=1e-05; two-tailed t-test. (e) The prevalence of micronuclei formation in HCT116 and HPT cells cultured under standard conditions or exposed to reversine (500nM) for 24hr. *, p=0.007; ***, p<0.001; two-tailed Fisher’s exact test. (f) Representative images of micronuclei formation in HPT2 cells exposed to reversine (500nM) for 24hr. Scale bar, 10μm. (g) Flow cytometry-based quantification of sub-G1 cell fraction induced by 48hr (left) or 14d (+3d of recovery; right) exposure to MPI-0479605 (250nM). **, p=0.002; ****, p=3e-05; two-tailed t-test. (h) Genome-wide copy number profiles of HCT116 and HPT single cells, as generated by the AneuFinder algorithm, at baseline (untreated; do), after 3d of MPI-0479605 (250nM) treatment (ongoing SACi; d3), and after recovery from 14d of treatment (recovered; d14+3). Individual cells are represented as rows, with chromosomes plotted as columns. In all plots, data represent the mean ± s.d. unless otherwise noted; n=3 biological replicates in all experiments unless otherwise noted.

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: Expressing, Generated, Two Tailed Test, Flow Cytometry, Cell Culture, Genome Wide

Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) Differential mRNA expression levels of mitotic kinesins between HCT116 and HPT cell lines. KIF18A is highlighted in red. (b) Imaging of metaphase spindle in HCT116-GFP and HPT1 cells, Scale bar, 10μm. (c) Spindle length (left), width (middle), and angle (right) in HCT116 and HPT cells. The definitions of length, width and angle are shown in Extended Data Fig. 9c. ***, p<0.001; two-tailed t-test. (d) The percentage of spindle microtubule-bound kinetochores in HCT116 and HPT cells. ***, p<0.001; two-tailed t-test. (e) The sensitivity of near-euploid and highly-aneuploid cancer cell lines to the knockdown of KIF18A in the RNAi-DRIVE dataset. The more negative a value, the more essential the gene is. ***, p=3e-04; two-tailed t-test. (f) Proliferation curves of HCT116-GFP and HPT2 cells cultured in the presence of a KIF18A-targeting siRNA, or a non-targeting control siRNA. (g) The sensitivity of cancer cell lines to the knockdown of KIF18A as a function of their AS. Spearman’s ρ = −0.66 (p=0.026; one-tailed test). (h) The prevalence of cell divisions with multipolar spindles in HCT116 and HPT2 cells treated with KIF18A-targeting siRNAs or a non-targeting control siRNA. n.s., p>0.05; *, p=0.03; two-tailed t-test. (i) Representative images of multipolar spindles in HPT2 cells following siRNA-mediated KIF18A knockdown. Scale bar, 10μm. (j) Proliferation curves of HPT2 cells before and after over-expression of KIF18A (KIF18A-OE), in the absence or presence of MPI-0479605 (250nM). KIF18A-OE increases the inhibitory effect of SACi. (k) A model of the evolving response of aneuploid cancer cells to SACi. For more details, see Supplementary Note 12. In all plots, data represent the mean ± s.d. unless otherwise noted; n=3 biological replicates in all experiments. In all box plots: bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range; circles, individual data points.

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: Expressing, Imaging, Two Tailed Test, Cell Culture, One-tailed Test, Over Expression

Journal: Nature

Article Title: Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition

doi: 10.1038/s41586-020-03114-6

Figure Lengend Snippet: (a) Left: Western blot of KIF18A protein expression levels in HCT116 and HPT cell lines. Right: Quantification of KIF18A expression levels (normalized to GAPDH). **, p=0.002; two-tailed t-test; n=5 biological replicates. (b) Left: Imaging kinetochore-bound KIF18A protein levels in HCT116-GFP, HPT1 and HPT2 cells, Scale bars, 10μm. Right: Immunofluorescence-based quantification of KIF18A protein levels. **, p<0.01, two-tailed t-test. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range. (c) Schematics of the definitions of spindle length, width and angle. (d) Left: Imaging-based quantification of microtubule polymerization rate in HCT116 and HPT cells cultured under standard conditions. Right: Imaging-based quantification of microtubule regrowth following complete depolymerization in HCT116 and HPT cells. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range; circles, individual cell lines. *, p<0.05; **, p<0.01; ****, p<1e-4; two-tailed t-test. (e) Imaging-based quantification of EB1α-tubulin co-localization in HCT116 and HPT cells cultured under standard conditions. **, p<0.01. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range. (f) The sensitivity of near-euploid and highly-aneuploid cancer cell lines to the knockout of KIF18A in the CRISPR-Achilles dataset. The more negative a value, the more essential the gene is in that cell line. *, p=0.034; two-tailed t-test. (g) The sensitivity of near-euploid and highly-aneuploid cancer cell lines to the knockdown of KIF18A in the DRIVE RNAi screen (top vs. bottom 10% of cell lines). The more negative a value, the more essential the gene is in that cell line. ****, p=3e-06; two-tailed t-test. (h) Comparison of the mRNA expression levels of KIF18A between near-euploid and highly-aneuploid cancer cell lines. n.s., p>0.05; two-tailed t-test. (i) Comparison of the protein expression levels of KIF18A between near-euploid and highly-aneuploid cancer cell lines. n.s., p>0.05; two-tailed t-test. (j) The correlation between KIF18A mRNA expression and the genetic dependency on this gene in the Achilles-RNAi screen. Spearman’s ρ = 0.17 (p=0.004) (k) The correlation between KIF18A protein expression and the genetic dependency on this gene in the Achilles-RNAi screen. Spearman’s ρ=0.25 (p=0.009). (l) Relative mRNA expression levels of KIF18A, confirming successful siRNA-mediated KD in all cell lines 72hr post-transfection. **, p=0.006, p=0.003 and p=0.002 for HCT116-GFP, HPT1 and HPT2, respectively; ***, p=0.0007; one-tailed t-test. (m) Proliferation curves of HCT116 and HPT1 cells cultured in the presence of a KIF18A-targeting siRNA, or a non-targeting control siRNA. (n) Comparison of the doubling times of HCT116 and HPT cells following siRNA-mediated KIF18A knockdown. **, p=0.001; two-tailed t-test. (o) Time-lapse imaging-based quantification of the time from nuclear envelope breakdown (NEBD) to anaphase onset in HCT116 and HPT cell lines exposed to non-targeting or KIF18A-targeting siRNAs for 72hr. n.s, p>0.05; **, p=0.003; two-tailed t-test. Bar, median; box, 25th and 75th percentile; whiskers, 1.5 X interquartile range; circles, individual cell lines. (p) The prevalence of micronuclei formation in HCT116 and HPT cells exposed to non-targeting or KIF18A-targeting siRNAs for 72hr. n.s., p>0.05; ***, p<0.001; two-tailed Fisher’s exact test. (q) Relative protein expression levels of KIF18A, confirming successful KIF18 overexpression in the highly-aneuploid HPT1 and HPT2 cell lines 48hr post-transfection. Left: Western blot of KIF18A protein expression levels in HPT1 and HPT2 before and after KIF18A overexpression. Right: Quantification of KIF18A expression levels (normalized to α-Tubulin). *, p=0.013, **, p=0.005; one-tailed t-test; n=2 biological replicates. In all bar plots and line plots, data represent the mean ± s.d. unless otherwise noted; n=3 biological replicates unless otherwise noted. (r) Proliferation curves of HPT1 cells before and after over-expression of KIF18A (KIF18A-OE), in the absence or presence of MPI-0479605 (250nM).

Article Snippet: MPI-0479605 was purchased from MedChem Express (Princeton, NJ, USA), reversine and mitoxantrone were purchased from Sigma-Aldrich (Saint-Louis, MO, USA, and).

Techniques: Western Blot, Expressing, Two Tailed Test, Imaging, Immunofluorescence, Cell Culture, Knock-Out, CRISPR, Transfection, One-tailed Test, Over Expression