R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts

Abstract

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer.

Significance: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations.

Figure 1.. SRSF2-mutant leukemia confers sensitivity to PARP1/2 inhibitors.

(A-D) Murine MLL-AF9 Srsf2^+/+ and MLL-AF9 Srsf2^P95H/+ leukemia cells were treated either with DMSO (–) or a panel of pharmacologic inhibitors for 72 hours (n=3–4 independent experiments per inhibitor). Cell viability relative to DMSO was analyzed to generate relative viability heatmaps in (A); differential sensitivity analysis of indicated inhibitors based on normalized area under the curve (AUC) in (B); representative cell viability curve in response to olaparib in (C); and IC₅₀ values for different PARP inhibitors (olaparib, rucaparib, talazoparib, veliparib) in the screen in (D). Statistical analysis was performed using unpaired two-tailed Student’s t-test (**, p<0.01). (E) MLL-AF9 Srsf2^+/+ and MLL-AF9 Srsf2^P95H/+ cells were treated with DMSO or PARPi (500 nM) for indicated days. Viable cell numbers were determined using trypan blue exclusion method. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis using 2-way ANOVA was performed (*, **** indicate p<0.05, p<0.0001, respectively). (F) Cells were treated with DMSO or olaparib (PARPi; 1 μM) for 24h for Annexin V analysis. Error bars represent standard deviation (n=4 independent experiments). One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to adjust for multiple comparison (*, ** indicate p<0.05 and p<0.01, respectively). (G) IC₅₀ of MLL-AF9 Srsf2^+/+ and MLL-AF9 Srsf2^P95H/+ cells for PARP1-specific inhibitor (AZD5305) treatment for 72 hours. Error bars represent standard deviation. Statistical analysis was performed using unpaired two-tailed Student’s t-test (**, p<0.01). (H) MLL-AF9 Srsf2^+/+ and MLL-AF9 Srsf2^P95H/+ cells were genetically depleted of Parp1 using CRISPR-Cas9. Viable cell numbers were determined using trypan blue exclusion method. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis using 2-way ANOVA was performed (n.s. and **** indicate not significant and p<0.0001, respectively). (I) Relative cell viability, left, and IC₅₀ values, right, of K562 SRSF2^WT and SRSF2^P95H cells in response to olaparib for 7 days (n=3–4 independent experiments). Statistical analysis was performed using unpaired two-tailed Student’s t-test (*, p<0.05). (J) K562 SRSF2^WT and SRSF2^P95H cells were treated with either DMSO or PARPi (10 μM) for indicated days and viable cell number was determined using trypan blue exclusion method. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis using 2-way ANOVA method was performed (***, **** indicate p<0.001, p<0.0001, respectively). (K) K562 SRSF2^WT and SRSF2^P95H cells were treated with DMSO or olaparib (10 μM, 24h) for Annexin V analysis. Error bars represent standard deviation (n=4 independent experiments). One-way ANOVA followed by Tukey’s post-hoc test was used to adjust for multiple comparison (n.s., **, ***, indicate not significant, p<0.01, p<0.001, respectively). (L) Kaplan-Meier survival curves of mice transplanted with MLL-AF9 Srsf2^+/+ and MLL-AF9 Srsf2^P95H/+ leukemia cells followed by treatment with vehicle or PARPi (olaparib). The median survival time for each group: MLL-AF9 Srsf2^+/+ + vehicle (22 days; n=9 mice), MLL-AF9 Srsf2^+/+ + PARPi (23.5 days; n=10 mice), MLL-AF9 Srsf2^P95H/+ + vehicle (37 days; n=9 mice), MLL-AF9 Srsf2^P95H/+ + PARPi (48 days; n=9 mice). Mantel-Cox log-ranked test was used to compare differences in survival.

Figure 2.. Spliceosome mutant leukemias confer sensitivity to PARP1/2 inhibitors.

(A-C) Murine MLL-AF9 U2af1^+/+ and MLL-AF9 U2af1^S34F/+ leukemia cells were treated with either DMSO (–) or indicated pharmacologic inhibitors for 72 hours (n=3 independent experiments). Cell viability relative to DMSO was analyzed to generate relative viability heatmaps in (A); differential sensitivity analysis of indicated inhibitors based on normalized area under the curve (AUC) in (B); representative cell viability curve in response to olaparib in (C). Error bars represent standard deviation. Statistical analysis was performed using unpaired two-tailed Student’s t-test (***, p<0.001). (D) Indicated murine cells were treated with DMSO or olaparib (PARPi; 1 μM) for 24 hours followed by Annexin V analysis. Error bars represent standard deviation (n=4 independent experiments). One-way analysis ANOVA followed by Tukey’s post-hoc test was used to adjust for multiple comparison (**, ****, p<0.01, p<0.0001, respectively). (E) Cells were treated with DMSO or PARPi (500 nM) for indicated days. Viable cell numbers were determined using trypan blue exclusion method. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis using 2-way ANOVA was performed (****, p<0.0001). (F) Normalized cell growth inhibition of K562 U2AF1^WT and U2AF1^S34F cells. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis was performed using unpaired two-tailed Student’s t-test (***, ****, p<0.001, p<0.0001, respectively). (G-H) Relative cell viability and IC₅₀ of indicated human leukemia cell lines that are either spliceosome wildtype or mutant were treated with olaparib (G) and rucaparib (H) for 7 days. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis was performed using unpaired two-tailed Student’s t-test (*, p<0.05).

Figure 3.. Increased PARP inhibitor sensitivity in spliceosome-mutant cells is HR-independent.

(A-B) Immunoblot analysis of total Mono-/poly-ADPribosylation (MAR/PAR) levels and PARP1 levels in K562 SRSF2^WT and SRSF2^P95H cells (A) and K562 U2AF1^WT and U2AF1^S34F cells (B) at steady state or following acute olaparib treatment (PARPi, 10 μM for 1 hour). (C-D) Assessment of total MAR/PAR levels following PARP1 knockout in K562 SRSF2^WT and SRSF2^P95H cells (C) and in K562 U2AF1^WT and U2AF1^S34F cells (D). (E-F) K562 SRSF2^WT and SRSF2^P95H cells were treated with DMSO or olaparib (PARPi, 10 μM) for 24 hours for γH2AX immunofluorescence. Representative images and foci number quantification per nucleus (n>2000) are shown in (E) and (F), respectively. Red bars represent the mean in the indicated groups. Statistical analysis was obtained using one-way ANOVA (****, p<0.0001, n.s., non-significant). (G-H) K562 SRSF2^WT and SRSF2^P95H cells expressing either shControl or shBRCA1 were treated with DMSO or olaparib (PARPi,10 μM for 24 hours). Representative images are shown in (G). In H, quantification of the Rad51 foci numbers per nucleus in EdU/γH2AX-double positive cells (n>110, top panel). Red bars represent the median in the indicated groups. Statistical analysis was done using ordinary one-way ANOVA (****, p<0.0001). Bottom, QBIC analysis of indicated cells treated with DMSO or olaparib. The gray boxes highlight EdU/γH2AX positive cells used for Rad51 analysis. (I) Relative cell viability of K562 SRSF2^WT and SRSF2^P95H cells expressing shControl (shCtl) or shBRCA1 cells treated with olaparib for 7 days. Error bars represent standard error of mean (n=9). Statistical analysis using two-way ANOVA was performed (***p<0.001, ****p<0.0001, n.s., non-significant). (J) Assessment of HR repair efficiency by mClover:BFP ratio in K562 SRSF2^P95H or U2AF1^S34F cells relative to their isogenic wildtype cells. Error bars represent standard deviation (n=3). Statistical analysis was performed using unpaired two-tailed Student’s t-test (**p<0.01, n.s., non-significant). (K) Assessment of the HR repair function using the DR-GFP reporter cassette in K562 SRSF2^WT and SRSF2^P95H cells. Error bars represent standard deviation (n=3 independent experiments). Statistical analysis was performed using unpaired two-tailed Student’s t-test (*, p<0.05).

Figure 4.. PARP1’s activity at R loops in SF-mutant cells is critical to prevent PARPi-induced genomic instability.

(A) Representative images of S9.6:PARP1 PLA in indicated cells (scale bar = 5 μm). (B) Quantification of PLA foci numbers per nucleus in (A) (n>900). Red bars represent the median in the indicated groups. Statistical analysis was obtained using ordinary one-way ANOVA (****, p<0.0001). (C) Representative images of S9.6:MAR/PAR PLA in K562 SRSF2^WT and SRSF2^P95H cells treated with either acute olaparib (PARPi, 10 μM for 1h) treatment or e.coli RNase H (eRH) in vitro (scale bar = 5 μm). (D) Quantification of PLA foci numbers per nucleus in (C) (n>500). Red bars represent the mean in the indicated groups. Statistical analysis was obtained using ordinary one-way ANOVA (****, p<0.0001). (E) Representative images of S9.6:MAR/PAR PLA foci in indicated cells, scale bar = 5 μm. (F) Quantification of PLA foci numbers per nucleus (n>500) for each condition in (E). Red bars represent the median in the indicated groups. Statistical analysis was obtained using ordinary one-way ANOVA (****, p<0.0001). (G) Immunoblot analysis of total MAR/PAR, PARP1 and GFP levels following doxycycline-inducible expression of nuclear, GFP-tagged RNase H1^WT in K562 SRSF2^WT and SRSF2^P95H cells (100 ng/mL, 24 hours). (H) K562 SRSF2^WT and SRSF2^P95H cells inducibly expressing RNase H1^WT (400 ng/mL dox) were treated with olaparib (PARPi, 5 μM) or DMSO for 3 days, and the cell growth was normalized to respective DMSO controls (n=3 independent experiments). Statistical analysis was performed using ordinary one-way ANOVA (**, ***, ****, n.s., indicate p<0.01, p<0.001, p<0.0001, non-significant, respectively). (I) Immunoblot analysis of total MAR/PAR and PARP1 levels following doxycycline-inducible expression of nuclear RNase H1^WT in K562 U2AF1^WT and U2AF1^S34F cells (400 ng/mL dox, 24h). (J) K562 U2AF1^WT and U2AF1^S34F cells inducibly expressing nuclear RNase H1^WT by addition of doxycycline (400 ng/mL) were treated with olaparib (PARPi, 5 μM) or DMSO for 5 days, and the cell growth was normalized to respective DMSO controls (n=3 independent experiments). Error bars represent standard deviation. Statistical analysis was performed using ordinary one-way ANOVA (*, **, ***, ****, n.s., indicate p<0.05, p<0.01, p<0.001, p<0.0001, non-significant, respectively). (K) RNase H1^WT and RNase H1^D210N were constitutively expressed in K562 SRSF2^WT and SRSF2^P95H cells and were treated with DMSO or olaparib (PARPi; 10 μM) for 24 hours followed by Annexin V analysis. Error bars represent standard deviation (n=3 independent experiments). Error bars represent standard deviation. Statistical analysis was performed using ordinary two-way ANOVA (*, **, ***, n.s., indicate p<0.05, p<0.01, p<0.001, non-significant, respectively).

Figure 5.. PARPi induces R-loop associated transcription-replication conflicts, rendering spliceosome-mutant cells more sensitive ATR inhibition.

(A-B) K562 SRSF2^P95H cells were treated with DMS or olaparib (PARPi, 3 μM) for 24h and subjected it S9.6 immunofluorescence. Representative images (scale bar = 5 μm) and quantification of S9.6 foci numbers per nucleus (n>1200) are shown in (A) and (B) respectively. Red bars represent the mean in the indicated groups. Statistical analysis was obtained using ordinary one-way ANOVA (****, p<0.0001, n.s., non-significant). (C-D) K562 SRSF2^WT and SRSF2^P95H cells were treated with DMSO or olaparib (PARPi, 3 μM) for 24 hours. RNase H1-GFP expression in SRSF2^P95H cells was induced by addition of doxycycline (100 ng/mL). Representative images of RNA Pol2-pS2:PCNA PLA foci (scale bar = 5 μm) and quantification (n>650) are shown in (C) and (D), respectively. Red bars represent the mean in the indicated groups. Statistical analysis was obtained using one-way ANOVA (****, p<0.0001). (E) K562 SRSF2^WT and SRSF2^P95H cells were treated with DMSO, PARPi (olaparib, 3 μM) alone for 24 hours. ATR inhibitor (AZD6738, 10 μM) was added at the last 1 hour, followed by immunoblot analysis. (F) K562 SRSF2^WT and SRSF2^P95H cells were treated with PARPi+ATRi (3 μM each) or PARPi+ATMi (3 μM each) for 24 hours, followed by immunoblot analysis. (G) K562 SRSF2^WT and SRSF2^P95H cells were treated with either DMSO, olaparib (PARPi, 3 μM), or ATRi (AZD6738, 3 μM) alone, or combined PARP and ATR inhibitors for 24 hours. Doxycycline (200 ng/mL) was added in SRSF2^P95H cells to induce nuclear RNase H1^WT expression for the whole drug treatment duration. (H) Relative cell survival of K562 SRSF2^WT and SRSF2^P95H cells treated with either with DMSO or ATRi (AZD6738, 625 nM) and increasing concentrations of PARPi (olaparib) for 7 days. (I) Synergy maps for PARPi and ATRi combination treatment across six different doses for 4 days human K562 SRSF2^WT and SRSF2^P95H cells (n=3 independent experiments). Synergy score was determined using SynergyFinder 3.0. Loewe score >10 was considered as drug synergism downstream of R-loop response pathways. (J) Heatmaps showing the relative numbers of colony forming units in methylcellulose-containing media using murine MLL-AF9 Srsf2^WT and MLL-AF9 Srsf2^P95H cells following treatment with DMSO, or increasing concentrations of PARPi (olaparib), ATRi (AZD6738) alone or combined for 7 days (n=4 independent experiments). (K) Heatmaps showing the relative cell viability of murine MLL-AF9 U2af1^WT and MLL-AF9 U2af1^S34F leukemia cells treated with varying concentrations of individual PARPi (olaparib), ATRi (AZD6738) or combined for 72 hours (n=3 independent experiments). (L) Relative cell viability measured by CellTiterGlo from Fig. 5K using murine MLL-AF9 U2af1^WT and U2af1^S34F leukemia cells treated either with DMSO or ATRi (AZD6738, 250 nM) and increasing concentrations of PARPi (olaparib) for 72 hours.

Figure 6.. Primary human spliceosome mutant AML cells exhibited increased PARP1 activity at R loops and transcription-replication conflicts, rendering cells sensitive PARP and ATR inhibition in vivo.

(A-B). A total of n=7 spliceosome-wildtype and n=10 spliceosome-mutant (n=5 with an SRSF2 mutation and n=5 with a U2AF1 mutation) human primary acute myeloid leukemia (AML) patient samples were treated with PARPi and ATRi for 96 hours. See Supplementary Table S4 for detailed genetic information from each AML sample. The IC₅₀ values for each drug are shown in (A and B). The top line of the whisker denotes the highest value in the dataset and the bottom line of the whisker denotes the lowest value. The box spans the interquartile range (from 25th-75th percentile) and the line represents the median. Statistical analysis was performed using a two-tailed Mann-Whitney test (**, *** indicate p<0.01, p<0.001, respectively). (C) Synergy maps for combined PARP and ATR inhibition treatment for human primary AML cells. A total of n=4 splicing-wildtype and n=7 splicing mutants were used to determine the Loewe synergy score using SynergyFinder 3.0. (D) Bone marrow engraftment analysis of human CD45⁺ cells in NSG-SGM3 mice in PDX models that were derived from patients that are either splicing wildtype or carry splicing factor mutations after treatment with vehicle or combination of olaparib and AZD6738 (PARPi+ATRi) for 6 weeks. (E) Bone marrow engraftment analysis of human CD33⁺ cells in NSG-SGM3 mice in PDX models that were derived from patients that are either splicing wildtype or carry splicing factor mutation after treatment with vehicle or combination of olaparib and AZD6738 (PARPi+ATRi) for 6 weeks. Statistical analysis was performed using unpaired two-tailed Student’s t-test (n.s., * indicate not significant and p<0.05, respectively). (F) Representative images of S9.6:MAR/PAR PLA in primary AML cells that are either splicing wildtype or carrying the splicing mutations. Scale bar = 5 μm. (G) Quantification of the number of S9.6:MAR/PAR PLA foci per nucleus (n>300) for each experimental condition illustrated in (F). Red bars represent the mean in the indicated groups. Statistical analysis was obtained using two-way ANOVA with Tukey’s multiple comparisons test (****, p<0.0001). (H) Representative images of RNA Pol2-pS2:PCNA PLA in in primary AML cells that are either splicing wildtype or carry splicing mutations. Scale bar = 5 μm. (I) Quantification of the number of RNA Pol2-pS2:PCNA PLA foci per nucleus (n>500) for each condition in (H). Red bars represent the median in the indicated groups. Statistical analysis was obtained using two-way ANOVA with Tukey’s multiple comparisons test (****, p<0.0001).

Figure 7.. Proposed model for PARP1 function as a sensor of R loops in preventing transcription-replication conflicts in SF-mutant leukemias.

SRSF2- and U2AF1-mutant leukemias exhibited R-loop accumulation, causing transcription-replication conflicts. PARP1 senses and mediates ADP-ribosylation at R loops to prevent R-loop-induced genomic instability (top). In the presence of PARPi (middle), PARP1 is inactive and accumulates further R loops in SF-mutant cells. Aberrant R-loop accumulation causes more transcription-replication conflicts, leading to enhanced ATR response (bottom). Consequently, combining PARP and ATR inhibitors synergistically kill SF-mutant leukemias.