Kub5-Hera RPRD1B Deficiency Promotes "BRCAness" and Vulnerability to PARP Inhibition in BRCA-proficient Breast Cancers

Abstract

Purpose: Identification of novel strategies to expand the use of PARP inhibitors beyond BRCA deficiency is of great interest in personalized medicine. Here, we investigated the unannotated role of Kub5-Hera^RPRD1B (K-H) in homologous recombination (HR) repair and its potential clinical significance in targeted cancer therapy.

Experimental design: Functional characterization of K-H alterations on HR repair of double-strand breaks (DSB) were assessed by targeted gene silencing, plasmid reporter assays, immunofluorescence, and Western blots. Cell survival with PARP inhibitors was evaluated through colony-forming assays and statistically analyzed for correlation with K-H expression in various BRCA1/2 nonmutated breast cancers. Gene expression microarray/qPCR analyses, chromatin immunoprecipitation, and rescue experiments were used to investigate molecular mechanisms of action.

Results: K-H expression loss correlates with rucaparib LD₅₀ values in a panel of BRCA1/2 nonmutated breast cancers. Mechanistically, K-H depletion promotes BRCAness, where extensive upregulation of PARP1 activity was required for the survival of breast cancer cells. PARP inhibition in these cells led to synthetic lethality that was rescued by wild-type K-H reexpression, but not by a mutant K-H (p.R106A) that weakly binds RNAPII. K-H mediates HR by facilitating recruitment of RNAPII to the promoter region of a critical DNA damage response and repair effector, cyclin-dependent kinase 1 (CDK1).

Conclusions: Cancer cells with low K-H expression may have exploitable BRCAness properties that greatly expand the use of PARP inhibitors beyond BRCA mutations. Our results suggest that aberrant K-H alterations may have vital translational implications in cellular responses/survival to DNA damage, carcinogenesis, and personalized medicine.

Figure 1:. K-H loss compromises homologous recombination (HR)-mediated DSB repair.

(A) K-H deficiency transcriptome signature obtained using Ingenuity’s Pathway Analysis (IPA) of all mRNAs altered at least 2-fold after K-H depletion. ^§Top five resulting pathways were similar to the top five transcriptome molecular and cellular pathway signatures obtained for loss of BRCA1, RAD51, or BRIT1 – termed HR Deficiency (HRD) Gene Signature. (B) shSCR 231 cells were treated with siK-H and whole cell lysates analyzed by Western blotting. (C, D) Western blot analyses of (C) HCC1569 or (D) 231 breast cancer cells treated with siSCR or siK-H. (E) Homologous recombination (HR) plasmid assays of HEK293 cells treated with siSCR, siRad51, siCDK1 or siK-H oligomers ± CDK1 cDNA using the DR-GFP reporter system. Data are %means GFP+ cells normalized to siSCR, ± %SEM from three independent experiments; *p<0.05, ns = not significant.

Figure 2:. K-H promotes transcription by recruiting RNAPII to the CDK1 promoter.

(A, B) K-H and CDK1 mRNA expression levels in (A) 231 or (B) HCC1569 cells 48 h after siSCR or siK-H (3’-UTR) treatments. (C) K-H and CDK1 mRNA levels in stable shK-H or shSCR 231 cells. Data are means ± SEM from three independent experiments. (D) Top, Western blot analyses of K-H and CDK1 protein levels in shK-H (ORF) or shSCR 231 cells. Lamin B, loading control for nuclear protein extracts. Bottom, ChIP assessment of RNAPII recruitment to CDK1 or actin promoter regions in shK-H vs shSCR 231 cells. Primers adjacent to indicated promoters are listed in Supplementary Table S6. Data are means, ± SEM from three separate experiments. Statistics: ***p<0.001; **p<0.01; ns = not significant.

Figure 3:. K-H associates with RNAPII CTD (pS2) to enhance CDK1 expression.

(A) Left, Co-IP pull-down of endogenous RNAPII by endogenous K-H protein; Right, Structural representation of K-H bound to a heptapeptide fragment of RNAPII C-terminal domain (CTD). Binding is attained via hydrogen bonding and electrostatic interactions between the negatively-charged phosphorylated Ser2 of RNAPII and positively-charged Arg106 side chain. (B) Co-IP pull-down of endogenous RNAPII by myc-tagged wild-type K-H protein. (C) Myc-tagged K-H R106A mutant co-IP shows reduced pull-down of RNAPII due to loss of crucial protein-protein interactions. (D) Western blot analyses of CDK1, total and pS1497 BRCA1 protein levels in stable shK-H 231 cells after forced ectopic expression of wild-type vs mutant R106A K-H, empty pCMV vector or mock-transfection. CDK1-dependent reporter activity assays in (E) shSCR 231 or (F) HCC1569 BRCA1-proficient breast cancer cells treated with siK-H, siSCR or transfected with vector alone CMV-K-H wild-type or K-H R106A mutant cDNAs. Unless specifically indicated, p-values were calculated relative to control (e.g. empty vector or siSCR). Data are means, ± SEM from three separate experiments; *p<0.05; ***p<0.001; ns = not significant.

Figure 4:. Kub5-Hera (K-H) depletion activates PARP1 activity, rendering synthetic lethality with PARP1 inhibition.

(A) Relative PARP enzymatic activity [PAR formation/Time (sec)] for parental and stable K-H (shK-H), PARP1 (shPARP1) or Scrambled (shSCR) knockdown 231 cells. All activities were inhibited by Rucaparib, a PARP inhibitor. (B) Clonogenic survival of parental and indicated stable shRNA knockdown 231 cells treated ± Rucaparib (15 μM, 24 h). Data are %means, ± SEM from three independent experiments. ***p<0.001. (C) Cell death (%Apoptosis) of 231 cells described in (B) assessed by TUNEL+ staining after DMSO or Rucaparib treatments. (D) Top, Clonogenic survival (CFAs) of 231 cells as indicated. Bottom, Western blot analyses of shK-H or shSCR 231 cells transiently transfected with siPARP1 or siSCR. (E) Top, Clonogenic Survival of 231 cells as indicated. Bottom, Western blot analyses of stable shSCR or shPARP1 231 cells transiently transfected with siK-H or siSCR. (F) Western blot analyses of shPARP1 or shSCR 231 cells transiently transfected with siK-H or siSCR at indicated times. (G) Apoptosis (%TUNEL+ staining) from cells in (F). Data are %means, ± SEM from three independent studies; *p<0.05; **p<0.01; ***p<0.001.

Figure 5:. Hypersensitivity of K-H-depleted cells to Rucaparib is caused by persistent DSBs.

(A) Rate of persistent DSB (co-localized γ-H2AX and 53BP1 foci/nuclei) formation in stable shK-H or shSCR 231 cells after Rucaparib or vehicle only treatment for 24 h. Data are %mean of nuclei with ≥5 co-localized γ-H2AX/53BP1 foci, ± SEM from two independent experiments. (B) Representative images for RAD51 and γ-H2AX foci formation, surrogate DSB markers, in stable shK-H or shSCR 231 cells after Rucaparib or vehicle (0.01% DMSO) treatment for 24 h. DAPI stained cell nuclei. Scale bars: 10 μm. (C) Quantification of cells (>50 nucleus) with ≥10 co-localized RAD51 and γ-H2AX foci formation following treatment with Rucaparib (5 μM, 24 h). (D) Western blot comparison of shK-H 231 cells transiently transfected with K-H wild-type versus K-H R106A mutant cDNA for 48 h. After transfection, cells were treated with vehicle (0.1% DMSO) or Rucaparib (5 μM) for 24 h. Total cellular proteins from each treatments were subjected to Western blot analysis using indicated antibodies. (E) Quantitation of relative amount of DSBs (relative γ-H2AX normalized to vehicle) in (D). (F) Top, Clonogenic survival (CFAs) of Rucaparib-treated shK-H 231 cells transiently overexpressed with empty vector, CDK1, K-H WT, or K-H R106A mutant cDNA as indicated. Bottom, representative plates of surviving colonies after treatment with Rucaparib (5 mM) or DMSO for 24 h. Data are means, ± SEM. *p<0.05; **p<0.01; ***p<0.001; ns = not significant.

Figure 6:. K-H loss as a potential functional biomarker for synthetic lethality in BRCA-proficient breast cancers due to PARP inhibition.

(A) Array-CGH (aCGH) analyses of K-H gene copy number variation in a panel of human breast cancer cells. (B) K-H mRNA expression vs aCGH. p=0.0089, R=0.88. (C) K-H mRNA expression vs K-H protein levels in a subset of breast cancer cells selected based on gain or loss of K-H copy number. p=0.007, R=0.89. (D) Western blot analyses of K-H, CDK1, and PARP1 protein levels in various breast cancer cells. Hypersensitivities to Rucaparib are summarized in Table S2. (E) Rucaparib LC₅₀ values vs relative K-H protein levels in BRCA-proficient breast cancer cell lines. p=0.008, R=0.81. (F) Rucaparib LC₅₀ values vs relative CDK1 protein levels in BRCA-proficient breast cancer cell lines. p=0.038, R=0.69. See Supplementary Table S2 for additional information.