Functional ex vivo assay to select homologous recombination-deficient breast tumors for PARP inhibitor treatment.

PURPOSE: Poly(ADP-ribose) polymerase (PARP) inhibitors are promising targeted treatment options for hereditary breast tumors with a homologous recombination (HR) deficiency caused by BRCA1 or BRCA2 mutations. However, the functional consequence of BRCA gene mutations is not always known and tumors can be HR deficient for other reasons than BRCA gene mutations. Therefore, we aimed to develop a functional test to determine HR activity in tumor samples to facilitate selection of patients eligible for PARP inhibitor treatment. EXPERIMENTAL DESIGN: We obtained 54 fresh primary breast tumor samples from patients undergoing surgery. We determined their HR capacity by studying the formation of ionizing radiation induced foci (IRIF) of the HR protein RAD51 after ex vivo irradiation of these organotypic breast tumor samples. Tumors showing impaired RAD51 IRIF formation were subjected to genetic and epigenetic analysis. RESULTS: Five of 45 primary breast tumors with sufficient numbers of proliferating tumor cells were RAD51 IRIF formation deficient (11%, 95% CI, 5%-24%). This HR defect was significantly associated with triple-negative breast cancer (OR, 57; 95% CI, 3.9-825; P = 0.003). Two of five HR-deficient tumors were not caused by mutations in the BRCA genes, but by BRCA1 promoter hypermethylation. CONCLUSION: The functional RAD51 IRIF assay faithfully identifies HR-deficient tumors and has clear advantages over gene sequencing. It is a relatively easy assay that can be performed on biopsy material, making it a powerful tool to select patients with an HR-deficient cancer for PARP inhibitor treatment in the clinic.

An ex vivo assay of XRT-induced Rad51 foci formation predicts response to PARP-inhibition in ovarian cancer.

OBJECTIVE: BRCA-positive ovarian cancer patients derive benefit PARP inhibitors. Approximately 50% of ovarian cancer tumors have homologous recombination (HR) deficiencies and are therefore "BRCA-like," possibly rendering them sensitive to PARP inhibition. However, no predictive assay exists to identify these patients. We sought to determine if irradiation-induced Rad51 foci formation, a known marker of HR, correlated to PARP inhibitor response in an ovarian cancer model. METHODS: Ovarian cancer cell lines were exposed to PARP-inhibitor ABT-888 to determine effect on growth. Rad51 protein expression prior to irradiation was determined via Western blot. Cultured cells and patient-derived xenograft tumors (PDX) were irradiated and probed for Rad51 foci. In vivo PDX tumors were treated with ABT-888 and carboplatin; these results were correlated with the ex vivo ionizing radiation assay. RESULTS: Three of seven cell lines were sensitive to ABT-888. Sensitive lines had the lowest Rad51 foci formation rate after irradiation, indicating functional HR deficiency. Approximately 50% of the PDX samples had decreased Rad51 foci formation. Total Rad51 protein levels were consistently low, suggesting that DNA damage induction is required to characterize HR status. The ex vivo IR assay accurately predicted which PDX models were sensitive to PARP inhibition in vitro and in vivo. ABT-888 alone reduced orthotopic tumor growth by 51% in A2780ip2 cell line, predicted to respond by the ex vivo assay. Three PDX models' response also correlated with the assay. CONCLUSIONS: The ex vivo IR assay correlates with response to PARP inhibition. Analysis of total Rad51 protein is not a reliable substitute.

Tissue-engineered mucosa is a suitable model to quantify the acute biological effects of ionizing radiation.

The aim of this study was to evaluate the suitability of tissue-engineered mucosa (TEM) as a model for studying the acute effects of ionizing radiation (IR) on the oral mucosa. TEM and native non-keratinizing oral mucosa (NNOM) were exposed to a single dose of 16.5Gy and harvested at 1, 6, 24, 48, and 72h post-irradiation. DNA damage induced by IR was determined using p53 binding protein 1 (53BP1), and DNA repair was determined using Rad51. Various components of the epithelial layer, basement membrane, and underlying connective tissue were analyzed using immunohistochemistry. The expression of cytokines interleukin-1ß (IL-1ß) and transforming growth factor beta 1 (TGF-ß1) was analyzed using an enzyme-linked immunosorbent assay. The expression of DNA damage protein 53BP1 and repair protein Rad51 were increased post-irradiation. The expression of keratin 19, vimentin, collage type IV, desmoglein 3, and integrins α6 and ß4 was altered post-irradiation. Proliferation significantly decreased at 24, 48, and 72h post-irradiation in both NNOM and TEM. IR increased the secretion of IL-1ß, whereas TGF-ß1 secretion was not altered. All observed IR-induced alterations in TEM were also observed in NNOM. Based on the similar response of TEM and NNOM to IR we consider our TEM construct a suitable model to quantify the acute biological effects of IR.

The receptor tyrosine kinase inhibitor amuvatinib (MP470) sensitizes tumor cells to radio- and chemo-therapies in part by inhibiting homologous recombination.

BACKGROUND AND PURPOSE: RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR. MATERIALS AND METHODS: Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay. RESULTS: Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells. CONCLUSIONS: Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.

Functional characterization of EMSY gene amplification in human cancers.

The 11q13-q14 locus is frequently amplified in human cancers, with a complex structure harbouring multiple potential oncogenic drivers. The EMSY gene has been proposed as a driver of the third core of the 11q13-q14 amplicon. This gene encodes a protein reported to be a BRCA2-binding partner, which when over-expressed would lead to impairment of BRCA2 functions and could constitute a mechanism for BRCA2 inactivation in non-hereditary breast and ovarian cancers. We hypothesized that if EMSY amplification abrogates BRCA2 functions, cells harbouring this aberration would be unable to elicit competent homologous recombination DNA repair and, therefore, may have increased sensitivity to genotoxic therapies and potent PARP inhibitors. Microarray-based comparative genomic hybridization of cell lines from distinct tumour sites, including breast, ovary, pancreas, oesophagus, lung and the oral cavity, led to the identification of 10 cell lines with EMSY amplification and 18 without. EMSY amplification was shown to correlate with EMSY mRNA levels, although not all cell lines harbouring EMSY amplification displayed EMSY mRNA or protein over-expression. RNA interference-mediated silencing of EMSY did not lead to a reduction in cell viability in tumour models harbouring EMSY amplification. Cell lines with and without EMSY amplification displayed a similar ability to elicit RAD51 foci in response to DNA damaging agents, and similar sensitivity to cisplatin and olaparib. Taken together, this suggests that EMSY is unlikely to be a driver of the 11q13-q14 amplicon and does not have a dominant role in modulating the response to agents targeting cells with defective homologous recombination.

Compensatory role for Rad52 during recombinational repair in Ustilago maydis.

A single Rad52-related protein is evident by blast analysis of the Ustilago maydis genome database. Mutants created by disruption of the structural gene exhibited few discernible defects in resistance to UV, ionizing radiation, chemical alkylating or cross-linking agents. No deficiency was noted in spontaneous mutator activity, allelic recombination or meiosis. GFP-Rad51 foci were formed in rad52 cells following DNA damage, but were initially less intense than normal suggesting a possible role for Rad52 in formation of the Rad51 nucleoprotein filament. A search for interacting genes that confer a synthetic fitness phenotype with rad52 after DNA damage by UV irradiation identified the genes for Mph1, Ercc1 and the Rad51 paralogue Rec2. Testing known mutants in recombinational repair revealed an additional interaction with the BRCA2 orthologue Brh2. Suppression of the rec2 mutant's UV sensitivity by overexpressing Brh2 was found to be dependent on Rad52. The results suggest that Rad52 serves in an overlapping, compensatory role with both Rec2 and Brh2 to promote and maintain formation of the Rad51 nucleoprotein filament.

DNA repair after irradiation in glioma cells and normal human astrocytes.

We examined DNA damage responses and repair in four human glioma cell lines (A7, U87, T98G, and U373) and normal human astrocytes (NHAs) after clinically relevant radiation doses to establish whether we could identify differences among them that might suggest new approaches to selective radiosensitization. We used phosphorylation of histone H2AX visualized by immunocytochemistry to assess DNA double-strand break (DSB) formation and resolution. Fluorescence immunocytochemistry was used to visualize and quantify repair foci. Western blotting was used to quantify repair protein levels in the different cell lines before and after irradiation and during different cell cycle phases. Mitotic labeling was used to measure cell cycle parameters after irradiation. We found that the glioma cell lines repaired DSBs more slowly and less effectively than did NHAs in the clinically relevant dose range, as assessed by induction and resolution of H2AX phosphorylation, and this was most marked in the three TP53-mutated cell lines (T98G, A7, and U373). The glioma cells also expressed relatively high repair-protein levels compared with NHAs that were not altered by irradiation. High levels of the repair protein Rad51 in these cells persisted throughout the cell cycle, and a marked increase in Rad51 foci formation, which was not restricted to cells in G2/S phase, occurred at early time points after irradiation. TP53-mutated glioma cell lines demonstrated a very prominent dose-responsive G2 checkpoint and were sensitized to radiation by caffeine, which inhibits G2/S phase checkpoint activation. In conclusion, DNA repair events differed in these four glioma cell lines compared with NHAs. In particular, the three TP53-mutated glioma cell lines exhibited markedly increased Rad51 protein levels and marked, dose-dependent Rad51 foci formation after low radiation doses. This suggests that agents that disrupt Rad51-dependent repair or prevent G2 checkpoint activation may selectively sensitize these cells.

The rad51-K191R ATPase-defective mutant is impaired for presynaptic filament formation.

The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. We suggest the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding.