Topoisomerase 1 Inhibition in MYC-Driven Cancer Promotes Aberrant R-Loop Accumulation to Induce Synthetic Lethality

Abstract

MYC is a central regulator of gene transcription and is frequently dysregulated in human cancers. As targeting MYC directly is challenging, an alternative strategy is to identify specific proteins or processes required for MYC to function as a potent cancer driver that can be targeted to result in synthetic lethality. To identify potential targets in MYC-driven cancers, we performed a genome-wide CRISPR knockout screen using an isogenic pair of breast cancer cell lines in which MYC dysregulation is the switch from benign to transformed tumor growth. Proteins that regulate R-loops were identified as a potential class of synthetic lethal targets. Dysregulated MYC elevated global transcription and coincident R-loop accumulation. Topoisomerase 1 (TOP1), a regulator of R-loops by DNA topology, was validated to be a vulnerability in cells with high MYC activity. Genetic knockdown of TOP1 in MYC-transformed cells resulted in reduced colony formation compared with control cells, demonstrating synthetic lethality. Overexpression of RNaseH1, a riboendonuclease that specifically degrades R-loops, rescued the reduction in clonogenicity induced by TOP1 deficiency, demonstrating that this vulnerability is driven by aberrant R-loop accumulation. Genetic and pharmacologic TOP1 inhibition selectively reduced the fitness of MYC-transformed tumors in vivo. Finally, drug response to TOP1 inhibitors (i.e., topotecan) significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids. Together, these results highlight TOP1 as a promising target for MYC-driven cancers.

Significance: CRISPR screening reveals topoisomerase 1 as an immediately actionable vulnerability in cancers harboring MYC as a driver oncoprotein that can be targeted with clinically approved inhibitors.

Figure 1.

CRISPR screen identifies R-loop factors as MYC-SLs. A, Schematic outlining the 10A.PE and 10A.PM MYC-driven and -dependent model of breast cancer. In the MCF10A cell line with an activating mutation in the PIK3CA gene, empty vector (10A.PE) or MYC (10A.PM) is ectopically expressed. Dysregulated MYC in 10A.PM cells initiates and sustains transformation in vivo (12). B, Pipeline of the genome-wide CRISPR knockout screen. C, GSEA of MYC-SL hits. Nodes represent clusters of genes with similar biological functions. Colors represent normalized enrichment scores (NES). Size indicates the number of genes per node. D, MYC-SLs include many proteins involved in R-loop regulation, identified as R-loop factors. E, GSEA identifies a significant enrichment of R-loop factors among CRISPR screen hits. The NES and corresponding P value are indicated. F, Mean PCC values between MYC gene expression and the expression of R-loop factors identified in our CRISPR screen. Data points represent PCC values in TCGA cancer cohorts. The color of the boxes represent the statistical significance of PCC values. (A, Created with BioRender.com.)

Figure 2.

Dysregulated MYC increases cotranscriptional R-loop formation. A, EU incorporation assay showing transcription activity following MYC induction. DRB was used as a specificity control. Representative images (left) and quantification (right) of biological replicates (N = 3 biological replicates) are shown. Scale bar, 20 μm. P values were calculated using one-way ANOVA with Tukey HSD post hoc test. B, Representative images (left) and quantification (right) of nuclear R-loops using the S9.6 immunofluorescence assay. N = 3 biological replicates. Scale, 40 μm. P values were calculated using the Student t test. C, Overlap between sites of R-loop formation and MYC-binding sites. Annotation of gene tracks identifies the genomic locations of overlapping peaks. D, STRING protein interaction network containing MYC-SL R-loop factors. Black border represents proteins that have been previously identified in MYC-BioID experiments as putative MYC interactors (38). ****, P < 0.0001.

Figure 3.

Validation of TOP1 as a MYC-SL vulnerability. A, Western blot analysis showing TOP1 knockdown in 10A.PE and 10A.PM. B, Clonogenic assay using 10A.PE and 10A.PM cells with inducible shRNAs. Quantification of relative colony forming units (CFU) between groups. N = 3 biological experiments. Error bars, SD. *, P < 0.05; **, P < 0.01, calculated using the Student t test. C, Clonogenic assay using 10A.PE and 10A.PM cells with inducible shRNAs and stable overexpression of wild-type FLAG-RNaseH1^WT or mutant FLAG-RNaseH1^WKKD. Quantification of relative CFUs between groups. N = 3 biological experiments. Error bars, SD. *, P < 0.05, calculated using the Student t test. D and E, γH2AX immunofluorescence in 10A.PE and 10A.PM cells treated with DMSO or CPT. N = 3 biological experiments. Scale bar, 40 μm. P values were calculated using one-way ANOVA followed by Tukey HSD post hoc test. ****, P < 0.0001. F, Seventy-two hour MTT assay of 10A.PE and 10A.PM cells in response to a concentration-range of CPT at 1:4 dilution starting at 4 μmol/L. IC₅₀ values are shown for each cell line. P values were calculated using the extra-sum-of-squares F test. N = 3 biological experiments. ns, nonsignificant.

Figure 4.

TOP1 is a MYC-SL vulnerability in vivo. A, Schematic of 10A.PM xenograft experiments to evaluate effect of TOP1 inhibition on tumor growth. B, Tumor weights and representative images in 10A.PM xenografts following 18 days of TOP1 knockdown by shRNA compared with control shRNA. *, P < 0.05; **, P < 0.01, calculated using one-way ANOVA followed by Bonferroni multiple comparisons test. C, Mouse tumors were fixed, paraffin embedded, and stained for Ki67. N = 6 mice per treatment group except shTOP1–1, where N = 7. *, P < 0.05, calculated using one-way ANOVA followed by Bonferroni multiple comparisons test. D, Tumor weights from 10A.PM xenografts after topotecan treatment. Representative excised tumors shown for each treatment group. N = 7 mice per treatment group. *, P < 0.05, calculated using the Student t test. (A, Created with BioRender.com.)

Figure 5.

MYC activity correlates with TOP1 inhibitor response in cancer cells. A, Schematic of pipeline to generate MYC target gene enrichment scores in 1,406 cancer cell lines annotated in the CCLE using single-sample GSEAs. Columns are sorted by aggregate row means from low to high. B, Pearson correlation analysis between ssGSEA scores for “HALLMARK MYC TARGETS V1” and topotecan response from the PRISM drug repurposing library. Data points represent cancer cell lines. Gray, SE of linear regression model. C, Pearson correlation coefficient values between drug response and MYC signature enrichment scores in cancer cell lines for each drug shown. Red, TOP1 inhibitors. P values were adjusted for FDR using the Holm–Bonferroni method. D, Bubble plot showing Pearson correlation coefficient values between ssGSEA scores for all HALLMARK gene sets and TOP1 inhibitor drug response by size. P values were adjusted for FDR using the Holm–Bonferroni method and visualized by color.

Figure 6.

Drug response to TOP1 inhibitors is MYC-driven in breast cancer. A, Western blot analysis showing MYC levels in a panel of breast cancer cell lines. Actin was used as a loading control. N = 3 biological replicates. B, Pearson correlation between MYC protein levels and log₁₀-transformed topotecan IC₅₀ values. Gray, SE of linear regression model. C, Heatmap showing MYC characteristics in breast cancer PDOs. Cells are pseudocolored from high (red) to low (white). D, Pearson correlation between MYC target gene signature enrichment scores and log₁₀-transformed topotecan IC₅₀ values. Biological replicates for each PDO are shown. Gray, SE of linear regression model. E, Western blot analysis showing MYC and TOP1 protein levels in a panel of breast cancer PDOs. Models are stratified into MYC-high and MYC-low categories based on relative detectable signal for MYC protein. F, Log₁₀-transformed topotecan and SN-38 IC₅₀ values in MYC-high and MYC-low PDOs. T tests were performed between groups. **, P < 0.01; ***, P < 0.001. G, Representative images of BPTO.95 (MYC-low) and BXTO.143 (MYC-high) PDOs following 100 nmol/L of topotecan. Scale bars, 100 μm. H, Left, representative images of MYC immunohistochemistry signal in MYC-high and MYC-low breast cancer PDOs. Scale bar values are shown per image. Right, quantification of proportion of MYC-positive nuclei across the panel of PDOs. Student t test was used to determine P values. **, P < 0.01. A minimum of 1,000 nuclei from at least 50 individual organoids was scored for each model.

Figure 7.

Working model highlighting the proposed mechanism of TOP1 as a MYC-SL vulnerability. Under nontransformed conditions, depletion of TOP1 results in the resolvable formation of unscheduled R-loops and DNA damage. In cells under MYC-transformed conditions harboring elevated levels of transcriptional activity, TOP1 depletion leads to the intolerable accumulation of R-loops, resulting in the observed synthetic-lethal phenotype.